Section 1 Biotechnology

### **Chapter 1**

## Inventoried Yeast Species in Algeria

*Abderrahmane Benkhalifa*

### **Abstract**

In Algeria, the study of yeasts remained marginalized for a long time. One of the supposed causes would be the reduction of examples in the school system. In medicine, species are identified because of their pathogenicity. But in food production and other fields, yeasts are mentioned as quantification in the quality-control process as well as molds. In addition to homemade breads, traditions of fermentation involving yeasts are the process of other local products, such as dairy products, vinegars, beverages, and authentic cheeses. Yeasts affect fruits and plants but also increase yields and protect species from other pathogenic microorganisms. Some conscious researchers have looked into the prospecting of yeast showing their properties and evaluating their interest, adopting biotechnology approaches, and covering several environments. 131 taxa are listed in this first compilation with 27 species in human health, 30 in animal health, 27 from dairy products, 24 taxa isolated from soils, 41 from agricultural products, and 17 concerned environmental purposes. Biotechnologies concern 44 taxa in varied topics of biomass, ethanol, vinegar, enzymes, and esters. Sixteen selected natural products inhibit 14 yeast species. Many isolated strains are promising in agriculture, agri-food, and biotechnologies and present new economic prospects. The idea of national depository is proposed.

**Keywords:** yeasts, biodiversity, Algeria, new economies, zymology

### **1. Introduction**

Inventory of yeasts went from around 500 species in the mid-1980s to over 1500 species in 2010–2011 [1]. Biosystematics approaches leave many taxa in the midst of perpetual updating of their phylogenetic affiliations. Almost all species are hectic in the panoply of appellations. Moreover, interest in yeasts has widened considerably, since their development has opened up new horizons in various biotechnological uses, including enzymes and biofuels [2, 3]. The inventory of yeasts and their outlets is enriched each year with new species and new applications. Specialists know that only a tiny part is discovered [4]. In Algeria, the yeast inventory has not yet been established. We do not pretend to do it in a such rapid trial, but we try to perfect our first issue [5]. Within the national reports implementing the Convention on Biodiversity (CBD), the inventory of mushrooms needs serious updating. That of yeasts is totally neglected because it is included within fungi and they stay much less explored. Fungi and yeast remain limited in view of the attractiveness of aromatic and edible plants or those mentioned in traditional care, which are potentially targeted for their beneficial impact on human health. But looking at cultural heritage, homemade bread, vinegar,

and drinks are very rich in know-how. Local peoples conserve the use of fermented foods [6], vinegar [7], and varied dairy products [8–11]. In addition to home beard, Matmoura is an exceptional preservation mode of fermented wheat [12]. It is used to prepare a famous couscous called Hamoum, whose probiotic property has been proven [13].

We are undertaking a review here to highlight the richness and interest of yeasts in order to align them with the rest of the inventories of fauna, flora, and other micro-organisms. We cover those of human and animal infections and those identified in research work devoted to food processes and fermented products as well as environmental studies. We seek to promote identified taxa or isolated strains within national research studies. It demonstrates the links between ancestral practices, interesting local products from traditions, and their socioeconomic issues. In parallel, we focus on the educational strategy to upgrade the standardization of taxa and revitalize their management as real segments of biodiversity at the service of the economy.

### **2. Methodology**

To enrich the list of inventoried yeast species in Algeria, we spontaneously explored scholar literatures using Research gate and Google Scholar providing references in varied disciplines. They let quick access to what Algerians publish because they are the first concerned with this awareness documentation. Keywords were; yeast, fermentation, fermented foods, enzyme production, selected yeast strains... We searched through free academic search engines; PubMed, Isidor Pascal-Archives, and Agritrop. To complete local documents, such as theses and masters, we used the Algerian theses website (https://www.theses-algerie.com/), which is an aggregator portal of national universities and research institutions. We try to constitute a common list with actual taxonomic names but also with respect to those used by authors. Thus, we mentioned in some case synonyms, or the link between anamorphic and teleomorph forms. To verify favorite appellations, we used the taxonomic browser of the NCBI or the Mycobank database web site.

### **3. Yeasts between anthropological practices and modern economy**

In addition to bread and vinegar, fundamental work is essential to enlighten the choice between traditions and modernity. There are major stakes in terms of impact on health and economy. This leads us to look into the inventory of *taxa* or even strains linked to the development of traditional leavens or the manufacture of baker's yeast in the face of the thorny problem of its importation.

### **3.1 Baker's yeast import**

According to the National News Agency [14], requirement of baker's yeast is around 150,000 tons and we import over 100 million tons. Available data are those reported by the World Bank [15]. Top exporters to Algeria are presented in **Table 1**. The decrease observed during 2019 and 2020 is recovered in 2021 with an increase in the total imported rate. Top six countries export to Algeria less of 50,000 tons what donot corroborate with media divulgation.


*Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

**Table 1.**

 *Top yeast exporters to Algeria (2018–2021) [15] compared to trade value from national customs reports [16].*

From the National Customs reports, data concerning the quantity are not available in the consulted references [16]. Trade values are higher due to the grouping under reported item yeasts with other micro-organisms and baking powders. Public opinion is surprised that national production does not meet our industrial and domestic needs. Yet, giant infrastructures are established in Algeria, including that of Bechegouf in Guelma which should produce 30% of the national need [17].

### **3.2 Homemade sourdough and traditional breading**

Through social media, there are more initiatives to promote homemade sourdough. Thus have benefited volunteers whose make efforts in showing incredible fantasies of traditions in bread making as well as other subjects of ancestral food process [18]. Sometimes, it is accepted to improve understanding and adherence with time respect and temperature control as the use of cold storage equipment. One of the YouTube descriptions is given by Y. Sellam-Benlemaalem, an agronomist basically. Having worked as a teacher of ecology for a long time, she become a recognized chef for defending local culinary traditions while accepting modernity. In her description of sourdough, she reminds us of a rare process of preserving sourdough by drying it after flattening and cutting it into thin slices, to be preserved for several years. https:// www.youtube.com/watch?v=lu\_FRmzBe-M&t=256s

Rural families prepare their own sourdough. This practice has been resumed in particular during recent years of the spread of COVID-19. Vernacular names are "Khemira Beldya" homemade sourdough; "Khemirat Dar," housemade sourdough; "Khemira Mahaliyah": local sourdough; "Khemira Arbiyah" Arabic Sourdough. All indicate traditional sourdough despite the fact that this practice oscillates, jealously, between maintenance and abandonment among young people and those in urban areas, particularly [19]. Homemade sourdough can be obtained automatically by soaking and emitting whole wheat flour from wheat and barley or by adding diverse sources of inoculum (Annexe I) like dates, figs, beans, watermelon juice, vinegar, whey, and curdled milk as well as fruits, leaves, and stems of some spontaneous plants, which are generally not toxic or have harmful effects such like sorrel plants *Rumex bucephalophorus* or *Rumex acetosa*. These species are widespread in rainy regions throughout North Africa and are known for their sour taste. This is the reason why it is called in Arabic "Hammeida or Hommaydha" meaning acid taste. Another originality concerns sourdough from beans inoculation, which is used in particular in Tlemcen and Oran (Western Algeria), to prepare remarkable rich soup. Unfortunately, this recipe was replaced with the use of manufactured yeast and lemon juice [20]. Actually, this tradition collapses day after day.

Sourdough starter still simple and easy to prepare—you need water and flour according to your preference. The spontaneous bacterial and yeast present in the air or that exist in the flour, especially wholemeal, will do the rest (**Figure 1**). If not, we add to it inoculum showed before but without manufactured yeast.

Under an optical microscope, the traditional sourdough shows different kind of cellular forms, oval and ovoid, and a few crescent shapes, lenticels (**Figure 2**). Smaller cylindrical shapes with interconnected spheroids in the form of rosaries are likely bacterial species. It is delicate to examine sourdough, but with dilution, we do not consider starch granules. Wild *Saccharomyces* cells can be with different diameters ranging from single to double. Sourdough is fermented by wild strains of yeast and bacteria spontaneously. *Saccharomyces cerevisiae* constitutes the species responsible for sourdough processing. It is shown that sourdough strains, which are diploid, have

### **Figure 1.**

*Sourdoughs obtained using (a) hard wheat flour (left) (b) hard wheat, soft wheat and barley flours (right).*

**Figure 2.** *Morphology of spontaneous cells observed from sourdough under a Carl Zeiss Axiostar microscope × 1000.*

high copy number of genes able to use maltose, whereas industrial bakery strains are tetraploid with a rapid fermentation onset and are more efficient in CO2 production [20]. This suggests an interest in the selection of spontaneous strains from an industry-oriented performance evaluation perspective.

In El-Oued (South-eastern Algeria) the sourdough is dried due to the favorable drying conditions in the Sahara. It is then stored in powder form like commercial yeast (**Figure 3**). This technique has become teached in recent years in the vocational training institute. The gritty brownish color is due to the use of dates in the sourdough flour mixture.

### **3.3 Traditional vinegar**

In Algeria, vinegar is a particular case of fermentation because it is obtained from diversified sources of fruits, such as dates, apple and sometimes from pomegranate (**Figures 4** and **5**). The new one is obtained from the Indian fig (known as prickly pear), the fruit of *Opuntia ficus-indica*. Between homemade vinegars, or theses

### **Figure 3.**

*Powder sourdough obtained from local wheat flour mixed with dates (prepared by Ali Menai at the professional training Institute in El-Oued, Algeria).*

**Figure 4.** *Left to right: Vinegar from apple (a), dates (b,c), and prickly pear (d).*

proposed by companies and chemical vinegars, there are choices to be favored. In the case of the date vinegar, the fruit biomass is added with a few of other ingredients (wheat seeds, barley seeds, harmel or wild rue seeds, coriander seeds, a pinch of salt and a pinch of chili, and also two iron nails). Then, it is emerged in double quantity of water and maintained in preservation within 40–45 days [7]. During this time is done the conversion of sugar into alcohol and with the presence of acetic bacteria it transform by oxidization into vinegar, mainly with a concentration of acetic acid and distinct flavors due to the fruit parameters and the presence of other acids (malic acid in case of apple). In the case of dates, in small quantities, acetic acid, butandiol, propanone [21], amino acids, vitamins, and formic acid are also present [7]. During meals, the consumption of vinegar gives a feeling of fullness and thus limits the

**Figure 5.** *Vinegar manufacturing in El-Oued (Algeria) with a capacity of 3000 L.*

quantities of food consumed or exaggerated. Vinegar has many properties with positive health impacts. It is reported that vinegar: 1) regulates blood sugar by improving insulin secretion, 2) curbs obesity by suppressing fats accumulation, 3) could increase HDL-cholesterol and diminishes LDL-cholesterol levels, and 4) inhibits proliferation and induces apoptosis in human cancer cells [22]. In traditional care, vinegar reduces fevers essentially. According to an ethnological survey of families in Ghardaia, more than 20 cases of date vinegar virtues are registered [23]. Like, in sourdough, we should focus also very carefully on the type of yeast that we should choose among the spontaneous yeasts, which could be observed in the traditional way or others among the strains showing better transformation of sugar into alcohol. Recently, it is demonstrated that a selected *Kasachstania unispora* strain showed performance in sourdough environments compared with commercial *S. ceverisiae,* in particular, under stress condition as acetic acid concentration, ethanol, or salinity [24].

### **3.4 Dairy products**

The government gives specific importance to milk importation in regard to the consumption needs of more than 1500 MUS\$ in 2020 (**Table 2**). This represents 15–20% of importation fees. Cheese and similar takes more than 7–8% of dairy products (**Figure 6**).

Algerian milk production is ensured by cows, sheep, goats, and a small part by camels. Cow's milk constitutes the highest share of production with more than 71% but due to the need for food, the production of cow's milk is decreasing (**Figure 5**). That of sheep and goats is maintained to ensure a little less than the third. Camel milk hardly exceeds 15,000 tons, but it remains essential to Bedouins in arid and Saharan regions. The total quantities expressed in tones are much lower than what is requested by a population that will reach 50 million inhabitants in the next few years. With 3.3 million tons per year, the maximum quota is less than 2 liters per person per week. Thus, we


**Table 2.**

*Trade values of imported milk and dairy products in Algeria (2018–2020) [16].*

### **Figure 6.**

*Production of milk in tons in Algeria 2016–2020 (source FAO stat).*

have to review the mode of consumption. Milk transformation to other products offers an economic best consumption. Fermentation is one of the success keys. Sure, there is a complexity between the import, supported by the government, and the development of local breeding. Sustained imports may not remain the ideal solution at all times. In both urban and rural areas, residents need to practice milk preservation and milk processing to prolong their nutritional components and balance their savings.

Like humans, animal health is essential. The mastitis is given as a prior problem. Dairy products are fragile and face contaminations risk. Safe row milk or fermented milk depends on respect of traditional processing like in control of modern cheeses. Cow's milk is essential to ensure the large needs but it decreases as the food load becomes more and more expensive. Algeria is mainly affected by drought and must do its utmost to fight against desertification. But, there are many types of waste that require transformation. Biomass is geared toward animal feed or other needs like energy. In addition to local knowledge, yeast should help us achieve attended goals of milk production. There are at least 20 dairy products (Annexe II). The richness of vocabulary proves how people depend on dairy products [8, 10, 25–36]. Named products have been repeatedly described in various works but they havenot been developed yet. We are proud of local products as nostalgia but we still have to deepen their social and economic characteristics to guarantee the legacy on a healthy and profitable basis. Even large-scale, family-based milk producers extract butter to be consumed or processed into cooked butter called Smen/Dhan [8, 10, 25, 26]. Those are the two products extracted from milk as lipid components, and they represent a heritage food tradition. Indication of non-identified species in the case of Smen from camel milk in western regions, Bechar, Moghrar, Ain Sefra, and Saida [26] need to make attention in analyzing dairy products more carefully. Even if it was found to contain a slight percentage of yeast (2.08–3.88 cu ft./g) despite the morphological description of the described isolates, it was not possible to identify them. This suggests to analyzed yeasts archive and products one by one in the future.

### *Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

The churning of milk also allows the production of L'Ben and in a quantity that often exceeds the need for daily consumption of families. It is therefore sold if not transformed in turn into several modes of fresh or dry cheese products according to the knowledge and know-how of the populations. The consumption of curdled milk and L'Ben, the compound obtained after churning, is very famous in Algeria as dairy products. L'Ben is usually flavored with Phoenician Juniper (*Juniperus phoenicea*) dry powder (**Figure 7a**). Next are the traditional cheese-making methods even in competition with the adoption of modern cheese techniques, which constitute a regional emblem not dissociated from tradition. Jben is so popular (**Figure 7b**) but the case of Jben Al-Gafs is endemic to the Boussaada district and has a real maturity during 2 weeks (**Figure 8a** and **b**).

### **3.5 Fermented wheat**

There is an interesting case related to ancestral food security is the technique of preserving wheat underground. Matmoura is a traditional method of preservation observed in many places in North Africa, even in Egypt. It consists in placing the wheat production crop underground, which is managed differently due to the nature

### **Figure 7.**

*Ancestral traditional dairy products: Left to right; (a). L'Ben sprinkled with Phoenician Juniper, (b). Jben presented on fig leaf.*

### **Figure 8.**

*A, b. Jben Al-Gafs, showing ripening effect as real authentic cheese from Bousâada, Algeria (photo is imported from the website vitaminedz.com).*

of the soil and the need of the families. The ground is leveled up next to the fields and the sides and bottom of the pit are covered with straw. Wheat crop is stored in the pit for several years. Because of the moisture and some water leakage, the sides ferment only. During the re-extraction of the wheat for use, the fermented dark grains are separated by themselves and used to prepare a special couscous known as Hamoum because of its dark color. This technique is still practiced in north-center and northwestern Algeria, but it has become a rare product. Therefore, revealing its health secrets [12, 13, 37] and distinctive taste preoccupies some researchers and those interested in re-considering it as a cultural heritage. The subject of this fermented wheat is an authentic case of fermented food which needs deep research in microorganism identification as well as yeast. First exploration showed fourth interesting species by decreasing numeric importance on occurring culture mediums: *Saccharomyces pastorianus* (50%), *Saccharomyces boulardii* (39%), *Schizosaccharomyces pombe* (5%), and *Saccharomyces cerevisiae* (1%) [38]. This step will encourage prospecting other fermented foods and beverages and allow registering all places where Matrmoura was used as a conservation technique.

### **3.6 Other ethnobiological cases**

We have not verified ethnological practices where yeasts are used in the care or in treatment of diseases, but in the Gourara region in the Algerian Sahara, there is a particular practice where the itching of the hands and the symptoms of fungal attacks are treated by dipping the patient's hands several times into the traditional poultry trough. This traditional material is made of pottery. It is assumed that in such a process there is probably the effect of particular yeasts but this hypothesis remains to be explored by survey in those regions where the same practice is done and try to isolate strains it should exist.

We note from some research works the use of yeast as fortifying in animal feed, in particular, dairy cows using *Saccharomyces cerevisiae* to improve milk production [39–41]. So far we cannot consider this as community adoption, but it will be in the future due to the increasing need and cost of feed. Also, because an experiment is to be done with farmers or breeders and considering the imports of many brands, it is clear that using yeast as an animal feed supplement will be a common practice. The first observation is the negligence in guiding veterinarians to adopt species other than *S. cerevisiae* the ones that should be valued as waste and be more beneficial to breeders. *Candida utilis* would be interesting to improve the yield of the degradation of cellulose of vegetable waste and to offer in addition to the energy a pre-digested animal feed. It would be a shame to not favorite the production of yeasts and choose its easier and direct use in animal feeding, knowing that it is imported. The production trials of *S. cerevisiae* are in favor of the valorization of date waste over molasses, thus recovering large quantities of date waste. In this way, we would promote the use of common dates and improve the productivity of biomass.

### **4. Yeasts in the school program and training strategy**

Yeasts are mentioned first in the medium cycle under the theme of fermentation. The strategy requires to be reviewed in its finality because objectives are not clearly specified even less the link between the disciplinary approaches and the means of implementation. The teaching of fermentation should not be reduced to the sole

### *Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

case of bread or baker's yeast. It should be extended to its practical vocation linked at least to diversified and rich traditions of foods, vinegar, and dairy products. We should start with healthy examples at this stage and encourage the discovery of a very interesting living world. The current textbook should push learners to make more practical efforts. We believe, this is where the curiosity flaws are etched in the minds of future generations. At the university, the subject of yeasts is treated in several fields, including Cytology, Botany, Microbiology, and Biotechnology. In general, the star remains the baker's yeast, *Saccharomyces cerevisiae,* from school levels to university and that during more than 150 years. Ultrastructure is described only to confirm the eukaryotic cell model without worrying too much about cell's composition or physiology. In some applied microbiological evaluation, *Candida albicans* constitutes the second example. Fortunately, in medical sciences and parasitology, the clinical needs cover several taxa: *Candida albicans*, *C. tropicalis*, *Candida parapsilosis*, *C. Krusie*, *C. glabrata*, *Naganichia alba*, *Sporothrix schenckii* and sometime others. Obviously, mastering epidemiology has an evident impact on the economy regarding the risk of mortality, duration of patient hospitalization, and cost of care conditions.

We have an interest to introduce the subject of yeasts as diversified living organisms. After testing this approach in cytology, as a fundamental course of graduation, we consider this as a crucial opportunity not to be overlooked in order to initiate learners to consider the case of yeasts like any other category of cells. Since the main objective is to essentially distinguish the cellular criteria of living organisms, we have proposed the adoption of the following flowchart (**Figure 9**). With a participatory approach based on the principle of biodiversity, we encourage learners to choose for each category their own examples of the previous basic culture as a prerequisite and also of their complementary curiosity according to extra muros efforts. We gain the advantage of quickly initiating young learners and even before university to channel their school culture to recognize cellular trains and thus create the need to prepare the ground for any learning of biosystematics. For yeasts, we solve from the first step their attachment to fungi and especially their complication of sometimes overlapping unicellular eukaryotes and multi-cellular eukaryotes.

**Figure 9.** *Diagram of living organisms.*

The curiosity of the young learners leads to favoring the choice of a few remarkable and captive criteria which distinguish yeasts from other organisms. There are three recent ones that should not be overlooked. That of the mode of division in budding or in fission without forgetting the recent discovery of the mixed mode by budding and fission at the same time and also the mode of star budding concerning the studied species [42]. The second consist of showing yeast diversity in their shape forms [4] and not focalize on the ovoid of elongated ones only. The third peculiarity is that of Ergosterol as a lipid compound in yeast membrane instead of cholesterol as in animal cells and phytosterols in plants. The rest of the sensitivity comes from the role to be discovered for each species and also from the possibilities of its appropriate culture to anticipate this or that biochemical or biotechnological exploitation.

### **5. Yeast inventories in Algerian research studies**

Historically, Algeria is mentioned as the soil origin of type stain CBS 277 of *Pseudosaccharomyces africanus* isolated from a locality named Akbari, cited in Mycobank database and other [43]. Currently, the preferred name is *Hanseniaspora vineae* (Syn. *Kloeckera Africana*). The interest of this species concerns the increasing of flavor complexity with neutral grape varieties. Similar strain of this species was isolated in Japan and showed capacity in improving bread flavor [44]. Another strain of *Hanseniaspora osmophila* (*Kloeckera Africana*) was deposed in the UK, National Collection of Yeast Cultures in April 1920 and reported isolated from the previous habitat (Soil Akbari, Algeria) as mentioned under the number NCYC 26 [45]. Likewise, under the original epithet *Kloeckera Africana*, the strain DBVPG 6792 was deposed in 1993 in the Yeasts Collection (DBVPG) of the Dipartimento di Biologia Vegetale of the Universita' di Perugia, Italy [46].

In The Algerian Annals of Agronomy, Bremond E. tested the fermentation of mature bananas coming from Guinea in West Africa and unvaluable for the consumption to produce alcohol for the military service [47]. Regarding the purpose, active sourdough was used without the precision of yeast or any other microorganism species. At that time, they suggested that Algiers Pasteur Institute, as a professional contribution, would conserve and purify the purity of the selected strains. From that, we retain two important lessons; archive of yeast prospections will be done correctly. Any work has real importance with a clear impact on application in wine processing.

Identification of yeasts as species first concerned medical purposes to control pathogens. In parallel but more fragmented come some works confirming the role of yeasts in fermented foods or drinks. Those who focus on discovering them in environments or describing their benefits are much more recent.

### **5.1 Yeasts within human health**

Human fungal infections including those caused by yeast and yeast-like fungi constitute a serious burden in Algeria [48–57]. At least 27 taxa are identified in care controls. Often in topic of medicine research, we observe in first *Candida* species with sometimes resistance phenomena to treatments [48, 54]. Inventoried species are: *Aureobasidium melanogenum* (*Aureobasidium pullulans* var. *melanogenum*), *C. albicans*, *Corynebacterium auris*, *C. dubliniensis*, *Candida famata*, *C. glabrata* (syn. *Torulopsis glabrata)*, *C. Kefyr, C. Krusie*, *Candida lusitaniae* (Syn. *Clavispora* 

*Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

*lusitaniae*), *C. orthopsilosis*, *C. parapsilosis*, *C. rugosa*, *C. tropicalis, Candida zeylanoides*, *Clavispora lusitaniae* (Teleomorph of *Candida lusitaniae*) *Cr. gattii*, *Cr. neoformans* var. *grubii*, *Cr. neoformans* var. *neoformans*, *Cr. neoformans*, *Lodderomyces elongiporous* (related to sex. Form of *Candida parapsilosis*), *Meyerozyma elongisporous*, *Naganishia albida* (*Cryptococcus albidus* var. *albidus*), *Naganishia liquefaciens, Pichia kudriavzevii* (Telomorph of *Candida krusei*), *Prototheca wickerhamii, Rhodotorula mucilaginosa*, *Saccharomyces cerevisiae* and *Trichosporon* sp*.*

This later was observed with tenia petis infection [49, 50]. *Saccharomyces ceverivisiae* was isolated from infant fees and showed that are safe that can be considered as probiotic [54] whereas *C. parapsilosis* and *C. albicans* have potential inhibitor effects on referential strains of *Escherichia coli* and *Staphylococcus aureus*. *Cr. gattii* [55] and *C. auris* [56] are reported for the first time in Algeria. In this later work, deep identification separated the subspecies level of Cr. neoformans into its two varieties *Cr. neoformans* var. *grubii* and *Cr. neoformans* var. *neoformans*. Only one case mentioned a denture contaminated by *C. albicans* [57], the *ex vivo* decontamination of this yeastcolonized denture was realized by iodine-thiocyanate.

The urban environment is considered as a potential risk of contamination by the inhalation of fungi spores, pigeon droppings, trees, and soils are surveyed some time in urban areas and around hospitals. *Candida* sp., *C. albidus*, *C. laurantii*, *Cryptococcus neoformans*, *Rhodotorula* sp., *Saccharomyces* sp., *S. cerevisiae*, and *Trichosporon* sp. are reported from Algiers the capital [55]. Also, in Annaba and from pigeon droppings, 6 isolated species were identified; *Cryptococcus albidus* and *Cryptococcus diffluens* (for the first time) were isolated, which represents an environmental risk for humans [58]. Prospected pigeon's droppings from Constantine showed *C. albicans*, *C. glabrata*, *C. parapsilosis*, *Cr. Terreus,* and *Cr. uniguttulatus* [59].

### **5.2 Yeasts in animal health**

From diagnosed cow cases [60–64], a total of 30 species have been identified mainly causing mastitis. Goats [7], sheep and camels are certainly affected but no isolates have been identified in the research consulted during the elaboration of this presentation. Species are; *C. albicans*, *C. colliculosa*, *C. famata*, *C. glabrata*, *Candida guilliermondii*, *C. inconspicua*, *C. kefyr*, *C. krusei*, *Candida lambica*, *C. lusitaniae* (Anamorph of *Clavispora lusitaniae*), *C. parapsilosis*, *C. pseudotropicalis, C. rugosa*, *C. tropicalis*, *C. zeylanoides, Cryptococcus albidus, Cr. laurantii, Cr. Neoformans, Cr. terreus* (Syn. *Solicoccozyma terrea*), *Cutaneotrichosporon cutaneum* (Syn. *Trichosporon cutaneum*), *Geotrichum capitatum*, *Metschnikowia pulcherrima (Syn. Torulopsis pulcherrima), Pichia kudriavzevii* (Telomorph of *Candida krusei), Rhodotorula glutinis, R. rubra, S. cerevisiae, S. fragilis, Trichosporon* sp., *T. capitatum,* and *T. fermentans* (Syn. to *Dipodascus fermentans*). With a chance that *Pichia kudriavzevii* were observed in rare cases for the moment. This species presents a most remarkable resistance to the antifungal agent fluconazole. The essential oils of *Origanum floribundum*, *Rosmarinus officinalis,* and *Thymus ciliates* were tested and shown to be favorable against *Candida albicans* and can be considered as alternatives in the control of mastitis fungi [62]. Also, essential oils of *Cinnamomum cassia* (cited as *Cinnamomum aromaticum*) and *Syzygium aromaticum* were tested against seven isolated species *C. albicans, C. lambica, C. tropicalis, C. zeylanoides, Cryptococcus albidus, Cr. Laurentii,* and *Rhodotorula glutinis* and shown strong effects to be proposed as an alternative solution to face the fungal mastitis risk [63, 64].

### **5.3 Yeasts from soil and agriculture products**

Logically, wine and soil were the past first subject of prospecting yeast, in particular, during colonial period. That was the reason to observe today's referenced strains, *Hanseniaspora* osmophila CBS 277 and *H. vineae* (syn. *Kloeckera africana*, cited as *Pseudosaccharomyces africanus*) and in gene banks [43, 45, 46]. *Hanseniaspora opuntiae* x *pseudoguilliermondii* DBVPG 5828 was isolated from Soil close to plum tree and deposited as a reference strain in 2010 [65]. Recently, 20 other taxa were isolated from soil and agriculture products in varied agriculture areas [66–74]. Isolated taxa are *Aureobasidium pullulans*, *Candida* sp., *C. glabra, Clavispora lusitaniae*, *Cryptococcus* sp., *Cr. aerius* (Yeast state of *Solicoccozyma aeria*), *Cr. magnus* (Syn. of *Filobasidium magnum*), *Debaryomyces hansenii*, *Hanseniaspora opuntiae*, *Hanseniaspora uvarum*, *Lipomyces* sp., *Meyerozyma guilliermondii* (Teleomorphic form of *C. guilliermondii* Syn. *P. guilliermondii*), *Phyllophorus anomalia, P. kluyveri*, *Rhodotorula mucilaginosa*, *Saccharomyces* sp., *S. cerevisiae*, *Schwanniomyces* sp. *Ustilago cynodontis*, *Yarrowia lipolytica.*

Selected promising strains are; *Schwanniomyces* sp. strain LB3 [67] for amylase production, *P. kluyveri* DBVPG 5826 showing killer activity [70], and *A. pullulans* for the evaluated activity of its polygalacturonase with admissible application to reduce the cloudiness of fruit juice [72], Compared with *Saccharomyces cerevisiae*, isolated from soil strains of *Debaryomyces hansenii*, *Meyerozyma guilliermondii,* and *Rhodotorula mucilaginosa* showed the preference for alkaline pH and interesting resistance to salinity and elevated temperature and have a potential of Plant Growth Promoting (PGP) function [69]. *Ustilago cynodontis* isolated from the Sebkha of Oran (Saline soil) has multi-enzyme activities as lipolytic, proteolytic, and cellulotic [71]. *C. glabrata* has an interesting & amylase activity [75].

Fruits naturally are a suitable yeast habitat. Grapes and dates are the first prospected fruits. Twelve isolated Species from grapes [76–78] are: *Candida pseudointermedia*, *C. solani*, *Hanseniaspora uvarum, Metschnikowia pulcherrima* (Syn. *Torulopsis pulcherrima*), *Pichia deserticola, P. fermentans, S. cerevisiae, Starmerella gropengiesseri* (cited with basioname: *Candida gropengiesseri*)*, Starmerella magnoliae* (cited as homotipic basioname *Candida magnoliae*), *Torulaspora delbrueckii*, *T. microellipsoides* (cited as *Zygosaccharomyces microellipsoides*), *T. pretoriensis*. From dates, 5 identified species were isolated; *S. cerevisiae* was isolated to select several adapted strains to produce alcohol or biomass [79, 80], *Clavispora lusitaniae*, *Hanseniaspora uvarum*, *Kodamaea ohmeri* were selected to test the production of Alcohol, flavor and amino acid [80, 81] and *Candida apicola* was isolated to be targeted as temperature resistant [82]. The natural fermented green Olives contain a least 4 taxa; *Candida* sp., *C. parapsilosis* and *Saccharomyces* sp. and *S. cerevisiae* [83]. *Candida boidinii* G5 (KF156789), isolated from spent olive (Chemlal variety), showed lipase activity [84].

Like dates must sugarcane molasses and fig were explored to isolate S. cerevisiae [85]. From other fruits and products, *Candida parapsilosis* was isolated from melon, *Zygosaccharomyces bailii* from Gherkin, and *Zygosaccharomyces rouxii* from honey [81]. To produce &-amylase, *Candida guilliermondii* (Syn. *Pichia guilliermondii*) and *C. tropicalis* were isolated from potatoes [86]. Recently, *Aureobasidium pullulans*, *Rhodotorula diobovata, Vishniacozyma tephrensis* were isolated from fruits and beet peels and showed extracellular enzyme synthesis [87].

Wheat seeds were explored to isolate 8 species; *Lipomyces kononenkoae*, *Rhodotorula mucilaginosa*, *Schwanniomyces occidentalis* (Syn. *Debaryomyces occidentalis*) [73], *Clavispora lusitaniae* ABS7 [88], *Meyerozyma caribbica*, *M. guilliermondii*, *Pichia guilliermondii* (Anamophic of *M. guilliermondii*), *Rhodotorula rubra* [89].

Fermented wheat conserved within Matrouma as ethnological technique permitted to silotae *Saccharomyces boulardii*, *S. cerevisiae*, *C. pastorianus*, *Schizosacchoromyces pombe* [38]. The latter is isolated for the first time in Algeria while it could exist in many products as well as those made with seeds or powder millet.

Research works are limited to the examination of efficiency in biomass production in particular to produce *S. cerevisiae* or oriented to valorize that in bioethanol. Those oriented to biotechnological was stay at the exploration stage and need more significant interest to go up the level of applied fields in industries.

In phytopathology, the case of fungi must be extended to yeasts because they are often associated with molds and yeasts. Yeasts are considered as sources for stopping fungal development. Two new strains, isolated from the Red Sea and identified belonging to the species *Candida orthopsilosis* and *Rhodotorula mucilaginosa*, showed improvement in wheat growth parameters and its resistance against *Fusarium oxysporum*, with a complete inhibition of zearalenone production in roots and ears [90]. This indicates an interesting and amazing perspective in testing yeasts against other fusariosis cases. Basically, *Metschnikowia pulcherrima* is an epiphytic species as others. Nectar and sweet fruits are welcoming to it. This species is must be appreciated for its strong antagonistic activities against pathogenic microorganisms without producing toxic components. Thus, it is used against plant pathogens like in breaking fruit invasion by *Botrytis cinerea* [91]*.*

### **5.4 Yeasts in dairy products**

Raw milk evaluated in North Western Algeria showed that yeast and fungi were detected in all ewes' raw milk samples [92]. The investigation of bovine mycotic mastitis in two departments of the northeast showed that 10.17% of the samples were positive [62]. Milk handling could be prior factor causing high yeast and mold loads. Other risk factors are due to the effect of the season and the distance between farm and dairy unit. When septic conditions are ensured we have to look for those spontaneous benefit species, in particular, if dairy products are conserved or transformed. 27 identified species were isolated from dairy products. *Y. lipolytica* is isolated from all king of milk [81, 93, 94]. From fermented bovine milk 7 identified species are *C. tropicalis, Issatchenkia orientalis* (Syn. *P. Kudriavezii*)*, K. marxianus*, *Saprochaete suaveolens, Trichosporon coremiiforme*, *Wickerhamomyces anomalus, Yarrowia lipolytica* [93]*.* The strain L2 (ACKF156787) of the last one species is isolated to evaluate the lipase enzyme in traitment of olive waste [84]. *Schizosaccharomyces* sp. with similar strain to *S. octosporus* was isolated from whey [95]. From yogurt, *K. fragilis, K. marxianus* and a similar strain to *Schizosaccharomyces malidevorans* were isolated [95]. Cheese contains *C. lactis* [95] and *Geotrichum candidum* [94]. From camel milk 16 species were isolated; *Issatchenkia orientalis* (Syn. *Pichia Kudriavezii*), *Trichosporon coremiiforme, Yarrowia lipolytica* [81], *C. maris, C. parapsilosis, C. tartarivorans, C. tropicalis, C. zylanoides, C. lusitaniae, K. marxianus, M. Farinosa, P. fermentans, P; galeiformis, manshurica, R. mucilaginosa* [96] *Candida kefyr* was isolated from cow milk, whey [95] and fermented camel milk [97]. Cooked Butter **(**S'men) from Camel milk showed also the contamination of Saccharomyces cerevisiae and other *Saccharomyces* sp. [26].

To conserve high volatile value in fermented cow milk (Rayeb), while this term can mean also coagulated milk, 7 species have been isolated [93]. Four selected species *S. suaveolens, I. orientalis, K. marxianus,* and *W. Anomalus* produce esters that influence the taste and ensure organoleptic parameters of the dairy product. The double-drying process of interesting yeast such as *S. suaveolens, W. Anomalus,* deserves to be popularized and taught as a way to innovate or improve traditional processes. Modern cheese

ripening is considered an exogenous process but traditional or local as soft or dried can be improved by understanding of the physiological strategy of the species involved. [98] leads with the commercial strain of *Geotrichum candidum* associated with *Penicillium camembertii* the choice of the amino acid that would be consumed most efficiently and the short peptides will be targeted. This experience showed clearly the interest to select from local spontaneous *G. candidum,* which is apparently very rarest. Stay with domesticated strains and remember that we are in the fourth group described previously that of species, basically beneficial but potentially harmful. Special attention is registered to valorise the famous case of *Candida kefyr* isolated from camel milk or yogurt [95, 97]. Another targeted strain of this species has highest enzyme activity (up to 5000 EU/ ml) and highest level of single-cell protein [95]. Other identified *taxa* in this study were *Candida* sp. (similar to *C. pseudotropicalis*), *Kluyveromyces fragilis, K. lactis, K. marxianus*, *Schizosaccharomyces* sp. (similar to *S. octosporus*) and *Schizosaccharomyces* sp. (similar to *S. Malidevorans*, syn. of *S. pombe*). *Yarrowia lipolytica* is isolated from sheep, goat, and camel milk [93, 96, 97]. *Candida kefyr* was isolated from Camel fermented milk in an isolated oasis; Sebseb far from Metlili [97] as an interesting safe case. Local conditions and know-how of fermentation of camel milk are to be documented from this region.

The presence of yeasts is notified in the traditional cheese Bouhezza [31, 32], as well as the local product Michouna [33, 34] but not identified. In those studies like others [27–35] the orientation is given to evaluate bacteria. Yeasts are sometimes only quantified as Yeasts and molds even in extensive studies to evaluate the sensitive properties or the chemical characteristics of the famous Bouhezza cheese [99, 100]. Like Dried cheese named Klila, the famous cheese Jbeen Al-Gafs is little studied and was analyzed only for bacteria [11]. Authors certify that yeast and molds are abundant in the final stage after 14 days of maturation.

### **5.5 Yeast for environmental purposes**

First attention to yeasts in the environment is commented previously under human health. 16 isolated species from soil, trees, and particularly drooping pigeons in and around hospitals in urban areas [55, 58, 59] are *Candida* sp., *C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. zeylanoides, Cr. albidus, Cr. diffluens* (Syn. of *Naganishia diffluens*)*, Cr. laurentii, Cr. neoformans*, *Cr. terreus* (Syn. *Solicoccozyma terrea*), *Cr. uniguttulatus*, *Rhodotorula* sp., *S. cerevisiae,* and others *Saccharomyces* sp. and *Trichosporon* sp. We also consider all species of yeast counted as food and dairy contaminants as environmental agents. From beaches, sand affected or under risk to be affected by polluted water or charge of waste were prospected in the western region near Oran [101]. Six registered taxa are; *C. albicans, C. zeylanoides, Cr. albidus, Geotrichum* sp., *Rhodotorula* sp., and *S. cerevisiae.* What remains to be done for this purpose? is to compare this with a clean beach. The wastewater facilities of Mascara in western Algeria were examined to isolate four potentially pathogenic yeasts, *C. albicans, C. glabrata*, *Cr. Neoformans,* and *Trichosporon* sp. [102]. At least 18 species are isolated from particular environments. Recently, *Geotrichum candidum* was isolated from soils that have been used as a depository of electronic and electrical waste for more than 20 years, around Annaba in the eastern part, and was described as able to degrade *in vitro* waste of batteries and circuit boards [103]. From lakes, Chott or sebkha (saline soils) previously cited studies were reported in the section of agriculture soils. Within Dayet Oum Ghellaz, a wetland affected by drought conditions near Oran and known as highly lead and cadmium polluted, three yeast species were isolated, *Clavispora lusitaniae*, *Rhodotorula mucilaginosa* and *Wickerhamomyces anomalus* [104] and shown

*Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

high tolerance to NaCl and growth with heavy metal concentrations. Isolated strains W02 (*W. anomalus*) and R07 (*R. Mucilaginosa*) gives a removal of 12.68 ± 0.91 and 15.55 ± 0.72 mg of lead/g of biomass respectively. This is a promising and interesting first result of bioremediation with should be perfected in order to be proposed in real applications. The percentage removal of heavy metals of *R. Mucilaginosa* was measured in Serbia after 48 h for Cd2+, Zn2+, and Ni2+ and showed 2.11%; 4.99%, and 29.25%, respectively [105]. In Algeria and from an oilfield soil near Hassi Messaoud, the isolated strain YBR of *Rhodotorula* sp. was tested using wastewater of olive mills as low coast substrates produce biosurfactant [106] and can be used to remove hydrocarbons from polluted soils. Microorganisms produce a large specter of surfactants as extracellular components which have potentially wide properties due to their different chemical structure. They have also antibacterial and antifungal activities as well as they offer a potential use in food processing. Bioconversion of olive mills wastewater was conducted on an experimental scale using to referenced yeast species *Yarrowia lipolytica* MUCL 28849 and *Cryptococcus curvatus* ATCC 20509 in order to produce biomass [107]. Even when Y. lipolytica decreases in biomass, the decrease in polyphenol concentration is more than 4 times lower compared to the baseline.

### **5.6 Research in biotechnology**

### *5.6.1 In applied genetics*

From the wild collection, two haploid strains of S. cerevisiae K10 and MYC5 were selected to produce a protoplast fusion with two other commercial strains VDH2 and FXX118 provided by the Swiss company PEC and two other strains S. cerevisiae LGI2 and *Kluyveromyces lactis* LGK1 selected by the Genetics Laboratory at the Faculty of Biological Sciences (FSB) of the University of Sciences and Technology Houari Boumediène (USTHB) in Algiers. The protoplastization was carried out using Algerian *Helix asperca digestive juice*. Three hybrids were obtained at three levels intra-generic, intra-specific and inter-specific and their physiological properties were examined in order to obtain strains that were viable and more tolerant to temperature and alcohol concentration [108]. This case study confirmed technology transfer in developed countries but should not be discontinued. The main reason is the lack of interconnection between research and yeast manufactures, which prefer or stay under the effect of imports. Yeast production units are called upon to be competitive and must have their own research laboratory which values research results instead of only focusing on overseas purchases. The university must also generalize this type of innovative research attempts so that it is taught in graduation and to all courses in the genetics module instead of increasingly theoretical or virtual teaching.

### *5.6.2 Yeast biomass production*

The example of selection of local strains like *K. fragilis* applied to valorize whey has now been more than 20 years. That of wild *S. cerevisiae* isolated to be compared with referenced strains. Some selected local strains are sometimes more efficient in biomass production than commercial strains [79, 109–113]. While, from the case of date palm, obtained yeast biomass is different due to the date varieties and the volume of the equipments [114–116]. The second advantage is to valorize the renewable low cost substrates and finding other interesting species like *Cr. curvatus* or *Y. lipolytica* [107]. The innovative test consists of valorizing olive mill wastewater and also the

*Opuntia ficus indica* peels [117]. Date must stay the lower cost available substrates which need serious industrial orientation, first in yeast production. Obtained yield is about 10% [115]. Whey is also targeted to produce biomass 11–22 g/l. with *Kluyveromyces lactis*, and 10–13 g/l. *K. marxianus and C. versatilis* [112].

### *5.6.3 BioEthanol production*

Actually, the effervescence of studies is due to real mutations under the constraints of COVID-19 and the accelerated need for ethanol at the national level. Two species were used; *S. uvarum* [118, 119] and *S. cerevisiae* [79, 85, 109, 110, 119–128] with varied substrates in particular dates and date wastes of several varieties. A newly selected strain showed a modest ethanol production but also phenyl ethanol, glycerol, acetates, and acetic acid [81, 129]. The ethanol yield of 30 ml/125 g from *Balanites* fruits [130, 131] is less than what we obtained with dates, which give 300–600 ml/1 kg dates [118]. Banana gives by far past 9 l/100 Kg [47]. To further increase bioconversion yield, a practical nature by trying to target other species of yeast such as other substrates, and even if dates are the most available, we must not forget the peelings of fruits and vegetables in the juice factories. The mixture of grape juice and date must improve yield at 160 g/L. [125]. Three major constraints need to be taken as strategic debates. First one concerns the varied substrates to be valorized. While common dates stay evident favorite due to their availability, other biomass, such as waste olive and fruit peels, are considered as well as whey. The second concerns the choice of yeast species and does not focalize only on *Saccharomyces* strains in particular because we know that waste dates and dry ones are rich in sucrose not only glucose. The test of mutant *K. marxiamus* on glucose substrate gives highly significant ethanol but lower on sucrose medium [132]. The third is that of fundamental cellular pathway of ethanol production. The selection of new wild strains gives a chance to tolerate ethanol concentration and thus can produce more efficiency [133, 134] but there are other ways to produce glycerol, acetate, and phenyl ethanol. We should know how we can concentrate on the way to produce ethanol only. We borrow from [133] the diagram of ethanol biosynthetic

### **Figure 10.**

*Ethanol biosynthetic pathway in Saccharomyces cerevisiae [133].*

### *Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

pathway (**Figure 10**) in which glycerol, 2,3-butanediol, and acetate are produced, although less than ethanol. To maximize the productivity of ethanol it is necessary to minimize the flux of carbon toward glycerol and 2,3-butanediol. The reality of ethanol production route is often masked by the classic rule of sugar transformation into ethanol as being a single theoretical scheme where nothing needs to be modified.

### *5.6.4 Vinegar production: the best way to improve traditional process*

Vinegar can be defined as a product obtained exclusively through biotechnological processes by double fermentation, alcoholic and acetic fermentation of liquids, or other substances of agricultural origin [134]. Traditional vinegar constitutes one of the natural products. It is obtained as biological vinegar from the fermentation of varied fruits. In addition to grapes and apples, dates are often presented in Algeria as ancestral ones using date varieties, and essentially those of low market values. Traditional way consists of double fermentation simultaneously and usually, has alcohol digress superior to what is accepted by standards and commercial legislation [7, 21]. Date vinegar is pronounced in flavor with a sweet taste due to esters produced by microorganisms. The initial sugar rate is not transformed totally at the end of maturation for 45 days. Thus the taste is usually sweeter [7, 21, 118, 135]. Due to the date varieties or to the process itself, sometime the total acidity of 2,5% is less than what is recommended. The spontaneous flora is showed more adequate but the resulting vinegar should be better with separating phases [118, 136].

Vinegar obtained from pomegranate, apple, and prickly pear are also made, but little research is done. Fruit vinegars have more reputation for health purposes. Experimentally biological vinegars have been shown to regulate lipid metabolism and decrease liver damage in high-fat-fed rats [137]. With apples the situation is similar and the main goal to prepare the vinegar is to valorise the huge losses of apples due to frost and convert them into producing a food health byproduct [138]. In addition to Ethanol and Acetic acid, traditional vinegar from dates contains [136]; Formic acid (to treat warts), Acetaldehyde (Ethanal), which gives ethanol by oxygenation, 1-hydroxy-2-propanone, 1–3 Butandiol, 2-Butanol, 1 Methyl Ester Formic acid (Fumigant and larvicide), 1–3 di-hydroxy propanone, 2,3-Dihydro-3,5-dihydroxy-6-methyl-4 h-pyran-4-one. The richness of those components explains or argues for the use of traditional vinegar in popular care methods and opens new perspectives for evaluating the nutritional properties of organic vinegars. The 1–3 di-hydroxy propanone is used in treatment of Vitiligo on exposed areas [98] and the 2,3-Dihydro-3,5-dihydroxy-6-methyl-4 h-pyran-4-one is an antioxidant [139].

### *5.6.5 Yeasts as the source of enzymes*

Twelve yeast species are tested to produce varied enzymes and also to valorise different substrates as well as fruit wastes. *Aureobasidium pullulans* on tomato pomace produces polygalacturonase [68] and also Cellulase, Amylase, protease, and lipase in an experimental purpose valorising fruit and beet peels [87]. Lipase is produced by *Candida boidinii* isolated from olive pomace [84], *Ustilago cynodontis* isolated from saline soil [71], *Vishniacozyma tephrensis* [87] and *Yarrowia lipolytica* [84]. α-Amylase is produced by *C. glabrata* isolated from saline soil and valorising whey [75], *C. guillierrnondii* and *C. tropicalis* [86], *Clavispora lusitaniae* [88, 140], *Schwanniomyces* sp. [67] and *Ustilago cynodontis* [71]. Cellulase is produced by *A. pullulans* [87] and *U. cynodontis* [71]. The protease is produced by *A. pullulans* [87], *Cr. neoformans*

[74], and *U. cynodontis* [71]. Maltase is produced by *C. lusitaniae* [88]. The first step of application is given by selected strains ABS7 of *C. lusitaniae* to purify the enzyme amylopullulanase in order to be used in laundry detergent [141]. A toxin from killer effect is targeted to be applied against food spoilage [142] and the third promising case came from the potential use of pectinase in clarification of fruit juices [143].

Particular attention should be given to cellulases. The case of Algerian Green energy progress plan has a potential of 0.67 Million Ton Oil Equivalent (Mtoe) from three sources of lignocellulosic; Alfa, olive pomace, and cereal straw [144]. Elsewhere, the author indicates that with the adoption of new energy crops and the strengthening of cereal technologies, Algeria would increase its bioprocesses energies up to 73.5 Mtoe and 58.9 Mtoe from these sources of biomass. We must consider more important, the lignocellulosic waste abandoned in palm groves with more than 20 Million date palms, giving at least 200.000 tons of waste, which is easy to use. This rate is suspected to increase by 4,5% by 2030 [145]. In addition to *S. cerevisiae,* selected strains concern also *K. marxianus* and *P. pastoris* [146].

### **5.7 Means of combating pathogenic yeasts**

Honey is logically the first natural product which was applied against *C. albicans* and *Rhodotorula* sp. [147]. Traditional vinegar shows an effect on *C. albicans* [23] that is probably due to the presence of formic acid, formic acid methyl ester, and or 1–3 dihydroxy propanone. Essential oils of *Citrus sinensis* and *Citrus lemonum* give a positive impact against *C. albicans* [148]*. Cymbopogon citratis* extract has an inhibitory activity on *C. albicans* and *C. tropicalis* [149]. Essential oils of *Myrtus communis* and *Myrtus nivellei* were applied against *C. albicans*, *C. parapsilosis*, *C. tropicalis*, *Cr. neoformans* and also those isolated from vulvo-vaginal candidose patients *C. guillermondii* et *C. krusei* [150, 151]. Essential oils of *Origanum floribundum, Rosmarinus officinalis,* and *Thymus ciliates* has an effect on *C. albicans* isolated from bovine mastitis [62]. That of *Cinnomomum aromaticum* and *Syzygium aromaticum* should encourage their use against *C. tropicalis, C. albicans, Cr. neoformans, Cr. salbidus,* and *Geotrichum capitatum. Terfezia claveryi* crud extract dissolved in ethyl acetate has an effect on *C. albicans* [152]. The lichen *Xanthoria parietina* extract was tested against 4 strains isolated on patients in Thenia hospital (Algeria); *C. albicans*, *C. parapsilosis*, *Trichosporon* sp., and *Malassezia* sp. Two cases; *C. parapsilosis* and *Malassezia* sp. were mildly sensitive [153]. The isolated strain codified E96 and cited as actinomycete showed an antagonistic effect against pathogenic yeast; *Cr. albidus, Cr. diffluens,* and *Cr. Neoformans* [58]*.* Other cases of microorganisms were tested against referenced strains of *C. albicans* but all of those results stay at the experimental step only. From the marine ecosystem, *Cystoseira stricta* extract inhibits the referenced strains *C. albicans* [154]. *Asparagopsis armata* extract has an inhibitory activity with 0,58 mg/ml on the *C. albicans* IP 444 but with 2,34 mg/ml on *C. albicans* ATCC 1023 [155] showing the contrast within strains.

### **6. Conclusion**

Yeast species inventoried in Algeria constitute a forgotten part of biodiversity. It aims to promote studies of yeasts oriented toward economic and cultural services. Derived from soils, plants, and animals, yeasts characterize fermented foods and drinks and can improve the quality of citizen's life. Those who refer to care in hospitals logically deserve special attention because they can be sources of complications for

### *Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

patients and require additional hospitalization costs. The yeasts inventoried reached 131 taxa (Annexe 1); 27 related to human health, 30 animal health, 27 from dairy products, 24 isolated from the soil, 41 taxa from agricultural products (grapes dates, olives, roots, ...), and 17 isolated from the environment (Trees, feints of pigeons, beaches, landfills). Biotechnonlogies concern 44 taxa (Biomass, Ethanol, enzymes, esters). 16 selected natural products inhibit 14 yeast species. This rich information should be used to establish a national network and help stakeholders assess and make their contributions. The inventory is not completely exhaustive because there are probably unlisted works. Anyway, the inventory will remain continuously open in view of the progress of studies which are increased by the adoption of molecular tools. Yeasts are closely linked to socio-economic purposes relating to traditions (baking, vinegars, dairy products, yeast food supplements for animals) or referred to in modern industrial processes (biomasses, ethanol, and enzymes). Their studies have socio-economic impacts linked to the benefits derived; 1) the mastery of human care with repercussions on the medical care of patients and on the strategy for the use of antibiotics, 2) in increasing milk and meat yields, 3) in environmental issues including waste treatment and depollution. The link between ancestral traditions and scientific explorations increases the chances of serving the national economy and should encourage people not to depend solely on imports. The use of biomass or ethanol is one of the most urgent needs to be supported in a national strategic plan which gives concrete form to renewable energies. Testing the production of ethanol and its mixture in the fuel is possible, but carefully choose the proprietary biomass to be transformed. Dates, waste from olive groves, and the lost share of cellulose in palm groves are most promising.

Updating the national directory of identified accessions is the first step in the collaboration between specialists to honor the standard of biodiversity inventories. A network of national skills should be set up to promote prospecting in various environments because what remains to be done undoubtedly represents 99%. We are keen to complete this initial list with a consortium of national specialists in this complex and diverse field. This can be considered an important step because it is necessary to establish a national repository of strains and listed species. The Algerian Pasteur Institute, with experience and capabilities, can fulfill this role, but it makes sense to be more specialized in pathogenic species and their antagonists. The strains prospected in other fields such as agriculture, environment, and food technology require greater attention in strengthening the technical capacities of other institutions. Whatever the applications, the repository justifies being targeted by the recently created National GenBank. The main reason is to standardize identifications, promote selected strains and establish the national register of biological resources.

### **Acknowledgements**

The author would like to thank Mr. A. Medjahed, Director Ge-neral of INESG for his encouragement and his patience during the period of writing this synthesis. Pr Salim Mokrane and Sonia Kaci of the LBSM, at the Ecole Normale Supérieure are kindly thanked for their help in taking photos and exchanging ideas.

### **Conflict of interest**

The author declares no conflict of interest.

### **Annexes**

Annexes I and II referred to in this chapter are available at: https://bit.ly/3wbo74l.

### **Author details**

Abderrahmane Benkhalifa Higher Normal School, Bachir El-Ibrahimi, Algiers, Algeria

\*Address all correspondence to: a.benkhalifa@hotmail.com; abderrahmane.benkhalifa@g.ens-kouba.dz

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Kurtzman CP, Fell JW, Boekhout T. The Yeasts, a Taxonomic Study. Fifth ed. Vol. 1. London, UK: Elsevier; 2011. p. 537

[2] Żymańczyk-Duda E, Brzezińska-Rodak M, Klimek-Ochab M, Duda M, Zerka A. Yeast as a versatile tool in biotechnology. In: Morata A, Loira I, editors. Yeast - Industrial Applications. London, UK, UK, London: InTech; 2017. DOI: 10.5772/intechopen.70130

[3] Mota MN, Múgica P, Sá-Correia I. Exploring yeast diversity to produce lipid-based biofuels from agro-forestry and industrial organic residues. Journal of Fungi. 2022;**8**:687. DOI: 10.3390/ jof8070687

[4] Boekhout T, Amend AS, El-Baidouri F, Gabald T, Geml J, Mittelbach M, et al. Trends in yeast diversity discovery. Fungal Diversity. 2022;**114**:491-537. DOI: 10.1007/s13225-021-00494-6

[5] Benkhalifa A, Toumi M. L'inventaire des levures pourrait-il booster l'économie en Algérie? In: 2ème Séminaire National d'Ethnobotanique et de Valorisation des Substances Naturelles. Algérie: Univ. Benyoucef Benkhedda, Alger I; 2021. pp. 9-12

[6] Merrad S, Kaabeche Z. État de l'art sur la conservation d'aliments par fermentation: Focus sur les aliments solides [thesis]. M'Sila, Algeria: Université M. Boudiaf; 2020. p. 50

[7] Ould El Hadj MD, Sebihi AH, Siboukeur O. Qualité hygiénique et caractéristiques physico-chimiques du vinaigre traditionnel de quelques variétés de dattes de la cuvette de Ouargla. Rev. Energ. Ren: Production et Valorisation – Biomasse. Special Issue. Alger: Centre de Développement des Energies Renouvelables (CDER); 2001. pp. 87-92

[8] Gast M. Lait. Encyclopedie berbère. 2008;**28-29**:4322-4330. DOI: 10.4000/ encyclopedieberbere.294

[9] Aissaoui-Zitoun O, Pediliggieri C, Benatallah L, Lortal S, Licitra G, Zitoune MN, et al. Bouhezza, a traditional Algerian raw milk cheese, made and ripened in goatskin bags. Journal of Food, Agriculture and Environment. 2012;**10**(2):289-295

[10] Leksir C, Boudalia S, Moujahed N, Chemmam M. Traditional dairy products in Algeria: Case of Klila cheese. Journal of Ethnic Foods. 2019;**6**:7. DOI: 10.1186/ s42779-019-0008-4

[11] Saidane Z, Dahou AA, Tahlaiti H, Daoudi M, Doukani K, Homrani A. Physico-chemical parameters with direct influence on the dynamism of the indigenous microflora of the traditional cheese "J'ben Elgafs". Asian Journal of Dairy and Food Research. 2021;**40**(2):157- 161. DOI: 10.18805/ajdfr.DR224

[12] Mokhtari S, Taghouti M, Saidi D, Kheroua O. Traditional Algerian fermented food: First data on nutritional characteristics of wheat (*Triticum durum*) fermented in underground silos Matmora (Mascara, Algeria) compared to unfermented wheat. Advances in Biology & Earth Sciences. 2020;**5**(3):176-192

[13] Benakriche B, Bousbahi S, Gérard P. Impact nutritionnel d'un blé fermenté type Hamoum sur la translocation bactérienne intestinale chez le rat malnutri en phase de réalimentation. Nutrition Clinique et Métabolisme. 2019;**33**(1):102. DOI: 10.1016/j.nupar.2019.01.416

[14] Algérie Presse Service [Internet]. 2021. Available from: https://www.aps. dz/economie/117637-la-relance-de-lusine-de-production-de-levure-deguelma-couvrira-30-du-marche-national. [Accessed: August 28, 2022]

[15] Word Integrated Trade Solution (WITS) – WorldBank. Yeasts; active exports to Algeria in 2019-2021. Available from: https://wits.worldbank.org/trade/ comtrade/en/country/All/year/2021/ tradeflow/Exports/partner/DZA/ product/210210. [Accessed: August 28, 2022]

[16] Direction des Etudes et de la Prospective. Statistiques du commerce extérieur d'Algérie, 2018-2019, 2019-2020. Algérie: Ministère des Finances, Direction Générale des Douanes, Multigr; 2021. p. 71

[17] Hana Saada (from Radio of Algeria) Yeast production. Available from: https:// www.dzbreaking.com/2021/02/18/yeastproduction-guelma-plant-to-cover-30 of-national-market/. [Accessed: August 28, 2022]

[18] Vincent. Matlouh "*matloa*"*,* an Algerian bread made out of hard wheat and semolina, leavened by either sourdough or fresh yeast. 2022. Available from: https://baking-history.com/ matlouh/. [Accessed: August 28, 2022]

[19] Benkhalifa A, Kehli K, Ould Mohammed A. Levure naturelle et levain maison: Richesse et déclin des traditions en Algérie. In: 2ème Séminaire National d'Ethnobotanique et de Valorisation des Substances Naturelles. Algérie: Univ. Benyoucef Benkhedda, Alger I; 2021. pp. 68-70

[20] Bigey F, Segond D, Friedrich A, Guezenec S, Bourgais A, Huyghe L, et al. Evidence for two main domestication trajectories in Saccharomyces cerevisiae linked to distinct breadmaking processes. Current Biology. 2021;**31**(4):722-732.e5. DOI: 10.1016/j. cub.2020.11.016

[21] Boukhiar A. Analyse du processus traditionnel d'obtention du vinaigre de dattes tel qu'appliqué au sud algérien: Essai d'optimisation [thesis]. Algérie: Université M'hammed Bouguera, Boumerdès; 2009

[22] Samad A, Azlan A, Ismail A. Therapeutic effects of vinegar: A review. Current Opinion in Food Science. 2016;**8**:56-61. DOI: 10.1016/j. cofs.2016.03.001

[23] Cherif B, Bouras N, Oumouna M, Ould El Hadj MD, Holtz MD, Sabaou N. Ethno-pharmacological use and antimicrobial activity of traditional date vinegar of Ghardaïa. Algerian. Journal of Arid Environment. 2014;**4**(1):83-93

[24] Korcari D, Ricci G, Capusoni C, et al. Physiological performance of *Kazachstania unispora* in sourdough environments. World Journal of Microbiology and Biotechnology. 2021;**37**:88. DOI: 10.1007/ s11274-021-03027-0

[25] Bendimerad N. Caractérisation phénotypique technologique et moléculaire d'isolats de bactéries lactiques de laits crus recueillis dans les régions de l'Ouest Algérien. Essai de fabrication de fromage frais type Jben [thesis]. Tlemcen, Algeria: Université Abou Bekr Belkaid; 2013. p. 149

[26] Kacem M, Karem NE. Physicochemical and microbiological study of "shmen", a traditional butter made from camel milk in the Sahara (Algeria): Isolation and identification of lactic acid bacteria and yeasts. Grasas y Aceites. 2006;**57**(2):198-204. DOI: 10.3989/gya.2006.v57.i2.37

[27] Boulkroune A, Debbah A. Valorisation du lactosérum pour la production d'une enzyme coagulante du lait [thesis]. Constantine, Algeria: Université Frère Mentouri; 2019

*Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

[28] Guetouache M, Guessas B. Characterization and identification of lactic acid bacteria isolated from traditional cheese (Klila) prepared from cow's milk. African Journal of Microbiology Research. 2015;**9**(2):71-77. DOI: 10.5897/AJMR2014.7279

[29] Meribai A, Djenidi R, Hammouche Y, Bensoltane A. Caracterisation physicochimique et qualité microbiologique du Klila: un fromage traditionnel sec des régions arides d'Algérie: Etude préliminaire. Journal of new sciences, Agriculture and Biotechnology. 2017;**40**(4):2169-2174

[30] Benamara RN, Benahmed M, Ibri K, Boudjemaa BM, Demarigny Y. Algerian extra hard cheese of Klila: A review on the production method, and microbial, organoleptic, and nutritional properties. Journal of Ethnic Foods. 2022;**9**:41. DOI: 10.1186/s42779-022-00157-0

[31] Aissaoui-Zitoun O, Benatallah L, Ghennam EL, Zidoune MN. Manufacture and characteristics of the traditional Algerian ripened Bouhezza cheese. Journal of Food, Agriculture & Environment. 2011;9(2): 96-100

[32] Boudalia S, Boudebbouz A, Gueroui Y, Bousbia A, Benada M, Leksir CH, et al. Characterization of traditional Algerian cheese Bouhezza prepared with raw cow, goat and sheep milks. Food Science and Technology. 2020;**40**(Suppl. 2):528-537

[33] Derouiche M, Zidoune MN. Caractérisation d'un fromage traditionnel, le *Michouna* de la région de Tébessa, Algérie. Livestock Research for Rural Development. 2015;**27**(11):229

[34] Bouaguel R, Bouguedah L. Caractérisation microbiologique des fromages traditionnels Michouna et Adghess préparés à partir du lait de

chèvre [thesis]. Oum El-Bouaghi, Algeria: Univ. Larbi Ben M'Hidi; 2020

[35] Khoualdia G. Caractérisation du Fromage Traditionnel algérien Medeghissa [Thesis]. Algeria: Institut de la Nutrition, de l'Alimentation et des Technologies Agro-Alimentaires (I.N.A.T.A.A.); 2017

[36] Ben Mohamed C, Bouamer N. Caractérisation Physico-Chimique du Colostrum Camelin (*Camelus dromedarius*) [Thesis]. Ouargla, Algeria: Univ. Kasdi Merbah; 2013

[37] Yssaad D, Benakriche B, Gérard G, Pochart P, Kheroua O. Fermented wheat Hamoum improves the recovery of intestinal mucosal and the short-chain fatty acids profile of colonic bacterial flora in malnourished rats. Bioscience Research. 2020;**17**(2):1166-1175

[38] Benstaali M, Benmamar KA. Approche microbiologique de la flore indigène d'un Blé fermenté type Hamoum: Les Levures comme caractère probiotique [thesis]. Mostaganem, Algeria: Univ. Abdelhamid Ibn Badis; 2021

[39] Boudjenah A. Effet d'une supplémentation de l'aliment en levure *Saccharomyces cerevisiae* sur les paramètres zootechniques de la vache laitière en Peripartum [thesis]. Alger: Ecole National Supérieure Vétérinaire; 2008

[40] Ayad MA, Benallou B, Saim MS, Smadi MA, Meziane T. Impact of feeding yeast culture on milk yield, milk components, and blood components in Algerian dairy herds. Journal of Veterinary Science and Technology. 2013;**4**:2. DOI: 10.4172/2157-7579.1000135

[41] Besseboua O, Benbarek H, Hornick JN, Ayad A. Effect of yeast *Saccharomyces cerevisiae* feed supplement on milk production and its composition

of lactating Holstein Friesian cow from northern Algeria. Journal of Veterinary Science. 2020, 2020;**4**(2):64-74

[42] Mitchison-Field LMY, Vargas-Muñiz JM, Stormo BM, Vogt EJD, Van Dierdonck S, Pelletier JF, et al. Unconventional cell division cycles from marine-derived yeasts. Current Biology. 2019;**29**(20):3439-3456. DOI: 10.1016/j. cub.2019.08.050

[43] Cadez N, Raspor P, de Cock AWAM, Boekhout T, Smith M. Th, molecular identification and genetic diversity within species of the genera *Hanseniaspora* and *Kloeckera*. FEMS Yeast Research. 2002;**1**(4):279-289. DOI: 10.1111/j.1567-1364.2002.tb00046.x

[44] Takaya M, Ohwada T, Oda T. Characterization of the yeast *Hanseniaspora vineae* isolated from the wine grape 'Yamasachi' and its use for bread making. Food Science and Technology Research. 2019;**25**(6):835- 842. DOI: 10.3136/fstr.25.835

[45] C.R. Lab. Carlsberg, 1911-1, 10, p. 285. Isono M, et al. Process for producing unsaturated steroids. US Patent 3,616,225. 1971. Available from: https://www.ncyc.co.uk/catalogue/ hanseniaspora-osmophila-26 [Accessed: September 30, 2022]

[46] Vaughan-Martini A, Angelini P, Cardinali G. Use of conventional taxonomy, electrophoretic karyotyping and DNA–DNA hybridization for the classification of fermentative apiculate yeasts. International Journal of Systematic and Evolutionary Microbiology. 2000;**50**:1665-1672

[47] Bremond E. L'Alcool de Bananes. Annales de l'Institut National Agronomique. 1941;**1**(2):126-134

[48] Chekiri-Talbi M, Denning DW. Burden of fungal infections in

Algeria. European Journal of Clinical Microbiology & Infectious Diseases. 2017;**36**(6):999-1004. DOI: 10.1007/ s10096-017-2917-8

[49] Djeridane A, Djeridane Y, Ammar-Khodja A. Epidemiological and aetiological study on tinea pedis and onychomycosis in Algeria. Mycoses. 2006;**49**:190-196

[50] Djeridane A, Djeridane Y, Ammar-Khodja A. A clinicomycological study of fungal foot infections among Algerian military personnel. Clinical and Experimental Dermatology. 2006;**32**(1):60-63. DOI: 10.1111/j.1365-2230.2006.02265.x

[51] Ait-Seddik H, Ceugniez A, Bendali F, Cudennec B, Drider D. Yeasts isolated from Algerian infants's feces revealed a burden of *Candida albicans* species, non-*albicans Candida* species and *Saccharomyces cerevisiae*. Archives of Microbiology. 2016;**198**(1):71-81. DOI: 10.1007/s00203-015-1152-x

[52] Arrache D, Madani K, Zait H, Achir I, Younsi N, Zebdi A, et al. Fongémies diagnostiquées au laboratoire de parasitologie mycologie du CHU Mustapha d'Alger. Algérie Journal of Mycology Medicine. 2016;**2016**:237- 238. DOI: 10.1016/j.mycmed.2015.06.049

[53] Bendjelloul M, Boucherit-Otmani Z, Boucherit K. Study of strains of *Candida* spp. isolated from catheters in UHC of Oran (Algeria): Identification and antifungal susceptibility. Journal of Médical Mycology. 2016;**26**(3):212-216. DOI: 10.1016/j.mycmed.2016.02.022

[54] Megri Y, Arastehfar A, Boekhout T, Daneshnia F, Hörtnagl C, Sartori B, et al. *Candida tropicalis* is the most prevalent yeast species causing candidemia in Algeria: The urgent need for antifungal stewardship. Antimicrobial Resistance

*Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

and Infection Control. 2020;**9**:50. DOI: 10.1186/s13756-020-00710-z

[55] Hamroune Z. Epidémiologie de la cryptococcose en Algérie [thesis]. Algeria: Université Benyoucef Benkhedda Alger I; 2020

[56] Zerrouki H, Ibrahim A, Rebiahi SA, Elhabiri Y, Benhaddouche DE, de Groot T, et al. Emergence of *Candida auris* in intensive care units in Algeria. Mycoses. 2022;**65**(7):753-759. DOI: 10.1111/ myc.13470

[57] Sebaa S, Faltot M, De Breucker S, Boucherit-Otmani Z, Bafort F, Perraudin JP, et al. Ex vivo decontamination of yeast-colonized dentures by iodine-thiocyanate complexes. Clinical, Cosmetic and Investigational Dentistry. 2018;**10**:149- 158. DOI: 10.2147/CCIDE.S165377

[58] Ouargli M, Gacemi-Kirane D, Mansouri R, Al-Yasiri MS, Ranque S, Roux V. Antifungal activity of Streptomyces sp. against environmental and clinical *Cryptococcus* spp. isolates. Journal of Chemical and Pharmaceutical Research. 2015;**7**(10):1019-1027

[59] Touati S, Touati M. *Cryptococcus*: Recherche de la levure dans l'Est Algérien [thesis]. Algeria: Univ. Frères Mantouri; 2021

[60] Akdouche L, Aissi M, Zenia S, Saadi A. Importance of yeasts in the mammary infection of the cattle in the region of Sidi M'Hamed ben Ali, Wilaya of Relizane, Algeria. Journal of Veterinary Science & Technology. 2014;**5**(2):172. DOI: 10.4172/2157-7579.1000172

[61] Ksouri S, Djebir S, Hadef Y, Benakhla A. Survey of bovine mycotic mastitis in different mammary gland statuses in two north-eastern regions of Algeria. Mycopathologia.

2015;**179**:327-331. DOI: 10.1007/ s11046-014-9845-2

[62] Ksouri S, Djebir S, Bentorki AA, Gouri A, Hadef Y, Benakhla A. Antifungal activity of essential oils extract from *Origanum floribundum* Munby, *Rosmarinus officinalis* L. and *Thymus ciliatus* Desf. Against *Candida albicans* isolated from bovine clinical mastitis. Journal of Medical Mycology. 2017;**27**(2):245-249. DOI: 10.1016/j. mycmed.2017.03.004

[63] Benbelkacem I. Suivi de quelques élevages bovins laitiers à l'ouest du pays: Etude des facteurs influençant l'apparition et/ou le maintien des mammites fongiques [thesis]. Alger: Ecole national Supérieur Vétérinaire; 2019

[64] Benbelkacem I, Selles SMA, Aissi M, Khaldi F, Ghaz K. In vitro assessment of antifungal and antistaphylococcal activities of *Cinnamomum aromaticum* essential oil against subclinical mastitis pathogens. Veterinária. 2019;**68**(1):31-37

[65] Saubin M, Devillers H, Proust L, Brier C, Grondin C, Pradal M, et al. Investigation of genetic relationships between *Hanseniaspora* species found in grape musts revealed interspecific hybrids with dynamic genome structures. Frontiers in Microbiology. 2020;**10**:2960. DOI: 10.3389/fmicb.2019.02960

[66] Labbani FZ. Activité Killer chez des levures isolées des sols du Nord-Est Algérien: Purification, caractérisation et effet sur les souches de levures indésirables [thesis]. Vol. 1. Algeria: Département de Biochimie et Biologie Cellulaire et Moléculaire, Faculté des Sciences de la Nature et de la Vie Univ. Frères Mentouri Constantine; 2015

[67] Toumi S, Tifrit A, Hadjazi D, Chama Z, Abbouni B. Production of a thermostable amylase by yeast strain

isolated from saharian soils cultivated in soft cheese whey. Der Pharmacia Lettre. 2016;**8**(17):32-41

[68] Bennamoun L. Isolement, sélection de souches levuriennes de sols arides sahariens (El-M'gheir) productrices de polygalacturonase: Purification et caractérisation enzymatique [thesis]. Algeria: Université Frères Mentouri Constantine I; 2017

[69] Bilek FN, Rezki MA, Grondin C, Yahia N, Bekki A. Plant growth promoting characteristics and stress tolerance of yeasts isolated from Algerian agricultural soils. South Asian Journal of Exprimental Biology. 2020;**10**(6):413-426

[70] Labbani F-Z, Turchetti B, Bennamoun L, Dakhmouche S, Roberti R, Corazzi L, et al. A novel killer protein from *Pichia kluyveri* isolated from an Algerian soil: Purification and characterization of its in vitro activity against food and beverage spoilage yeasts. Antonie Van Leeuwenhoek. 2015;**107**(4):961-970. DOI: 10.1007/s10482-015-0388-4

[71] Chamekh R, Deniel F, Donot C, Jany JL, Nodet P, Belabida L. Isolation, identification and enzymatic activity of halotolerant and halophilic fungi from the great Sebkha of Oran in northwestern of Algeria. Mycobiology. 2019;**47**(2):230-241. DOI: 10.1080/12298093.2019.1623979

[72] Bennamoun L, Hiligsmann S, Dakhmouche S, Ait-Kaki A, Labbani F-ZK, Nouadri T, et al. Production and properties of a thermostable, pH-stable Exo-Polygalacturonase using *Aureobasidium pullulans* isolated from Saharan soil of Algeria grown on tomato pomace. Foods. 2016;**5**(4):72. DOI: 10.3390/foods5040072

[73] Laiche AT. Production d'enzymes amylolytiques chez des souches levuriennes isolées à partir de deux

origines (sol saharien et blé) et cultivées sur milieu à base de lactosérum [thesis]. Ouargla: Univ. K. Merbah; 2019. p. 181 (+Annexes)

[74] Maamra F, Maissa N. Caractérisation des enzymes protéolytiques des souches fongiques isolées à partir du sol saharien [thesis]. El-Oued: Univ. H. Lakhdar; 2017

[75] Laiche AT, Siboukeur O. Characterization of partially purified extracellular α-amylase from *Candida glabrata* grown in whey based medium. Ponte Academic Journal. 2018;**74**(4):163-173

[76] Boukhennoufa A, Berber N, Aissaoui R, Bekada AMB. Microbiological, biochemical and molecular identification (PCR-RFLP-ITS) of the yeast from sultana grape cultivated in Ain Merane (Wilaya of Chlef). Current Research on Biological Sciences. 2016;**1**(1):7-12

[77] Berber N, Aissaoui R, Bekada AMA, Coarer M. Isolation and molecular identification (PCR-Delta and PCR-RFLP-ITS) of yeasts from the black Muscat grape cultivated in El-Malah (Wilaya of Ain Temouchent, Algeria). Advances in Environmental Biology. 2016;**10**(12):55-61

[78] Berber N. Caractérisation biomoléculaire et biotechnologique des souches de *Saccharomyces cerevisiae* issues des cépages Algériens [thesis]. Mostaganem: Univ. Abdelhamid Ibn-Badis; 2018

[79] Açourene S, Ammouche A, Djaafri K. Valorisation des rebuts de dattes par la production de la levure boulangère, de l'alcool et du vinaigre. Sciences & Technologie. C, Biotechnologies. 2008;**28**:38-45

[80] Rezki-Bekki MA. Production de métabolites par les levures: Caractérisation et identification des *Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

arômes et des alcools, Faculté des Sciences, de la Nature et de la Vie [thesis]. Algeria: Univ. d'Oran; 2014

[81] Rezki MA, Benbadis L, DeBillerbeck G, Benbayer Z, François JM. Isolation and physiological characterization of indigenous yeasts from some Algerian agricultural and dairy products. Journal of Yeast & Fungal Research. 2013;**4**(6):75-83. DOI: 10.5897/JYFR2013.0117

[82] Belbahi A, Bohuon P, Leguérine I, Meot JM, Loiseau G, Madani K. Heat resistance of *Candida apicola* and *aspergillus Niger* spores isolated from date fruit surface. Journal of Food, Processing &. Engineering. 2015;**40**(1):e12272. DOI: 10.1111/jfpe.12272

[83] Kacem M, Karam N-E. Microbiological study of naturally fermented Algerian green olives: Isolation and identification of lactic acid bacteria and yeasts along with the effects of brine solutions obtained at the end of olive fermentation on *lactobacillus plantarum* growth. Grasas y Aceites. 2006;**57**(3):292-300

[84] Bataiche I. Recherche de nouvelles potentialités de *Yarrowia lipolytica*, isolé de différents milieux naturels pour des applications biologiques [thesis]. Algeria: Université Frères Mentouri; 2014

[85] Kechkar M, Sayed W, Cabrol A, Aziza M, Ahmed ZT, Amrane A, et al. Isolation and identification of yeast strains from sugarcane molasses, dates and figs for ethanol production under conditions simulating algal hydrolysate. Brazilian Journal of Chemical Engineering. 2019;**36**(1):157-169. DOI: 10.1590/0104-6632.20190361s20180114

[86] Bessalem S, Laraba-Djebari F, Bellal M. Isolement, Purification et Caractérisation de l'a-amylase de levures isolées à partir de Pomme de terre. Recherche Agronomique. 2001;**5**(9):75-90

[87] Labbani F-ZK, Dakhmouche S, Bennamoun L, Ait-Kaki A, Nouadri T. Extracellular hydrolytic enzymes of yeasts isolated from fruit and beet peels in Algeria. International Journal of Ecosystems and Ecology Science (IJEES). 2022;**12**(1):7-16. DOI: 10.31407/ijees12.1 Abstract

[88] Dakhmouche-Djekrif S, Gillmann L, Bennamoun L, Ait-Kaki A, Labbani K, Nouadri T, et al. Amylolytic yeasts: Producers of α-amylase and Pullulanase. International Journal of Life Science & Scientific Research. 2016;**2**(4):339-354

[89] Dakhmouche-Djekrif S. Production et caractérisation de l'amylopullulanase de la levure *Clavispora lusitaniae* ABS7 isolée de blé cultivé et stocké en zones arides [thesis]. Constantine, Algeria: Université des Frères Mentouri; 2016

[90] Abdel-Kareem MM, Zohri AA, Nasr SA. Novel marine yeast strains as plant growth-promoting agents improve defense in wheat (*Triticum aestivum*) against *Fusarium oxysporum*. Journal of Plant Disease Protocol. 2021;**128**:973-988. DOI: 10.1007/s41348-021-00461-y

[91] Sipiczki M. *Metschnikowia pulcherrima* and related Pulcherrimin-producing yeasts: Fuzzy species boundaries and complex antimicrobial antagonism. Microorganisms. 2020;**8**(7):1029. DOI: 10.3390/microorganisms8071029

[92] Beldjilali AF, Belgroun F, Benlahcen K, Bettache G, Aggad H, Kihal M. Evaluation of microbiological and sanitary quality of ewe's raw milk in Western of Algeria and detection of antibiotic residue by Delvotest. Advances in Environmental Biology. 2013;**7**(6):1027-1033

[93] Hamoudi-Belarbi L, Nouri L, Belkacemi K. Effectiveness of convective drying to conserve indigenous yeasts with high volatile profile isolated from Algerian fermented raw bovine milk

(Rayeb). Food Science & Technology, Campinas. 2016;**36**(3):476-484. DOI: 10.1590/1678-457X.00416

[94] Aziza M, Amrane A. Diauxic growth of *Geotrichum candidum* and *Penicillium camembertii* on amino acids and glucose. Brazilian Journal of Chemical Engineering. 2012;**29**(2):203-210

[95] Kebbouche-Gana S, Gana ML. Algerian yeast strains: Isolation, identification and production of single cell protein from whey with strain *Candida kefyr*. International Journal of Bioscience, Biochemistry and Bioinformatics (IJBB). 2014;**4**(3):162- 165. DOI: 10.7763/IJBBB.2014.V4.331

[96] Ider S, Belguesmia Y, Coucheney F, Kihal M, Drider D. Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria. Archives of Microbiology. 2019;**201**(3):399-407. DOI: 10.1007/ s00203-019-01626-y Epub 2019 Feb 4

[97] Hamza H, Hinana R. Étude du potentiel biotechnologique des levures isolées du lait de chamelle dans la région de Ghardaïa [thesis]. Algeria: Université de Ghardaïa; 2020

[98] Fesq H, Brockow K, Strom K, Mempel M, Ring J, Abeck D. Dihydroxyacetone in a new formulation a powerful therapeutic option in vitiligo. Dermatology. 2001;**203**(3):241-243. DOI: 10.1159/000051757

[99] Derouiche N, Medjouj H, Aissaoui-Zitoune W, Zidoune MN. Some traditional cheeses manufactured in Algeria. In: Henriques MHF, Pereira CJD, editors. Cheese Production, Consumption and Health Benefits. New York: Food Science and Technology, Nova Science Publisher; 2017. pp. 225-242

[100] Medjoudj H, Aouar L, Zidoune MN, Hayaloglu AA. Proteolysis, microbiology, volatiles and sensory evaluation of Algerian traditional cheese Bouhezza made using goat's raw milk. International Journal of Food Properties. 2017;**20**(3):S3246-S3265. DOI: 10.1080/10942912.2017.1375515

### [101] Matallah-Boutiba A,

Benmessaoud N, Messaoui N, Boutiba Z. Microbiological Sandy Beach quality in Western Algeria. Journal of Marine Biology & Oceanography. 2016;**6**:1. DOI: 10.4172/2324-8661.1000171

[102] Benfreha-Benyelles M, Mokrani S, Belgharbi AA, Meddah A, Moussa-Boudjemaa B. Enquête microbiologique et possibilité de réutilisation des eaux usées pour la production végétale. Un cas de la station d'épuration d'El-Kouwaer pendant la période de dysfonctionnement. Algérie, Revue Agrobiologia. 2021;**11**(2):2566-2578

[103] Bourzama G, Iratni N, Ennaghra N, Ouled-Haddar H, Soumati B, Amour K, et al. *In vitro* removal of electronic and electrical wastes by fungi isolated from soil at Annaba area in northeast of Algeria. Environment and Natural Resources Journal. 2021;**19**(4):302-309

[104] Aibeche C, Selami N, Zitouni-Haouar FE, Oeunzar K, Addou A, Kaid-Harche M, et al. Bioremediation potential and lead removal capacity of heavy metal-tolerant yeasts isolated from Dayet Oum Ghellaz Lake water (northwest of Algeria). International Microbiology. 2022;**25**(1):61-73. DOI: 10.1007/s10123-021-00191-z

[105] Grujić SM, Radojević ID, Vasić SM, Čomić LR, Ostojić AM. Heavy metal tolerance and removal efficiency of the *Rhodotorula mucilaginosa* and *saccharomyces boulardii* planktonic cells and biofilm. Kragujevac Journal of Science. 2018;**40**:217-226

[106] Derguine-Mecheri L, Kebbouche-Gana S, Djenane D.

### *Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

Biosurfactant production from newly isolated *Rhodotorula* sp. YBR and its great potential in enhanced removal of hydrocarbons from contaminated soils. World Journal of Microbiology and Biotechnology. 2021;**37**:18. DOI: 10.1007/ s11274-020-02983-3

[107] Dermeche S, Moulti-Mati F. Assessment of olive mill wastewaters bioconversion potential into biotechnological and health interest microbial biomass, Algerian. Journal of Environmental Science and Technology. 2019, 2019;**5**(1):898-908

[108] Belhocine H. Contribution à l'amélioration génétique des levures d'intérêt industriel par la technique de fusion des protoplastes [thesis]. Vol. 2011. Alger: Université des sciences et de la technologie Houari Boumediène; 2011. p. 89

[109] Chibi S, Rabet S, El-hadi D. Etude des paramètres environnementaux sur la croissance de *Saccharomyces cerevisiae* isolée de rebuts de dattes. Algerian Journal of Environmental Science and Technology. 2016;**2**(3):62-69

[110] Chibi S, El-Hadi D. The isolation and characterization of yeast strains *Saccharomyces cerevisiae* cultivated on the musts of rebuts of dates: Physiological studies of the adaptation and the resistance to ethanol. Algerian Journal of Environmental Science & Technology. 2019;**5**(2):937-946

[111] Gana S. et Touzi A. Valorisation du lactosérum par la production de levures lactiques avec les procédés de fermentation discontinue et continue. Rev. Energ. Ren. Special Issue: Production et Valorisation– Biomasse. Alger: Centre de Développement des Energies Renouvelables (CDER); 2001. pp. 51-58

[112] Moeini H, Nahvi I, Tavassoli M. Improvement of SCP production and BOD removal of whey with mixed yeast culture. Electronic Journal of Biotechnology. 2004;**7**(3):249-255

[113] Ould El Hadj MD, Bitour Z, Siboukeur O. Etude de la production de levure boulangère (*Saccharomyces cerevisiae*) cultivée sur mout de rebuts de dates. Courrier du Savoir Scientifique et Technique. 2006;**7**(7):13-18

[114] Açourene S, Ammouche A. Optimization of culture medium for Baker's yeast, ethanol, citric acid and &-amylase production from dates syrup. Research Journal of Agriculture & Biological Sciences. 2010;**6**(6):846-860

[115] Oulad-Belkhir A. Fabrication de la levure boulangère à base de rebuts de dates Ghars [thesis]. Ouarlga: Univ. Kasdi Merbah; 2016

[116] Kara AM, Outili N, Ait KA, Cherfia R, Benhassine S, Benaissa A, et al. Optimization of Baker's yeast production on date extract using response surface methodology (RSM). Food. 2017;**6**(8):64. DOI: 10.3390/ foods6080064

[117] Diboune N, Nancib A, Nancib N, Anibal J, Boudrant J. Utilization of prickly pear waste for baker's yeast production. Biotechnology and Applied Biochemistry. 2019;**66**(5):744-754. DOI: 10.1002/bab.1753

[118] Boughnou S. Essai de production du vinaigre à partir de déchets de dattes. Ann. Inst. Nat. Agro., El-Harrach. 1988;**12**(2):65-83

[119] Kaidi F, Touzi A. Production de Bioalcool à partir des déchets de dattes. Rev. Energ. Ren. Special Issue: Production et Valorisation – Biomasse. Alger: Centre de Développement des Energies Renouvelables (CDER); 2001. pp. 75-78

[120] Ould E-HMD, Cheick M, Hamdi W, Sayah Z, Bouaziz S. Etude comparative de la production d'éthanol brut à partir de trois variétés de dattes communes (Degla Beida, Tacherwit et Hamraya) reparties dans les différentes classes de dattes (molle, demi-molle et sèche) de la cuvette de Ouargla (Sahara septentrional Est algérien). Algerian. Journal of Arid Environment. 2012;**2**(2):78-87

[121] Mehani I, Boulal A, Bouchekima B. Biofuel production from waste of starting dates in South Algeria. International Journal of Environmental and Ecological Engineering. 2013;**7**(8):572-574

[122] Oucif-Khaled MTO, Segni L. Production of bioethanol from dates of poor quality. African Journal of Agriculture Research. 2014;**9**(37):2814-2818

[123] El-Hadi D, Korteby S, Chibi S. Production de bioethanol à partir de deux variétés de dates (Deglet Nour et Hamraya). Revue Agrobiologia. 2016;**6**(1):111-120

[124] Boulal A, Kihal M, Khelifi C. Fermentative strength of yeasts strain, naturally isolated using common date in south-west of Algeria. International Journal of ChemTech Research. 2017;**10**(1):180-188

[125] Mansour A, Rihani R, Bentahar F. Etude de la production de bioethanolbiocarburant à partir de sous produits agricoles: Effet de l'aération. Algerian Journal of Environmental Science ALJEST. 2019;**5**(3):1087-1093

[126] Laouar A. Caractérisation physicochimique et microbiologique de deux variétés de dattes "Hmira, Feggous" et production de bioéthanol à partir de rebuts de dattes "Hmira" [thesis]. Béchar: Univ. Mohamed Tahri; 2020

[127] Edjekouane M, Lansari F, Khelifi O, Boukheteche I, Laksaci H. Production

of Bioéthanol from a local natural resource. Algerian Journal of Renewable Energy and Sustainable Development. 2020;**2**(1):56-59. DOI: 10.46657/ ajresd.2020.2.1.8

[128] El-Bey S, Ouakil A, Zoubiri F-Z, Rihani R, Bentahar F. Production de bioéthanol à partir de jus d'orange de Rouïba. Revue Nature et Technologie. 2022;**14**(2):1-7

[129] Rezki MA, Aouad L, Bekki A. Production of ethanol and polyethanol by yeasts isolated from date (*Phoenix dactylifera* L.) wastes. African Journal of Biotechnology. 2015, 2015;**14**(50):3288- 3294. DOI: 10.5897/AJB2015.14979

[130] Hadri K, Boulal A, Cheikh N. Ethanol production by *Balanites aegyptiaca* fruits valorization in the Adrar region of Algeria. Journal of Renewable and Sustainable Energies. 2021;**1**(1):109-115

[131] Hadri K, Cheikh N, Ammar M, Messaoudi F-Z, Boulal A. Valorization of *Balanites aegyptiaca* fruits by production of bioethanol: Study and optimization. Current Trends in Natural Sciences. 2022;**11**(21):290-303. DOI: 10.47068/ ctns.2022.v11i21.032

[132] Aziz S. Production of Ethanol from Molasses Using Thermotolerant *Kluyveromyces Marxianus* [Thesis]. Jamshoro: Mehran University of Engineering & Technology; 2010

[133] Macedo N, Brigham CJ. From beverages to biofuels: The journeys of ethanol-producing microorganisms. International Journal of Biotechnology for Wellness Industries. 2014;**3**:79-87

[134] Dabija A, Hatnean CA. Study concerning the quality of apple vinegar obtained through classical method. Journal of Agroalimentary Processes and Technologies. 2014;**20**(4):304-310

*Inventoried Yeast Species in Algeria DOI: http://dx.doi.org/10.5772/intechopen.109694*

[135] Bouaziz S., Ould El-hadj M. D., Contribution a l'étude des caractéristiques physico-chimiques et biochimiques de quelques types de vinaigres traditionnels de dattes obtenues a partir de quelques variétés de la région de Ouargla, Annales des Sciences et Technologie. 2010;**2**(1):80-86

[136] Benahmed-Djilali A, Benrachedi K, Benamara S. La qualité du vinaigre de dattes obtenu par le procède traditionnel du sud algérien; composés volatils. In: X colloque international, Sécurité alimentaire: Réalités et perspectives. Adrar: Univ. Ahmed Draia; 2007. pp. 443-460

[137] Bouazza A, Bitam A, Amiali M, Bounihi A, Yargui L, Koceir E. Ahmed, effect of fruit vinegars on liver damage and oxidative stress in high-fat-fed rats. Pharmaceutical Biology. 2016;**54**(2):260- 265. DOI: 10.3109/13880209.2015.1031910

[138] Ameur D, Heleili N. Comparative study on physico-chemical and sensory properties of vinegar produced from apple varieties. Agricultural Science Digest. 2022;**42**:534-540. DOI: 10.18805/ ag.DF-401

[139] Yu X, Zhao M, Liu F, Zeng S, Hu J. Identification of 2,3-dihydro-3,5 dihydroxy-6-methyl-4H-pyran-4-one as a strong antioxidant in glucose–histidine Maillard reaction products. Food Research International. 2013;**51**(1):397- 403. DOI: 10.1016/j.foodres.2012.12.044

[140] Dali NS, Hamame A. Recherche de levures productrices d'enzymes glycolytiques exocellulaires thermostables: Production (sur boite de Pétri et en batch) et Caractérisation des enzymes produites. Constantine, Algeria: Univ. F. Mantouri; 2016

[141] Dakhmouche-Djekrif S, Bennamoun L, Labbani FZK, Ait-Kaki A, Naouadri T, Pauss A, et al. An alkalothermophilic amylopullulanase from the yeast *Clavispora lusitaniae* ABS7: Purification, characterization and potential application in laundry detergent. Catalysts. 2021;**11**:1438. DOI: 10.3390/catal11121438

[142] Labbani F-ZK, Bennamoun L, Dakhmouche S, Ait-Kaki A, Nouadri T. Production, characterization and in vitro evaluation of a yeast killer toxin against some food and beverage spoilage yeasts, in the abstracts of the 1st international webinar on innovative applications of biotechnologies in the food industry: From the laboratory to the enterprise. Algerian Journal of Nutrition and Food Sciences (AJNFS). 2021;**1**(4):26-27

[143] Bennamoun L, Dakhmouche S, Ait-Kaki A, Labbani F-ZK, Nouadri T, Meraihi Z. Production et caractérisation de pectinase levurienne: Essais d'application dans la clarification de jus de citron in the abstracts of the 1st international webinar on innovative applications of biotechnologies in the food industry: From the laboratory to the enterprise. Algerian Journal of Nutrition and Food Sciences (AJNFS). 2021;**1**(4):3

[144] Gares M, Hiligsmann S, Chaouche NK. Lignocellulosic biomass and industrial bioprocesses for the production of second generation bioethanol, does it have a future in Algeria? SN Applied Sciences. 2020;**2**:1680. DOI: 10.1007/s42452-020-03442-2

[145] Mihi A. Dynamic simulation of future date palm plantation (Phoenix dactylifera L.) growth using CA–Markov model and FAO-LCCS data in Algerian dryland oases desert. Modeling Earth Systems and Environment. 2022;**8**:3215- 3230. DOI: 10.1007/s40808-021-01289-z

[146] Šuchová K, Fehér C, Ravn JL, Bedő S, Biely P, Geijer C. Cellulose- and xylan-degrading yeasts: Enzymes, applications and biotechnological potential. Biotechnology Advances. 2022;**59**:107981. DOI: 10.1016/j. biotechadv.2022.107981

[147] Moussa A, Djebli N, Aissat S, Meslem A, Benhalima A. Antifungal activity of four honeys of different types from Algeria against pathogenic yeast: *Candida albicans* and *Rhodotorula* sp. Asian Pacific Journal of Tropical Biomedicine. 2012;**2**(7):554-557. DOI: 10.1016/S2221-1691(12)60096-3

[148] Saadoune Z, Boutoumi Y. Impact of the composition of the essential oils of *Citrus sinensis* (Orange) and *Citrus limonum* (lemon) on the microbiological activity. Algerian Journal of Engineering Research, AJER. 2018;**3**:1-8

[149] Boukhatem MN, Ferhat MA, Kameli A, Saidi F, Kebir HT. Lemon grass (*Cymbopogon citratus*) essential oil as a potent anti-inflammatory and antifungal drugs. Libyan Journal of Medicine. 2014;**19**(9):25431. DOI: 10.3402/ljm. v9.25431

[150] Bouzabata A, Cabral C, Gonçalves MJ, Cruz MT, Bighelli A, Cavaleiro C, et al. *Myrtus communis* L. as source of a bioactive and safe essential oil. Food and Chemical Toxicology. 2015;**75**:166-172. DOI: 10.1016/j.fct.2014.11.009

[151] Bouzabata A, Bazzali O, Cabral C, Gonçalves MJ, Cruz MT, Bighelli A, et al. New compounds, chemical composition, antifungal activity and cytotoxicity of the essential oil from *Myrtus nivellei* batt. & Trab., an endemic species of Central Sahara. Journal of Ethnopharmacology. 2013;**149**(3):613-620. DOI: 10.1016/j. jep.2013.06.054

[152] Neggaz S, Fortas Z. Tests of antibiotic properties of Algerian Desert truffle against bacteria and fungi. Journal of Life Sciences. 2013;**7**(3):259-266

[153] Bouchenak O, Boumaza S, Yahiaoui K, Benhabyles N, Laoufi R, Toubal S, et al. Evaluation de l'activité antioxydante et l'effet antimicrobien des composes phénoliques extraits du lichen *Xanthoria parietina* de la région de Boumerdes. Revue des BioRessources. 2020;**10**(2):93-103

[154] Boukhatem T, Chadli R, Bouchama A, Hamed D. Cytotoxicity test on *Artemia salina*, antibacterial and antifungal activities of *Cystoseira stricta* extracts from the coast of Mostaganem, western Algeria. Journal of Materials and Environmental Sciences. 2018;**9**(12):3190-3196

[155] Messahli I, Gouzi H, Sifi I, Chaibi R, Rezzoug A, Rouari L. Anticandidal activity of dichloromethane extract obtained from the red algae a. armata of the Algerian coast. Acta Ecologica Sinica. 2022;**42**(5):461-466. DOI: 10.1016/j. chnaes.2021.08.005

### **Chapter 2**

## Upgrading Non-Conventional Yeasts into Valuable Biofactories

*Kevin Castillo-Mendieta, Jimmy Arias and Fernando Gonzales-Zubiate*

### **Abstract**

The use of synthetic biology on yeasts has enhanced the production of commercially relevant chemicals, from biofuels to recombinant therapeutic proteins, to name just a few. Despite most of these advances had already been studied and described in *Saccharomyces cerevisiae*, during the last years the attention has turned to the use of alternative expression systems with a higher yield and quality such as non-conventional yeasts. Recently, there has been an increase in studies about non-conventional yeasts due to advantages based on their natural capacity to tolerate harsh conditions or the wide range of carbon sources they need during the generation of specific products. This chapter, therefore, aims to describe the current status of the most used non-conventional yeasts in metabolite production as well as the engineering behind them in order to optimize or regulate protein expression: *Pichia pastoris*, *Kluyveromyces marxianus*, *Kluyveromyces lactis* and *Yarrowia lipolytica*.

**Keywords:** non-conventional yeasts, bioengineering, synthetic biology, CRISPR-Cas9, Golden Gate cloning, TALENs

### **1. Introduction**

Yeast is probably one of the oldest domesticated organisms, since it was used for beer brewing already in Sumer and Babylonia around 6000 BC [1]. Not surprisingly, yeast cells were among the first microorganisms seen after the invention of the microscope in the seventeenth century, but their recognition as a living organisms did not come until two centuries later [2]. Yeasts, as such, do not form a single taxonomic or phylogenetic group in the kingdom fungi, rather, they occur in different subdivisions belonging to *Ascomycota and Basidiomycota*. Moreover, they are unicellular with budding and binary fission as the main asexual reproduction, and sexual spore production in stress conditions [3, 4].

Interestingly, yeast cells can exhibit a variety of cell sizes, shapes and colors. Even individual cells from a pure strain of a single species can display morphological heterogeneity. Moreover, yeast cell size varies widely from 2 up to 50 μm in length. Many yeast species are ellipsoidal or ovoid, but other cell shapes can be also observed as in *Candida albicans* and *Yarrowia lipolytica* which are mostly filamentous [1].

With respect to their diversity, there are around 2000 accepted yeast species included in the Yeast Trust Database (theyeasts.org) [4]. They have been isolated from highly diverse environments such as insect guts, food products, soil, oceans and even ancient ice fields [1]. However, it seems that we have just scratched the tip of the iceberg. According to Fell's estimation, what we have found represents only 1% of the species that might exist in nature [4].

Furthermore, yeasts as a whole are interesting because they are capable of metabolizing a wide variety of carbon sources including glucose, fructose, lactose, xylose and arabinose [3]. Besides, the metabolic activity of some yeasts can be dependent on the sugar concentration present in the medium: fermentation in high sugar concentration and aerobic respiration in low sugar concentrations (Crabtree effect) which can be advantageous in some industrial processes [3, 5].

### **2. Industrial applications of yeasts**

Industrially, yeasts possess many attractive features that confer them some benefits in relation to bacteria such as *Escherichia coli*. For instance, yeasts have the capacity to grow on a wide variety of carbon sources, perform post-translational modifications, and compartmentalize reactions in organelles, they also present high secretion capacity, and are less susceptible to infectious agents like bacteriophages [6].

For these reasons, natural yeasts have been used in a lot of industrial processes. For example, in the food industry, the alcoholic fermentation of *S. cerevisiae* is used for the production of bread, beer and wine [7]. Furthermore, other yeasts species take different roles in the elaboration of food products such as yoghurts, in which *Torulopsis candida*, and *Kluyveromyces fragilis* are used for the improvement of aroma, texture and addition of nutrients by fermenting lactose with hydrolysis of milk casein [8].

Another important application of *S. cerevisiae* is the production of biofuels such as bioethanol, which is a result of sugar fermentation under anaerobic conditions. *S. cerevisiae* catabolizes sugars by glycolysis until it produces pyruvate that is then converted to acetaldehyde and carbon dioxide, which is reduced to ethanol by an alcohol dehydrogenase [9]. In addition, other yeast species also have the capacity to produce bioethanol. In fact, *Kluyveromyces marxianus*, *Dekkera bruxellensis* and *Scheffersomyces stipitis* are capable of producing bioethanol by fermentation of polyfructan substrates, hexoses and lignocelluloses substrates respectively [9]. Some yeasts are able to naturally produce bioethanol using lignocellulose resources (cheap, abundant and renewable) making them of great interest in second-generation biofuels, thus providing a clear advantage over first-generation biofuels that employ large cultivated areas [9–11].

On the other hand, despite *S. cerevisiae* has been widely studied and its industrial applications being countless, other yeast species, known as non-conventional yeasts (NCYs1 ) are becoming more popular in industrial applications. Several NCYs have diverse advantages compared to *S. cerevisiae*, mainly they are more suitable for a big number of biotechnological processes since they present natural tolerance to stresses like extreme pH, temperatures and osmolarity conditions [12]. Some of the most studied NCY species that are capable to withstand harsh conditions are *Yarrowia lipolytica*, *Hanensula polymorpha*, *Pichia pastoris* and *Kluyveromyces lactis* [13]. In fact, *K. lactis* is widely used in the cheese industry, replacing the conventional rennet, due

<sup>1</sup> Currently, there is not an accepted definition of NCYs, but many scientists consider NCYs as "non-*Saccharomyces*" yeasts [7].

### *Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

to its ability to produce lactic acid from lactose. On the other hand, *Y. lipolytica* is used for the production of biosurfactants, carotenoids and lipids and *K. marxianus* for the production of bioethanol, aroma compounds and biosurfactants [6, 12].

Furthermore, some of the most interesting features of NCYs are their capacity to accumulate metabolites, synthesize and secrete recombinant proteins and enzymes [12]. For instance, yeasts like *Y. lipolytica* and *K. lactis* are able to secrete high titers of proteins extracellularly better than *S. cerevisiae* [13]. Moreover, some useful industrial enzymes like amylases, cellulases, proteases and lipases, have been reported to be produced by several strains of the NCY *Aureobasidium pullulans* [14]. These interesting properties have led to a proliferation of studies in NCYs aiming to improve their performance in the production of important metabolites and proteins. Hence, this review focuses on providing a clear description and analysis of the use of synthetic biology tools and strategies at the expression level that helps enhance four of the most popular NCYs: *P. pastoris*, *K. marxianus*, *K. lactis* and *Y. lipolytica* into valuable biofactories.

### **3. Use of synthetic biology in NCYs**

Synthetic biology relies on the premise that a biological system can be built using a collection of previously described parts and subsystems [6]. This is achieved by standardization and modularization of useful biological parts, mechanisms and systems, or redesign the existing ones to provide new and better qualities [15]. Therefore, it is capable to define building blocks at various levels such as expression, protein and pathway levels [15]. The ability to control the dynamics at each level is important in order to establish unique and robust expression and production platforms for biomanufacturing [6]. For example, in synthetic biology-inspired therapies, the regulation of gene expression is important to determine the amount of the therapeutic and allows for accurate control over the design of synthetic cells [16].

Furthermore, with the emergence of modern genome editing tools, the synthetic biological capabilities to rewire and engineer organisms for production purposes have enabled the application of engineering efforts in non-conventional yeast of interest to industrial biotechnology [6].

This section will focus on the description of synthetic biology tools at the expression level, covering the engineering of genetic parts which include promoter, terminator and signal peptide, as well as codon optimization. In addition, available genome editing tools like CRISPR-Cas and cloning methods such as Golden Gate are also discussed. Their applications will be described in detail, later on, in Section 4.

### **3.1 Engineering of genetic parts**

**Codon optimization.** The degeneracy of the genetic code means that several amino acids can be encoded by more than one codon (e.g. Leu = CUU, CUC, CUA, CUG), thus a random codon usage would be expected for those amino acids [17]. However, the expression of the same gene is different depending on the organism, because of the availability of host's tRNA pool. This is known as codon bias and is thought to affect the translation efficiency [18]. As a result, codon optimization is an important strategy when considering the expression of heterologous proteins.

**Promoters.** Selecting an adequate promoter is an important step since it can affect the level of expression of the desired protein. There can be constitutive or inducible

promoters, the latter more advantageous since they allow researchers to separate cell growth from the production of the desired protein. This avoids potential toxic effects due to a constitutive expression of heterologous proteins [19]. In addition, having a variety of promoters available is desired to fine-tune and optimize pathways that involve the de-expression of several proteins [20].

**Terminators.** It not only plays a critical role in transcription but is also able to influence mRNA stability and lifetime. This provides a new level of regulating protein expression; however, the impact of terminators is sometimes underestimated compared to promoters [21, 22]. In *S. cerevisiae* expression vectors, some endogenous terminators such as TCYC1 and TADH1 are commonly used. Nevertheless, it has been demonstrated that *S. cerevisiae* terminators can show a high degree of transferability across other non-conventional yeasts [23].

**Signal peptides.** If a protein of interest is desired to be secreted, it is only required to add a secretion signal peptide at the N-terminus of the nascent polypeptide [24]. However, selecting an appropriate signal peptide is crucial since the protein quality and yield may vary widely depending on the heterologous protein being expressed [25]. Therefore, screening and characterizing many signal peptide sequences is a good approach to have adequate expression levels of different proteins.

### **3.2 Cloning methods and genome editing tools**

**Golden Gate.** Golden Gate cloning method uses type II restriction enzymes (*Bsa*I and *Bpi*I) to precisely assemble multiple genetic parts by simultaneous restriction and ligation steps. These restriction enzymes cut outside their recognition sequences leaving 4-letter overhangs which can be freely designed; hence this method offers important benefits as it is cheaper than other advanced techniques, it does not require long flanking DNA and it allows scar-less cloning [26].

**TALEN.** Transcription-activator-like effector (TALE) nuclease is a genome editing tool based on type II effector proteins from bacterial plant pathogens of the genus *Xanthomonas* fused with the non-specific nuclease domain of the restriction endonuclease *Fok*I [27]. These TALENs are designed such that they bind separate targets in opposition to each other with an appropriate spacer between them allowing the *Fok*I nucleases to cause a double-strand break (DSB) and subsequently allowing the editing of the genome [28]. This genome editing tool has many advantages such as easy assembly, availability of powerful resources, cross-species flexibility and a high rate of success [27].

**CRISPR-Cas9.** This tool revolutionized the field of gene editing, since it is capable of creating a DSB in a specific DNA site by just using a single-guide RNA (sgRNA) complementary to the targeted region and an endonuclease (Cas9). This makes CRISPR-Cas9 a better genome editing tool compared to TALENs, which require laborious protein engineering steps for each new editing target. This editing tool has been successfully used for knock-out of a gene, but it can be adapted for other applications such as regulating transcription and facilitating metabolic engineering [29, 30].

### **4. Engineering non-conventional yeasts (NCYs)**

This section will discuss the application of synthetic biology tools and strategies in four of the most popular NCYs: *P. pastoris*, *K. marxianus*, *K. lactis* and *Y. lipolytica*, yeasts that have been selected for several reasons. First, they naturally present

*Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

### **Figure 1.**

*Description of the non-conventional yeasts (NCYs):* P. pastoris*,* K. marxianus*,* K. lactis *and* Y. lipolytica *and the synthetic biology tools and strategies applied to each of them. Descriptions of the same color represent a specific yeast. Example: All text in red belongs to* Y. lipolytica*.* **\***K. marxianus *is classified as Crabtree-negative, although some reports consider this species as Crabtree-positive due to strain variability.*

interesting characteristics with potential benefits in industrial applications such as the use of "waste products" as substrates, thermotolerance, high capacity to store lipids and the capacity to efficiently secrete desired compounds (**Figure 1**).

Second, they have their genomes sequenced and available at the National Center for Biotechnology Information (NCBI) website [31] and several metabolomic and transcriptomic studies have been reported [32–37]. This information is key when searching for new sources of BioBricks, since novel genetic parts or metabolites can be discovered under different contexts such as carbon sources or stress conditions. Third, information about regulatory associations between transcription factors (TFs) and target genes in these four species is available at the N. C. Yeastract database [38]. Last but not least, all the referred species have genome-scale metabolic models reported in the literature [39–42], allowing researchers to predict metabolic fluxes, and subsequently, optimize the production of relevant compounds in these microorganisms.

### **4.1** *Pichia pastoris*

*Pichia pastoris*<sup>2</sup> was initially developed by Phillips Petroleum Company for the production of single-cell protein for feedstock, but it was then repurposed as a promising expression system for the production of recombinant proteins [44] due to its many advantages such as high cell density cultivation, an efficient secretory capacity with a low background of endogenous proteins, the absence of protease secretion, the presence of alternative constitutive and inducible promoters and the ability to perform post-translational modifications to proteins [24, 45].

*P. pastoris* is a Crabtree-negative yeast that is able to utilize a variety of carbon sources including glucose, glycerol, fructose, sorbitol, methanol, alanine and cadaverine [43, 44]. As a matter of fact, it has the ability to use methanol as a sole carbon source (methylotrophic) due to several adaptations such as the expression of enzymes involved in methanol metabolism (e.g. alcohol oxidase) and the proliferation of peroxisomes (reaching over 80% of the cell volume) [44].

For these reasons, *P. pastoris* has become an industrially important microorganism. This is evidenced by the over 300 industrial processes that have been licensed and more than 70 commercial products that are currently on the market [46]. The use of synthetic biology tools in this yeast has led to a whole new level of potential industrial applications for this yeast. Some examples of the application of synthetic biology strategies in *P. pastoris* are provided below.

**Codon Optimization.** A clear application of this strategy in this yeast is the heterologous expression of enzymes such as phytases which are important enzymes that once included in animal feed can help not only increase the absorption of phosphorus in monogastric animals (e.g. pigs and horses) but at the same time reduce phosphorus levels in manure, thus finding a cheaper and more efficient way of producing this enzyme is desired. For instance, Xiong et al. compared the expression of a recombinant *Aspergillus niger* phytase in *P. pastoris* before and after codon optimization of both the phytase gene and the *S. cerevisiae* signal sequence α mating factor (α-MF). They obtained a phytase activity of 865 Units/ml, resulting in a 14.5-fold increase in the production/activity of phytase in comparison with the non-optimized gene and signal sequence [47]. The following year the same group applied this strategy to express a *Peniophora lycii* phytate enzyme achieving a phytase activity of 10,540 Units/ml and a 13.6-fold yield increase compared with the non-optimized gene and signal sequence [48]. In the last years, heterologous expression of other industrially

<sup>2</sup> Although initially named *Pichia pastoris* in the 1950s, it was then reclassified into the genus *Komagataella* in 1995, splitting up into the two species *K. pastoris* and *K. phaffii* [43]. Here, we still use the name *P. pastoris* for simplicity.

relevant proteins has been enhanced using this method, including but not limited to keratinases, endoinulinases, α-amylases, lipases, xylanases, fibases, pectinases, IFN-ω and hydroxynitrile lyases [49–57].

**Promoters.** Since all gene promoters of the methanol utilization (MUT) pathway are strongly repressed by carbon sources such as glucose, they can be a useful tool for the induced expression of heterologous proteins [58]. In fact, most of the heterologous expression of proteins in *P. pastoris* is carried out using one of those promoters, P*AOX*1 [59]*.* In addition, other orthologous MUT promoters from related species have been evaluated in this species such as *Hansenula polymorpha* PFMD which showed a 3.5 fold higher expression compared to the strongest endogenous MUT promoters [58].

On the other hand, using methanol (a flammable and toxic compound) in a large-scale fermentation process can be potentially dangerous [60]. For this reason, researchers have tried to find alternatives to overcome this limitation using different approaches such as employing orthologous promoters from related methylotrophic yeasts [58] or even engineering cis- and trans-acting elements in the PAOX1 [60]. For instance, using a trans-acting approach, Wang et al. developed a methanol-free method for protein expression using the PAOX1 promoter. They developed a strain that overexpressed the transcription activator Mit1 and repressed glucose- or glyceroldependent transcriptional repressors Mig1, Mig2 and Nrg1. Then they evaluated the performance of both the methanol-free system and the wild-type system under their respective optimal culture conditions. Although the expression level of the recombinant insulin precursor protein in the methanol-free system was only 58.6% of the wild-type, they claim that it can be further improved by overexpressing unfolded protein response activators, protein foldases or chaperons [60].

**Terminators.** Knowledge about terminators is rather limited in *P. pastoris*, although in recent years it has been gaining more attention. In 2020 Ito et al. developed a terminator catalog of 72 sequences including synthetic, endogenous and heterogenous (*S. cerevisiae*) terminators with a 17-fold range of expression when using red fluorescent protein and *Aspergillus aculeatus* β-glucosidase as reporter proteins. Moreover, interesting results were found from this study including independence of terminator activity from the upstream gene and high degree of transferability of *S. cerevisiae* terminators to *P. pastoris* [61].

**Signal Peptides.** The *S. cerevisiae* signal sequence α-MF is widely used for secreted expression of recombinant proteins in *P. pastoris* [62], where a peptidic pre-region leads the translocation from the cytoplasm to the ER lumen. Additionally, it has a peptidic pro-region which facilitates the proper transit of target polypeptides from the ER to the Golgi apparatus; unfortunately, this pro-region tends to aggregate in the ER, impeding a proper secretion of the target protein [63]. Several strategies can be applied to overcome this issue such as codon optimization or directed evolution of the signal sequence α-MF [63]. Lately, new endogenous signal peptides could outperform signal sequence α-MF. For instance, in 2019 Duan and collaborators were able to identify four endogenous signal peptides (Dan4, Gas1, Msb2 and Fre2) in *P. pastoris*. All of them showed more than fourfold enhancement of total β-galactosidase activity over the signal sequence α-MF, with being Gas1 the one that showed the best results (230-fold increase). Moreover, Msb2 signal peptide had a better performance in the expression of β-galactosidase, yEGFP and cephalosporin C acylase; therefore, it could be considered as a more effective signal for heterologous protein secretion in *P. pastoris* [62].

**Golden Gate.** Prielhofer et al. developed GoldenPiCS a modular cloning system that facilitates the engineering of *P. pastoris* by generating episomal plasmids with up to 8 expression units. The main feature of this method is that researchers can easily

exchange genetic parts and quickly create and test new variants. The feasibility of this method was demonstrated with the optimization of a CRISPR-Cas9 system for *P. pastoris* using different combinations of humanized Cas9 and sgRNA on one single episomal plasmid [26].

In a more recent study, Cheng et al. used the Golden Gate cloning method to develop a versatile and easy way of assembling eukaryotic gene exons into both prokaryotic and eukaryotic plasmids in a one-step reaction [64]. Thus, this new approach enables researches to rapidly identify the optimal expression host for the production of specific proteins, overcoming some disadvantages of traditional methods to obtain intron-free eukaryotic genes (e.g. whole-gene synthesis or reverse transcription methods) which are time-consuming, expensive and complicated to operate [64].

**CRISPR-Cas9.** In *P. pastoris* exogenous cassettes with long homology arms are integrated ectopically and homologous recombination (HR) occurs only at variable frequencies of <0.1 to 30% [29]. However, high efficiency for gene insertion by HR can be achieved by deleting the protein Ku70 which is involved in nonhomologous end-joining (NHEJ) repair [65]. Weninger et al. used this strategy to develop integration cassettes of CRISPR-Cas9 marker-free with close to 100% efficiency [66]. Yang et al. developed a high-efficiency CRISPR-Cas system in *P. pastoris* synthetizing Cas9 (codon-optimized for *Homo sapiens)* and the sgRNA on different plasmids. They validated the editing efficiency in gene deletion of six genes, reaching or exceeding 75% of efficiency for each target gene. This system eliminates the episomal sgRNA plasmid through sub-culture to allow editing of another gene with a consistent single gene editing efficiency [65]. However, it was demonstrated that this system performed poorly when editing multiple genes. Finally, even though huge progress has been made to improve CRISPR-Cas9 efficiency in *P. pastoris*, it is still lower than other yeasts such as *S. cerevisiae* and *Kluyveromyces lactis* [65].

### **4.2** *Kluyveromyces marxianus*

*K. marxianus* is phylogenetically related to *S. cerevisiae* and more closely related to *K. lactis*. It is a hemiascomycetous yeast that can exist as stable haploid or diploid cells and is able to spontaneously switch its mating type (homothallic) [67, 68]. *K. marxianus* strains have been isolated from a great variety of habitats including dairy products, soil, sugarcane bagasse, insects and fruits. Therefore, this yeast presents a high metabolic diversity and a significant degree of intraspecific polymorphism [68–71], in point of fact, several industrially relevant compounds have been found to be naturally produced by *K. marxianus* including pectinase, aroma compounds, inulinase, lipase and lactase [69, 72]. Furthermore, some strains have been described to exhibit multi-stress resistance [68].

*K. marxianus* is classified as facultative fermentative and Crabtree negative, although some reports consider this species as Crabtree positive due to strain variability [69, 73]. *K. marxianus* cannot naturally grow under strictly anaerobic conditions [69], but it can be genetically modified to grow under such conditions. However, growth rates are still lower than anaerobic growth in *S. cerevisiae* [74]. *K. marxianus* is capable of using non-conventional sugars such as xylose, arabinose and inulin as carbon sources [40]. In addition, this yeast has the ability to use lactose which cannot be accomplished by *S. cerevisiae* and can grow at higher temperatures with a wider range of substrates than *K. lactis* [67, 69].

All these features make *K. marxianus* a promising biofactory, having a wide range of applications such as host for the production of heterologous proteins;

alternative to baker's yeast; bioremediation of textile dyes, cheese whey and copper; biomass for animal feeding; probiotics and high-temperature bioethanol production [67, 69, 72, 73, 75]. For instance, Nonklang et al. found remarkable differences in high-temperature ethanol production between *S. cerevisiae* and *K. marxianus* as *K. marxianus* DMKU3-1042 was the fastest to convert glucose to ethanol at 45°C whereas none of the *S. cerevisiae* strains were able to grow at this temperature [67].

In the last years, some progress in synthetic biology has been accomplished in this species; however, it is still limited compared to other NCYs such as *P. pastoris* since *K. marxianus* still lacks efficient genetic tools, there are also limited auxotrophic markers and very few constitutive and inducible promotes have been described [21]. Despite that, some examples and applications are discussed below.

**Codon Optimization.** This strategy is currently used to improve the expression of recombinant proteins in *K. marxianus*, especially vaccines. For instance, codon optimization has been used for the heterologous expression of the porcine circovirus type 2 (PCV2) Cap protein in *K. marxianus* as an alternative to produce PCV2 viruslike particle vaccines to treat porcine circovirus disease and help reduce economical losses in the swine industry. Duan et al. reported in their experiment higher yields compared with *E. coli* and *P. pastoris* as host vectors [76]. Other examples where codon optimization has been applied to *K. marxianus* include heterologous expression of single-chain antibodies, overproduction of inulinase, expression of the dengue virus type 1 nonstructural protein 1 and porcine parvovirus-like particles [77–80].

**Promoters.** In the last years, several promoters have been identified for this species which can be induced by heat, xylose, lactose or inulin [22]. In addition, several strong endogenous promoters of genes such as purine-cytosine permease, inulinase, enolase and glyceraldehyde 3-phosphate have been characterized [21, 69]. More recently, Kumar et al. identified two new strong promoters (PIMTCP1 and PIMTCP2) which are more efficient at different temperatures and carbon sources than previously known promoters in this species [21].

Interestingly, the relative strength of promoters can change depending on the carbon source provided [81]. For instance, Kumar et al. showed that the *K. marxianus* inulinase promoter has relatively higher activity in the presence of xylose than dextrose [21]. In addition to finding new promoters, already described promoters have been also engineered to improve their features. For example, Zhou et al. improved the expression of lignocellulolytic enzymes in *K. marxianus* by a mutation inside the inulinase promoter and a deletion of an A-T-rich region inside the 5'UTR [82].

Notably, if thermotolerance of this yeast is to be exploited when expressing heterologous proteins, it is not enough to only focus on utilizing thermostable proteins but also identifying thermotolerant promoters because promoter activity tends to decrease with elevated temperature as it was demonstrated by Yang et al. [81]. Despite the fact that lower promoter activity is observed when increasing the temperature, *K. marxianus* promoters have been found to be stronger than their corresponding promoters from *S. cerevisiae* at such temperatures. In fact, the *K. marxianus* constitutive promoter PPGK has been shown to retain relatively strong activity with an increase in temperature [81].

**Terminators.** Only a limited number of terminators have been examined in *K. marxianus* including terminators from *S. cerevisiae* such as TCYC1, TPGK1 and TADH1 [21, 22]. Additionally, new recently described *K. marxianus* terminators have widened the range of regulation of protein expression in this species. For instance, in a recent study researchers found an increase in EGFP expression (fourfold increase

of mRNA level) in *K. marxianus* when using the endogenous terminators TIMTT1 or TIMTT2 instead of *S. cerevisiae* TCYC1 [21].

**Signal Peptides.** Research about signal peptides in this species is still limited since only a few signal sequences have been characterized and employed in heterologous protein expression on *K. marxianus* including signal sequences from *K. marxianus* Inu1, *P. pastoris* Pho1, *S. cerevisiae* α-MF and *K. marxianus* α-MF [21]**.** Moreover, some experiments of signal sequence engineering conducted to improve its activity have been reported, for instance, Yarimizu et al. developed a synthetic signal sequence in the yeast *K. marxianus* by redesigning the hydrophobic core of *Gaussia princeps* secretory luciferase signal sequence. The hydrophobic sequence was replaced by a repeat of 16 methionine residues, resulting in 20-fold higher activity than that from the wild type [83].

**CRISPR-Cas9.** Cernak et al. first used a CRISPR-Cas9 system to inactivate genes responsible for spontaneous mating-type switching (common phenomenon in *K. marxianus*), enabling the production of stable heterothallic haploid strains which can mate. As a result, they combined three complex traits found in different strains (ability to take up exogenous DNA, thermotolerance capacity and higher lipid production) into single *K. marxianus* clones [71].

Li et al. developed a one-step multigene integration system based on CRISPR-Cas9, which is capable of integrating up to three cassettes in a single, targeted genomic locus in *K. marxianus.* It consists of the CRISPR plasmid (expression of the sgRNA and Cas9) and the homology donor plasmid (700 bp up- and down-stream homology to the targeted site). This system has been proven to have an efficiency comparable to single-gene integration and it can be performed within 4 days from transformation to confirmation [84].

In 2022 Bever et al. developed a highly efficient CRISPR-Cas9 system in *K. marxianus* that allows editing of multiple genes which can be used in both NHEJ-functional and -deficient strains showing nearly 100% efficiency of gene disruption in those two strains, whereas 100% efficiency of donor integration was observed only in NHEJdeficient strains*.* In addition, this system achieved a dual integration efficiency of 25.5% in an NHEJ-deficient strain [85].

### **4.3** *Kluyveromyces lactis*

*Kluyveromyces lactis* is an NCY known for its capacity to assimilate lactose and convert it into lactic acid. *K. lactis* is a respiratory Crabtree-negative yeast highly used in industries due to its ability to secrete the protein β-galactosidase, used for making lactose-free products [23]. Moreover, this yeast is also capable to produce methionol, which is a flavor-active compound important in the overall aroma of soy sauce and cheese [7].

Some advantages of working with *K. lactis* yeasts are the capacity of producing heterologous proteins in simple growth medium, complete knowledge of their genome and more importantly, they can be easily genetically manipulated [75]. Due to its similarity in biosynthetic capacities to *S. cerevisiae*, *K. lactis* toolkits for heterologous gene expression are mostly the same. However, *K. lactis* presents many attributes that make it more suitable for protein expression and extracellular secretion than *S. cerevisiae* [13]. In fact, *K. lactis* uses an inducible promoter PLAC4 which is commercially available due to its capacity to secrete recombinant proteins in the culture fluid under lactose presence, a very useful feature for protein purification [13].

**Promoters.** In general, the promoters used for heterologous protein production strategies in *K. lactis* are the same as *S. cerevisiae*, PGAL1 or PPGK, which, due to their

### *Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

high level of transferability, have shown the potential of promoter engineering in *S. cerevisiae* to be applied in the *K. lactis* expression system [6]. However, other engineering strategies involving *K. lactis* promoters have also been developed. For example, Sakhtah et al. have recently developed a novel auto-inducible promoter system in *K. lactis*. For this, portions of two promoters, the constitutive PGAP1 and the carbon source-sensitive PICL1, were combined to form a hybrid promoter called P350 [86]. This novel promoter is induced by the depletion of glucose or glycerol in the medium, making it auto-inducible as the carbon sources are consumed by the growing cells. The development of this hybrid promoter promises to be useful for the implementation of one-step protein expression methods for small- and large-scale bioprocesses [86]. Moreover, another hybrid promoter approach used in K*. lactis* involves the combination of core promoter elements of *Trichoderma reesei* PCBH1 and *K. lactis* PLAC4, which showed an increase in protein production in this yeast [13].

**CRISPR-Cas9.** For the implementation of CRISPR-Cas9 in *K. lactis*, Horwitz et al. adapted an *S. cerevisiae* system by exchanging the 2 μ element with the *K. lactis* specific pKD1 vector-stabilizing element and the constitutive promoter PFBA1 at a *GAL80* site [87–89]. Moreover, sgRNA expression was driven by the typical PSNR52 pol III promoter and a T*SUP*4 terminator, and the deletion of the *KU80* gene was performed to reduce NHEJ [6, 29, 87–89]. The implementation of this system resulted in the successful integration of three donor six-gene-DNA parts into three separate loci (*DIT1, ADH1 and NDT80*) with a triple integration efficiency of 2.1% [6, 29, 87, 88]. Despite this low efficiency, the speed and ability to screen strains reduced the design-builttest cycle for this non-conventional yeast, and further improvements in targeting this efficiency could enhance genome editing for wild-type or industrial strains [6].

On the other hand, CRISPR-Cas9 genome editing was used by Burghardt et al. in order to increase the enzymatic production of the prebiotic fructo-oligosaccharides (FOS) in *K. lactis*. For this, the fructosyltransferase gene (*FFT)*, needed for forward reactions, from *Aspergillus terreus* NIH2624 was integrated with a *K. lactis* GG799 production host. Furthermore, a CRISPR-Cas9 system was used to delete a native invertase gene, involved in reverse reactions. The results showed an increase in transferase activity by 66.9% when grown in a fed-batch process [90].

### **4.4** *Yarrowia lipolytica*

*Yarrowia lipolytica* belongs to the *Ascomycota*, *Dipodascaceae* family. It is naturally found in lipid and protein-rich substrates such as soil, sewage and oil-polluted environments due to their capacity to hydrolyse lipids, assimilate hydrocarbons and fatty acids and secrete extracellular proteases [91]. *Y. lipolytica* is a Crabtree-negative haploid, heterothallic yeast with mating types Mat A and Mat B, and low mating frequency in nature [92]. In a laboratory setting, cells appear spherical, ellipsoidal or elongated and arranged singly, in pairs or clustered in groups. Furthermore, colonies present a creamy texture and a convoluted pale white matte surface [91]. About the carbon sources, *Y. lipolytica* is capable to assimilate hydrophobic substrates like alkanes, alkenes, fatty acids, fatty acid methyl esters, triglycerides and hydrophilic substrates like glucose, fructose, some alcohols, many polyols and many organic acids [91]. Some important characteristics of this yeast are its efficient secretion pathway and lipid storage capacity, two qualities that have made it a research model for protein secretion and lipid metabolism [92]. Moreover, due to its lipogenic metabolism, *Y. lipolytica* has been studied for the biosynthesis of acetyl-CoA-derived molecules such as terpenes [93]. In the last years, because of its production capacity of industrial

interest compounds and the ability to grow at high cell densities, different synthetic biology tools have been developed and applied in *Y. lipolytica* [94]. As matter of fact, Wong and colleagues designed a collection of BioBricks for *Y. lipolytica* called YaliBricks, which contains compatible restriction enzyme sites that allows modular genetic engineering [95].

**Promoters.** In *Yarrowia lipolytica*, two important promoters have been isolated and characterized; the promoter from the *XRP2* gene, which codes for an alkaline extracellular protease and the constitutive promoter from the *TEF* gene, which codes for translational elongation factor-1 [96]. Furthermore, recent studies are focusing on the development of hybrid promoters that could increase the strength of the available ones. Early approaches to promoter hybridization led to the characterization of upstream activating sequences (UASs), native to *XPR2*, which resulted in an increase on promoter activity when hybridized in several tandem repeats [97]. Madzak et al. engineered four hybrid promoters (named hp1d, hp2d, hp3d and hp4d) containing up to four copies of one of its upstream activation sequences (UAS1XPR2) fused upstream from a PLEU2 promoter [98]. The resulting promoters showed an increase in their strength depending on the number of tandem UAS1XPR2 elements, with hp4d being the strongest hybrid promoter and therefore used widely for heterologous gene expression in *Y. lipolytica* [94, 99]. On the other hand, Schwarts et al. constructed a synthetic hybrid promoter, using *GAL1* UAS from *S. cerevisiae* and the *TEF* core promoter from *Y. lipolytica*, that achieved a slightly higher expression than P*UAS1B8*- *TEF*, hybrid promoter that has been widely used [93]. Moreover, other native promoters like PTDH1, PGPM1, PEXP1, PFBAINm, PGPAT, PGPD and PYAT have been characterized and used in expression of heterologous genes with promising results [94].

**Terminators.** The most commonly used terminators for expression of heterologous genes in *Y. lipolytica* are derived from the native *XPR2* and *LIP2* genes [99]. Moreover, some *S. cerevisiae* terminators have shown a high degree of transferability in *Y. lipolytica* [100]. Indeed, synthetic terminators designed for *S. cerevisiae* have been used in *Y. lipolytica* with an increase of 60% in expression level over some wild-type terminators [56]. Additionally, these synthetic terminators are commonly smaller than the natural ones, conferring them an advantage for transcription units (TU) and vector design since they show low risk of undesired HR between TU or with the genome, contributing high stability to genetically modified strains [50]. However, despite these advances, the number of studies of terminators in *Y. lipolytica*, in comparison to promoters, is still scarce [96].

**Golden Gate.** Larroude and collaborators have developed a modular toolkit based on the Golden Gate strategy that allows assembly in one step of three transcript units together with integration into *Y. lipolytica* genome. This approach comes with a collection of six selective markers and sequences for random or specific integration, nine promoters of variable strength and five terminators [100]. In such manner, the heterologous production of β-carotene is possible with the expression of three genes involved in the carotenoid pathway after a single transformation [101], making *Y. lipolytica* a competitive biotechnological producer of β-carotene.

**CRISPR-Cas9.** The use of CRISPR-Cas9 in *Y. lipolytica* has been widely studied. In recent years, a system from *Streptococcus pyogenes* has been adapted, with a synthetic RNA polymerase III promoter and an optimized Cas9 to perform a marker-free gene disruption and integration in *Y. lipolytica*. In fact, five loci have been recently identified that could serve as hotspots for targeting marker-free gene integration [97]. This system resulted in a single-gene disruption and HR with a 90 and 70% of efficiency

*Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

respectively when Cas9 and the sgRNA were co-transformed using donor DNA [94]. In addition, systems like CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have been developed for controlling gene expression in *Y. lipolytica* [99]. Here, a deactivated Cas9 (dCas9) is fused to transcriptional repressors or activators, allowing binding to sgRNA-complementary DNA without cleavage that could result in DSBs [91]. An implementation of the CRISPRa system was performed by Schwarts et al. in order to upregulate *BGL1* and *BGL2* (β-glucosidase genes that are transcriptionally silent), and allow *Y. lipolytica* strains to use cellobiose as a carbon source. For this, a VPR activator was identified and fused to dCas9 to enable gene activation [93, 96].

Other CRISPR-Cas9 strategies have been developed, for example, a paired sgRNA, consisting of two vectors, each containing Cas9 gene and a sgRNA cassette, was used in order to target areas upstream and downstream the start and stop codon, respectively, and allow a complete gene knockout via gene excision with a 20% of efficiency [94].

**TALEN.** Used to direct DNA DSBs to occur at a specific target site, was applied in *Y. lipolytica* to produce structure-based mutagenesis of a fatty acid synthase (FAS) domain, and allow the synthesis of fatty acids with shorter chain lengths [99]. Moreover, site-directed mutagenesis improved in efficiency when homologous exogenous DNA was added to the targeted site, resulting in HR-mediated repair in 40% of clones [94].

### **5. Future perspectives**

To date, only about 1% of the yeast species found in nature have had their genomes fully characterized. Thus, it is not surprising, with the accessibility of new sequencing technologies, the complete genome analysis of many newly discovered yeast species with unique characteristics will be available. This will greatly expand the catalog of genetic parts allowing a more sensitive fine-tuning of desired economically relevant compounds and the discovery of new genes of interest. In addition, new yeast host vectors with desirable characteristics such as faster growth rates, stress-tolerance, efficient secretion systems and desired metabolic pathways will be engineered and domesticated to facilitate their use in industrial applications.

Moreover, in silico simulations will play a crucial role in the efficient design of new synthetic yeast biofactories since more accurate predictions will be made. Nevertheless, there are still limitations that have to be overcome such as the absence of gene regulatory information, lack of accurate metabolic models at genomic scale [102], or missing experimental design and testing of potential NCY biofactories. For instance, CRISPR-Cas9 still performs poorly regarding the adequate sgRNA production in NCYs. sgRNA expression is normally accomplished using RNA polymerase III (RNAP III) promoters (not well characterized in NCYs), implying more studies are needed for effective genetic engineering. Wagner and Alper suggest two approaches to overcome this issue: optimization of heterologous RNAP III promoters or the screening of native RNAP III promoters [6, 13] which are currently being tested in some NCYs such as *Y. lipolytica* [97].

Finally, a quite positive outcome of the use of NCYs as biofactories is the production of industrially relevant compounds in an economical manner. Synthetic biology helps to search and engineer strains capable of utilizing cheaper substrates, including "waste products" (e.g. whey and molasses), supporting a sustainable circular economy which in the future will certainly have a more relevant role.

### **6. Conclusions**

For several decades yeasts have proven to be of great importance for the development of modern society, contributing to industrial processes including food and pharmaceutics. In addition, the current application of synthetic biology techniques in these organisms has given them a greater potential to be used as substitutes for organisms commonly used in the industry, such as bacteria, given the benefits they present. The use of these techniques in unconventional yeasts such as *P. pastoris*, *K. marxianus*, *K. lactis* and *Y. lipolytica* has increased very rapidly in recent years. For example, the genome editing technique CRISPR-Cas9 has been developed in these four species to improve the production of compounds, such as the prebiotic fructooligosaccharides in the case of *K. lactis*. Other techniques, despite being recently applied, have shown promising results in improving the expression of genes and the production of compounds of interest. This is the case of the use of TALENs in *Y. lipolytica* and Golden Gate in *P. pastoris*.

On the other hand, the engineering of genetic parts has also been developed in these unconventional yeasts. Codon optimization in *P. pastoris* and *K. marxianus* has allowed the production of heterologous enzymes such as phytases, important in animal feed, and recombinant proteins such as PCV2 Cap protein, which can be used in vaccines against porcine circovirus disease. Modifications of promoters and terminators have also been investigated, with promoter studies being the most common, as shown by the recent literature existing on the four species described in this chapter. In sum, the advances shown here demonstrate the potential of non-conventional yeasts as alternatives to traditionally used organisms, or even for the discovery of new systems with potential industrial use, capable of improving the quality of people's life.

### **Author details**

Kevin Castillo-Mendieta1 , Jimmy Arias1 and Fernando Gonzales-Zubiate1,2\*

1 School of Biological Sciences and Engineering, Yachay Tech University, Urcuquí, Ecuador

2 MIND Research Group, Model Intelligent Networks Development, Urcuquí, Ecuador

\*Address all correspondence to: fgonzales@yachaytech.edu.ec

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Feldmann H. Yeast Molecular and Cell Biology. Weinheim, Germany: Wiley-Blackwell; 2012

[2] Nicholas P. Money. The Rise of Yeast: Oxford University Press; 2018

[3] Satyanarayana T, Kunze G. Yeast Diversity in Human Welfare. Singapore: Springer; 2017. DOI: 10.1007/978-981-10-2621-8

[4] Boekhout T, Amend AS, el Baidouri F, Gabaldón T, Geml J, Mittelbach M, et al. Trends in yeast diversity discovery. Fungal Diversity. 2021;**114**:491-537. DOI: 10.1007/S13225-021-00494-6

[5] de Deken RH. The Crabtree effect: A regulatory system in yeast. Journal of General Microbiology. 1966;**44**:149-156. DOI: 10.1099/00221287-44-2-149

[6] Wagner JM, Alper HS. Synthetic biology and molecular genetics in nonconventional yeasts: Current tools and future advances. Fungal Genetics and Biology. 2016;**89**:126-136. DOI: 10.1016/j. fgb.2015.12.001

[7] Forti L, Cramarossa MR, Filippucci S, Tasselli G, Turchetti B, Buzzini P. Nonconventional yeastpromoted biotransformation for the production of flavor compounds. Handbook of Food Bioengineering. 2018;**7**:165-187. DOI: 10.1016/ B978-0-12-811518-3.00006-5

[8] Nandy SK, Srivastava RK. A review on sustainable yeast biotechnological processes and applications. Microbiological Research. 2018;**207**:83- 90. DOI: 10.1016/j.micres.2017.11.013

[9] Parapouli M, Vasileiadis A, Afendra AS, Hatziloukas E. *Saccharomyces*  *cerevisiae* and its industrial applications. AIMS Microbiology. 2020;**6**:1-31. DOI: 10.3934/microbiol.2020001

[10] Unrean P, Khajeeram S. Modelbased optimization of *Scheffersomyces stipitis* and *Saccharomyces cerevisiae* co-culture for efficient lignocellulosic ethanol production. Bioresources and Bioprocessing. 2015;**2**:1-11. DOI: 10.1186/ S40643-015-0069-1

[11] Lignocellulose SK. A chewy problem. Nature. 2011;**474**:S12-S14. DOI: 10.1038/474s012a

[12] Geijer C, Ledesma-Amaro R, Tomas-Pejo E. Unraveling the potential of non-conventional yeasts in biotechnology. FEMS Yeast Research. 2022;**22**:1-6. DOI: 10.1093/femsyr/ foab071

[13] Madhavan A, Jose AA, Binod P, Sindhu R, Sukumaran RK, Pandey A, et al. Synthetic biology and metabolic engineering approaches and its impact on non-conventional yeast and biofuel production. Frontiers in Energy Research. 2017;**5**:1-12. DOI: 10.3389/ fenrg.2017.00008

[14] Prasongsuk S, Lotrakul P, Ali I, Bankeeree W, Punnapayak H. The current status of Aureobasidium pullulans in biotechnology. Folia Microbiologia (Praha). 2018;**63**:129-140. DOI: 10.1007/s12223-017-0561-4

[15] Liu Z, Zhang Y, Nielsen J. Synthetic biology of yeast. Biochemistry. 2019;**58**:1511-1520. DOI: 10.1021/acs. biochem.8b01236

[16] Bojar D, Scheller L, El HGC, Xie M, Fussenegger M. Caffeine-inducible gene switches controlling experimental

diabetes. Nature Communications. 2018;**9**:1-10. DOI: 10.1038/ s41467-018-04744-1

[17] Kurland CG. Codon bias and gene expression. FEBS Letters. 1991;**285**:165- 169. DOI: 10.1016/0014-5793(91)80797-7

[18] Quax TEF, Claassens NJ, Söll D, van der Oost J. Codon bias as a means to fine-tune gene expression. Molecular Cell. 2015;**59**:149-161. DOI: 10.1016/J. MOLCEL.2015.05.035

[19] Türkanoğlu Özçelik A, Yılmaz S, Inan M. *Pichia pastoris* promoters. Methods in Molecular Biology. 2019;**1923**:97-112. DOI: 10.1007 /978-1-4939-9024-5\_3

[20] Vogl T, Sturmberger L, Kickenweiz T, Wasmayer R, Schmid C, Hatzl AM, et al. A toolbox of diverse promoters related to methanol utilization: Functionally verified parts for heterologous pathway expression in *Pichia pastoris*. ACS Synthetic Biology. 2016;**5**:172-186. DOI: 10.1021/acssynbio.5b00199

[21] Kumar P, Sahoo DK, Sharma D. The identification of novel promoters and terminators for protein expression and metabolic engineering applications in *Kluyveromyces marxianus*. Metabolic Engineering Communications. 2021;**12**:1- 13. DOI: 10.1016/J.MEC.2020.E00160

[22] Rajkumar AS, Varela JA, Juergens H, Daran JMG, Morrissey JP. Biological parts for *Kluyveromyces marxianus* synthetic biology. Frontiers in Bioengineering and Biotechnology. 2019;**7**:97. DOI: 10.3389/ fbioe.2019.00097

[23] Gomes AMV, Carmo TS, Carvalho LS, Bahia FM, Parachin NS. Comparison of yeasts as hosts for recombinant protein production. Microorganisms. 2018;**6**:38. DOI: 10.3390/microorganisms6020038

[24] Obst U, Lu TK, Sieber V. A modular toolkit for generating *Pichia pastoris* secretion libraries. ACS Synthetic Biology. 2017;**6**:1016-1025. DOI: 10.1021/ acssynbio.6b00337

[25] Dou W, Zhu Q, Zhang M, Jia Z, Guan W. Screening and evaluation of the strong endogenous promoters in *Pichia pastoris*. Microbial Cell Factories. 2021;**20**:1-12. DOI: 10.1186/ S12934-021-01648-6

[26] Prielhofer R, Barrero JJ, Steuer S, Gassler T, Zahrl R, Baumann K, et al. GoldenPiCS: A golden gate-derived modular cloning system for applied synthetic biology in the yeast *Pichia pastoris*. BMC Systems Biology. 2017;**11**:1- 14. DOI: 10.1186/S12918-017-0492-3

[27] Wright DA, Li T, Yang B, Spalding MH. TALEN-mediated genome editing: Prospects and perspectives. Biochemical Journal. 2014;**462**:15-24. DOI: 10.1042/BJ20140295

[28] Li T, Wright DA, Spalding MH, Yang B. TALEN-based Genome Editing in Yeast. Cham, Switzerland: Springer 2015. pp. 289-307. DOI: 10.1007/978-3-319-10142-2\_27

[29] Raschmanová H, Weninger A, Glieder A, Kovar K, Vogl T. Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects. Biotechnology Advances. 2018;**36**:641-665. DOI: 10.1016/J. BIOTECHADV.2018.01.006

[30] Yip BH. Recent advances in CRISPR/Cas9 delivery strategies. Biomolecules. 2020;**10**:839. DOI: 10.3390/ BIOM10060839

[31] Sayers EW, Bolton EE, Brister JR, Canese K, Chan J, Comeau DC, et al. Database resources of the national center *Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

for biotechnology information. Nucleic Acids Research. 2022;**50**:D20-D26. DOI: 10.1093/NAR/GKAB1112

[32] Love KR, Shah KA, Whittaker CA, Wu J, Bartlett MC, Ma D, et al. Comparative genomics and transcriptomics of *Pichia pastoris*. BMC Genomics. 2016;**17**:1-17. DOI: 10.1186/ S12864-016-2876-Y

[33] Nurcholis M, Lertwattanasakul N, Rodrussamee N, Kosaka T, Murata M, Yamada M. Integration of comprehensive data and biotechnological tools for industrial applications of *Kluyveromyces marxianus*. Applied Microbiology and Biotechnology. 2020;**104**:475-488. DOI: 10.1007/S00253-019-10224-3

[34] Becerra M, González-Siso MI, Cerdán ME. A transcriptome analysis of *Kluyveromyces lactis* growing in cheese whey. International Dairy Journal. 2006;**16**:207-214. DOI: 10.1016/J. IDAIRYJ.2005.03.005

[35] Morin N, Cescut J, Beopoulos A, Lelandais G, le Berre V, Uribelarrea JL, et al. Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast yarrowia lipolytica. PLoS One. 2011;**6**:e27966. DOI: 10.1371/JOURNAL. PONE.0027966

[36] Pomraning KR, Wei S, Karagiosis SA, Kim YM, Dohnalkova AC, Arey BW, et al. Comprehensive metabolomic, lipidomic and microscopic profiling of yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis. PLoS One. 2015;**10**:1-17. DOI: 10.1371/JOURNAL. PONE.0123188

[37] Alvim MCT, Vital CE, Barros E, Vieira NM, da Silveira FA, Balbino TR, et al. Ethanol stress responses of *Kluyveromyces marxianus* CCT 7735 revealed by proteomic and metabolomic analyses. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology. 2019;**112**:827-845. DOI: 10.1007/ S10482-018-01214-Y

[38] Godinho CP, Palma M, Oliveira J, Mota MN, Antunes M, Teixeira MC, et al. The N.C.yeastract and communityyeastract databases to study gene and genomic transcription regulation in non-conventional yeasts. FEMS Yeast Research. 2021;**21**:1-10. DOI: 10.1093/FEMSYR/FOAB045

[39] Ye R, Huang M, Lu H, Qian J, Lin W, Chu J, et al. Comprehensive reconstruction and evaluation of *Pichia pastoris* genome-scale metabolic model that accounts for 1243 ORFs. Bioresour Bioprocess. 2017;**4**:1-12. DOI: 10.1186/ S40643-017-0152-X

[40] Marcišauskas S, Ji B, Nielsen J. Reconstruction and analysis of a *Kluyveromyces marxianus* genome-scale metabolic model. BMC Bioinformatics. 2019;**20**:1-9. DOI: 10.1186/ S12859-019-3134-5

[41] Dias O, Pereira R, Gombert AK, Ferreira EC, Rocha I. iOD907, the first genome-scale metabolic model for the milk yeast *Kluyveromyces lactis*. Biotechnology Journal. 2014;**9**:776-790. DOI: 10.1002/BIOT.201300242

[42] Xu Y, Holic R, Hua Q. Comparison and analysis of published genomescale metabolic models of yarrowia lipolytica. Biotechnology and Bioprocess Engineering. 2020;**25**:53-61. DOI: 10.1007/S12257-019-0208-1

[43] Peña DA, Gasser B, Zanghellini J, Steiger MG, Mattanovich D. Metabolic engineering of *Pichia pastoris*. Metabolic Engineering. 2018;**50**:2-15. DOI: 10.1016/J.YMBEN.2018.04.017

[44] Sreekrishna K, Kropp KE. *Pichia pastoris*. In: Nonconventional Yeasts in Biotechnology. 1996. pp. 203-253. DOI: 10.1007/978-3-642-79856-6\_6

[45] Kang Z, Huang H, Zhang Y, Du G, Chen J. Recent advances of molecular toolbox construction expand *Pichia pastoris* in synthetic biology applications. World Journal of Microbiology and Biotechnology. 2016;**33**:1-8. DOI: 10.1007/S11274-016-2185-2

[46] García-Ortega X, Cámara E, Ferrer P, Albiol J, Montesinos-Seguí JL, Valero F. Rational development of bioprocess engineering strategies for recombinant protein production in *Pichia pastoris* (Komagataella phaffii) using the methanol-free GAP promoter. Where do we stand? New Biotechnology. 2019;**53**:24-34. DOI: 10.1016/J. NBT.2019.06.002

[47] Xiong AS, Yao QH, Peng RH, Han PL, Cheng ZM, Li Y. High level expression of a recombinant acid phytase gene in *Pichia pastoris*. Journal of Applied Microbiology. 2005;**98**:418-428. DOI: 10.1111/J.1365-2672.2004.02476.X

[48] Xiong AS, Yao QH, Peng RH, Zhang Z, Xu F, Liu JG, et al. High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast *Pichia pastoris*. Applied Microbiology and Biotechnology. 2006;**72**:1039-1047. DOI: 10.1007/S00253-006-0384-8

[49] Hu H, Gao J, He J, Yu B, Zheng P, Huang Z, et al. Codon optimization significantly improves the expression level of a keratinase gene in *Pichia pastoris*. PLoS One. 2013;**8**:1-8. DOI: 10.1371/JOURNAL.PONE.0058393

[50] Wang Y, Jiang S, Jiang X, Sun X, Guan X, Han Y, et al. Cloning and codon optimization of a novel feline interferon

omega gene for production by *Pichia pastoris* and its antiviral efficacy in polyethylene glycol-modified form. Virulence. 2022;**13**:297-309. DOI: 10.1080/21505594.2022.2029330

[51] He M, Wu D, Wu J, Chen J. Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in *Pichia pastoris* and its application in inulooligosaccharide production. Journal of Industrial Microbiology & Biotechnology. 2014;**41**:105-114. DOI: 10.1007/S10295-013-1341-Z

[52] Wang JR, Li YY, Liu DN, Liu JS, Li P, Chen LZ, et al. Codon optimization significantly improves the expression level of α-Amylase gene from Bacillus licheniformis in *Pichia pastoris*. BioMed Research International. 2015;**2015**:1-9. DOI: 10.1155/2015/248680

[53] Ahn J, Jang MJ, Ang KS, Lee H, Choi ES, Lee DY. Codon optimization of Saccharomyces cerevisiae mating factor alpha prepro-leader to improve recombinant protein production in *Pichia pastoris*. Biotechnology Letters. 2016;**38**:2137-2143. DOI: 10.1007/ S10529-016-2203-3

[54] Hu H, Dai S, Wen A, Bai X. Efficient expression of xylanase by codon optimization and its effects on the growth performance and carcass characteristics of broiler. Animals. 2019;**9**:65. DOI: 10.3390/ANI9020065

[55] Che Z, Cao X, Chen G, Liang Z. An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (*Pichia pastoris*). BMC Biotechnology. 2020;**20**:1-13. DOI: 10.1186/S12896-020-00654-7

[56] Karaoğlan M, Erden-Karaoğlan F. Effect of codon optimization and

*Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

promoter choice on recombinant endo-polygalacturonase production in *Pichia pastoris*. Enzyme and Microbial Technology. 2020;**139**:1-9. DOI: 10.1016/J. ENZMICTEC.2020.109589

[57] Zhai Z, Nuylert A, Isobe K, Asano Y. Effects of codon optimization and glycosylation on the high-level production of hydroxynitrile lyase from Chamberlinius hualienensis in *Pichia pastoris*. Journal of Industrial Microbiology & Biotechnology. 2019;**46**:887-898. DOI: 10.1007/S10295-019-02162-W

[58] Vogl T, Fischer JE, Hyden P, Wasmayer R, Sturmberger L, Glieder A. Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of *Pichia pastoris*. AMB Express. 2020;**10**:1-9. DOI: 10.1186/S13568-020-00972-1

[59] Nong L, Zhang Y, Duan Y, Hu S, Lin Y, Liang S. Engineering the regulatory site of the catalase promoter for improved heterologous protein production in *Pichia pastoris*. Biotechnology Letters. 2020;**42**:2703-2709. DOI: 10.1007/ S10529-020-02979-X

[60] Wang J, Wang X, Shi L, Qi F, Zhang P, Zhang Y, et al. Methanol-independent protein expression by AOX1 promoter with trans-acting elements engineering and glucose-glycerol-shift induction in *Pichia pastoris*. Scientific Reports. 2017;**7**:1-12. DOI: 10.1038/srep41850

[61] Ito Y, Terai G, Ishigami M, Hashiba N, Nakamura Y, Bamba T, et al. Exchange of endogenous and heterogeneous yeast terminators in *Pichia pastoris* to tune mRNA stability and gene expression. Nucleic Acids Research. 2020;**48**:13000- 13012. DOI: 10.1093/NAR/GKAA1066

[62] Duan G, Ding L, Wei D, Zhou H, Chu J, Zhang S, et al. Screening endogenous signal peptides and protein folding factors to promote the secretory expression of heterologous proteins in *Pichia pastoris*. Journal of Biotechnology. 2019;**306**:193-202. DOI: 10.1016/J. JBIOTEC.2019.06.297

[63] Ito Y, Ishigami M, Hashiba N, Nakamura Y, Terai G, Hasunuma T, et al. Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in *Pichia pastoris*. Microbial Biotechnology. 2022;**15**:2364-2378. DOI: 10.1111/1751-7915.14061

[64] Cheng J, Wu M, Zhong R, Si D, Meng G, Zhang R, et al. Rapid golden gate assembly of exons from genomic DNA for protein expression in Escherichia coli and *Pichia pastoris*. BioTechniques. 2021;**71**:445-450. DOI: 10.2144/BTN-2021-0039

[65] Yang Y, Liu G, Chen X, Liu M, Zhan C, Liu X, et al. High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in *Pichia pastoris*. Enzyme and Microbial Technology. 2020;**138**:109556. DOI: 10.1016/J. ENZMICTEC.2020.109556

[66] Weninger A, Fischer JE, Raschmanová H, Kniely C, Vogl T, Glieder A. Expanding the CRISPR/Cas9 toolkit for *Pichia pastoris* with efficient donor integration and alternative resistance markers. Journal of Cellular Biochemistry. 2018;**119**:3183-3198. DOI: 10.1002/JCB.26474

[67] Nonklang S, Abdel-Banat BMA, Cha-aim K, Moonjai N, Hoshida H, Limtong S, et al. High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast *Kluyveromyces marxianus* DMKU3- 1042. Applied and Environmental Microbiology. 2008;**74**:7514-7521. DOI: 10.1128/AEM.01854-08

[68] Lane MM, Burke N, Karreman R, Wolfe KH, O'Byrne CP, Morrissey JP. Physiological and metabolic diversity in the yeast *Kluyveromyces marxianus*. Antonie Van Leeuwenhoek. 2011;**100**:507-519. DOI: 10.1007/ S10482-011-9606-X

[69] Fonseca GG, Heinzle E, Wittmann C, Gombert AK. The yeast *Kluyveromyces marxianus* and its biotechnological potential. Applied Microbiology and Biotechnology. 2008;**79**:339-354. DOI: 10.1007/S00253-008-1458-6

[70] Morrissey JP, Etschmann MMW, Schrader J, de Billerbeck GM. Cell factory applications of the yeast *Kluyveromyces marxianus* for the biotechnological production of natural flavour and fragrance molecules. Yeast. 2015;**32**:3-16. DOI: 10.1002/YEA.3054

[71] Cernak P, Estrela R, Poddar S, Skerker JM, Cheng YF, Carlson AK, et al. Engineering *Kluyveromyces marxianus* as a robust synthetic biology platform host. MBio. 2018;**9**:1-16. DOI: 10.1128/ MBIO.01410-18

[72] Karim A, Gerliani N, Aïder M. *Kluyveromyces marxianus*: An emerging yeast cell factory for applications in food and biotechnology. International Journal of Food Microbiology. 2020;**333**:108818. DOI: 10.1016/J. IJFOODMICRO.2020.108818

[73] Lane MM, Morrissey JP. *Kluyveromyces marxianus*: A yeast emerging from its sister's shadow. Fungal Biology Reviews. 2010;**24**:17-26. DOI: 10.1016/J.FBR.2010.01.001

[74] Dekker WJC, Ortiz-Merino RA, Kaljouw A, Battjes J, Wiering FW, Mooiman C, et al. Engineering the thermotolerant industrial yeast *Kluyveromyces marxianus* for anaerobic growth. Metabolic Engineering.

2021;**67**:347-364. DOI: 10.1016/J. YMBEN.2021.07.006

[75] Bilal M, Ji L, Xu Y, Xu S, Lin Y, Iqbal HMN, et al. Bioprospecting *Kluyveromyces marxianus* as a Robust Host for Industrial Biotechnology. Frontiers in Bioengineering and Biotechnology. 2022;**10**:562. DOI: 10.3389/ FBIOE.2022.851768

[76] Duan J, Yang D, Chen L, Yu Y, Zhou J, Lu H. Efficient production of porcine circovirus virus-like particles using the nonconventional yeast *Kluyveromyces marxianus*. Applied Microbiology and Biotechnology. 2018;**103**:833-842. DOI: 10.1007/S00253-018-9487-2

[77] Yang D, Chen L, Duan J, Yu Y, Zhou J, Lu H. Investigation of *Kluyveromyces marxianus* as a novel host for largescale production of porcine parvovirus virus-like particles. Microbial Cell Factories. 2021;**20**:1-13. DOI: 10.1186/ S12934-021-01514-5

[78] Bragança CRS, Colombo LT, Roberti AS, Alvim MCT, Cardoso SA, Reis KCP, et al. Construction of recombinant *Kluyveromyces marxianus* UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1). Applied Microbiology and Biotechnology. 2015;**99**:1191-1203. DOI: 10.1007/ S00253-014-5963-5

[79] Nambu-Nishida Y, Nishida K, Hasunuma T, Kondo A. Genetic and physiological basis for antibody production by *Kluyveromyces marxianus*. AMB Express. 2018;**8**:1-9. DOI: 10.1186/ S13568-018-0588-1

[80] Zhang Y, Li YF, Chi Z, Liu GL, Jiang H, Hu Z, et al. Inulinase hyperproduction by *Kluyveromyces marxianus* through codon optimization, selection of the promoter, and high-celldensity fermentation for efficient inulin *Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

hydrolysis. Annales de Microbiologie. 2019;**69**:647-657. DOI: 10.1007/ S13213-019-01457-8

[81] Yang C, Hu S, Zhu S, Wang D, Gao X, Hong J. Characterizing yeast promoters used in *Kluyveromyces marxianus*. World Journal of Microbiology and Biotechnology. 2015;**31**:1641-1646. DOI: 10.1007/S11274-015-1899-X

[82] Zhou J, Zhu P, Hu X, Lu H, Yu Y. Improved secretory expression of lignocellulolytic enzymes in *Kluyveromyces marxianus* by promoter and signal sequence engineering. Biotechnology for Biofuels. 2018;**11**:1-14. DOI: 10.1186/S13068-018-1232-7

[83] Yarimizu T, Nakamura M, Hoshida H, Akada R. Synthetic signal sequences that enable efficient secretory protein production in the yeast *Kluyveromyces marxianus*. Microbial Cell Factories. 2015;**14**:1-14. DOI: 10.1186/ S12934-015-0203-Y

[84] Li M, Lang X, Moran Cabrera M, de Keyser S, Sun X, da Silva N, et al. CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in *Kluyveromyces marxianus*. Biotechnology for Biofuels. 2021;**14**:1-15. DOI: 10.1186/S13068-020-01852-3

[85] Bever D, Wheeldon I, da Silva N. RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growthbiased metabolic engineering of *Kluyveromyces marxianus*. Metabolic Engineering Communications. 2022:1-10. DOI: 10.1016/J.MEC.2022.E00208

[86] Sakhtah H, Behler J, Ali-Reynolds A, Causey TB, Vainauskas S, Taron CH. A novel regulated hybrid promoter that permits autoinduction of heterologous protein expression in *Kluyveromyces lactis*. Applied and Environmental

Microbiology. 2019;**85**:1-12. DOI: 10.1128/AEM.00542-19

[87] Horwitz AA, Walter JM, Schubert MG, Kung SH, Hawkins K, Platt DM, et al. Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas. Cell Systems. 2015;**1**:88-96. DOI: 10.1016/j.cels.2015.02.001

[88] Cai P, Gao J, Zhou Y. CRISPRmediated genome editing in nonconventional yeasts for biotechnological applications. Microbial Cell Factories. 2019;**18**:1-12. DOI: 10.1186/ s12934-019-1112-2

[89] Bisschoff E. The Development of a Wide Range CRISPR-Cas9 Gene Editing System. Bloemfontein, South Africa: University of the Free State; 2019

[90] Burghardt JP, Fan R, Baas M, Eckhardt D, Gerlach D, Czermak P. Enhancing the heterologous fructosyltransferase activity of *Kluyveromyces lactis*: Developing a scaled-up process and abolishing invertase by CRISPR/Cas9 genome editing. Frontiers in Bioengineering and Biotechnology. 2020;**8**:1-15. DOI: 10.3389/fbioe.2020.607507

[91] Abdel-Mawgoud AM, Markham KA, Palmer CM, Liu N, Stephanopoulos G, Alper HS. Metabolic engineering in the host Yarrowia lipolytica. Metabolic Engineering. 2018;**50**:192-208. DOI: 10.1016/J.YMBEN.2018.07.016

[92] Madzak C. Yarrowia lipolytica strains and their biotechnological applications: How natural biodiversity and metabolic engineering could contribute to cell factories improvement. Journal of Fungi. 2021;**7**:1-67. DOI: 10.3390/jof7070548

[93] Schwartz C, Curtis N, Löbs AK, Wheeldon I. Multiplexed CRISPR

activation of cryptic sugar metabolism enables Yarrowia Lipolytica growth on cellobiose. Biotechnology Journal. 2018;**13**:1-7. DOI: 10.1002/biot.201700584

[94] Larroude M, Rossignol T, Nicaud JM, Ledesma-Amaro R. Synthetic biology tools for engineering Yarrowia lipolytica. Biotechnology Advances. 2018;**36**:2150-2164. DOI: 10.1016/j. biotechadv.2018.10.004

[95] Wong L, Engel J, Jin E, Holdridge B, Xu P. YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica. Metabolic Engineering Communications. 2017;**5**:68-77. DOI: 10.1016/J. METENO.2017.09.001

[96] Larroude M, Celinska E, Back A, Thomas S, Nicaud JM, Ledesma-Amaro R. A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β-carotene. Biotechnology and Bioengineering. 2018;**115**:464-472. DOI: 10.1002/bit.26473

[97] Markham KA, Alper HS. Synthetic biology expands the industrial potential of Yarrowia lipolytica. Trends in Biotechnology. 2018;**36**:1085-1095. DOI: 10.1016/j.tibtech.2018.05.004

[98] Madzak C, Tréton B, Blanchin-Roland S. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. Journal of Molecular Microbiology and Biotechnology. 2000;**2**:207-216

[99] Ma J, Gu Y, Marsafari M, Xu P. Synthetic biology, systems biology, and metabolic engineering of Yarrowia lipolytica toward a sustainable biorefinery platform. Journal of Industrial Microbiology &

Biotechnology. 2020;**47**:845-862. DOI: 10.1007/s10295-020-02290-8

[100] Larroude M, Park YK, Soudier P, Kubiak M, Nicaud JM, Rossignol T. A modular golden gate toolkit for Yarrowia lipolytica synthetic biology. Microbial Biotechnology. 2019;**12**:1249-1259. DOI: 10.1111/1751-7915.13427

[101] Larroude M, Nicaud JM, Rossignol T. Golden gate multigene assembly method for Yarrowia lipolytica. Methods in Molecular Biology. 2022;**2513**:205-220. DOI: 10.1007/978-1-0716-2399-2\_12

[102] Patra P, Das M, Kundu P, Ghosh A. Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts. Biotechnology Advances. 2021;**47**:1-29. DOI: 10.1016/j.biotechadv.2021.107695

Section 2
