**2. Ecology of oleaginous fungi**

Oleaginous microorganisms are able to accumulate lipids above the 20% of their biomass, on dry basis. Several species of yeasts and filamentous fungi are regarded as oleaginous, since they have the capability to synthetize and accumulate high amounts of TAG within their cells, up to 70% of the biomass weight. These lipids have similar composition and energy

Getting Lipids for Biodiesel Production from Oleaginous Fungi 73

ascomycetes (van der Walt, 1992). *Lipomyces* are true soil inhabitants and have a worldwide distribution. The oleaginous species *Lipomyces starkeyi* has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions, and can produce lipid on xylose, ethanol, and L-arabinose, or using a mixture of glucose and xylose (Zhao et al., 2008),

*Cryptococcus curvatus* is a yeast with industrial potential as single-cell oil because it can grow and accumulate lipid on a very broad range of substrates. It requires minimal nutrients for growth, accumulating up to 60% of its cellular dry weight (DW) as intracellular lipid (Meesters et al., 1996; Zhang et al., 2011). Yeasts of the *Cryptococcus* genus are widely distributed in nature and may be isolated from various substrates such as air, soil, bird excreta, water, animal surfaces and mucosae, leaves, flowers, and decomposing wood. Most species are considered as free-living (non-symbiotic) and only a few have medical importance being responsible for disease in man and animals (*C. neoformans* and *C. gattii*). *C. curvatus* is recognized as an opportunistic pathogen of animals, including humans (Findley

Species belonging to the genus *Rhodosporidium*, and to its asexual counterpart *Rhodotorula*, have been claimed as oleaginous yeasts. They belong to one of the three main lineages of the *Basidiomycota*, the *Pucciniomycotina*. *Rhodotorula* is a common environmental inhabitant. The synthesis of different commercially important natural carotenoids by yeast species belonging to the genus *Rhodotorula* has led to consider these microorganisms as a potential pigment sources. Within this genus, the mesophilic red yeast *Rhodotorula glutinis* is able to synthetize and store lipids also growing on glycerol, whereas the psychrophilic species *Rhodotorula glacialis,* that are not red yeasts, accumulates lipids in a range of temperature between -3 and 20°C (Amaretti et al., 2010). The red yeast *Rhodosporidium toruloides* is an oleaginous mesophilic species. *Rhodosporidium* are able to carry out a number of diverse biochemical reactions such as biodegradation of epoxides, biphenyls and oxiranes (Smit, 2004), biosynthesis of carotenoids (de Miguel et al., 1997) and other types of biotransformations, but a major biotechnological exploitation is associated to their ability to convert glycerol and lignocellulosic biowastes into lipids (Hu et al., 2009; Yu et al., 2011). Among the oily yeasts, two novel species of the anamorphic basidiomycetous genus *Trichosporon* have been recently identified (*T. cacaoliposimilis* and *T. oleaginosus*) (Gujjari et al., 2011), despite lipid accumulation has not yet explored in the perspective of biodiesel production. *Trichosporon* are basidiomycetous yeasts widely distributed in nature, consisting of soil- and water-associated species, predominantly found in environmental substrates, such as decomposing wood. They present distinct morphological characteristics of budding yeast cells and true mycelia that disarticulate to form arthroconidia. Some species are causative agents of diseases in man and cattle. They can occasionally belong to the gastrointestinal microbiota of humans as well as transiently colonize the skin and

Exploitation of oleaginous filamentous fungi for biodiesel production has a more recent history, which, with few exceptions, derives from studies focused to poly-unsaturated fatty acid production (PUFA), such as arachidonic acid and γ-linolenic acid. The most relevant example of this biotechnological application is represented by exploitation of *Mortierella alpina* to produce oils containing n-1, n-3, n-4, n-6, n-7, and n-9 PUFAs (Sakuradani et al., 2009). Among the major lipid producers there is *Mucor circinelloides,* a zygomycete fungus, which is emerging as opportunistic pathogen in immunocompromised patients (Li et al., 2011). *M. circinelloides* has been used for the first

as well as other wastes (Angerbauer et al., 2008).

et al., 2009).

respiratory tract.

value to plant and animal oils, but their production do not compete for food resources, in particular if it is based on inexpensive carbon sources, such as raw materials, by-products, and surplus. Furthermore, fungal SCO have a short process cycle, and their production is not subjected to seasonal and cyclical weather variations.

The study of oleaginous yeasts has a long history: their ability to accumulate lipids has been known from the 70s, but only in the last years the attention has been focused on exploitation of SCO for biodiesel production. The yeasts represent a part of the microbiota in all natural ecosystems, such as soils, freshwaters and marine waters, from the ocean surface to the deep sea. Widely distributed in the natural environment, they colonize also more extreme environments, such as low temperatures, low oxygen availabilities, and oceanic waters (Butinar et al., 2007). Approximately 1500 species of yeasts belonging to over 100 genera have been described so far (Satyanarayana & Kunze, 2010). Although the vast majority of yeasts are beneficial to human life, only a few are opportunistic human pathogens. As a whole, they play a pivoltal role in the food chain, and in the carbon, nitrogen and sulphur cycles. Among the huge number of species that have been described, only 30 are able to accumulate more than 25% of their dry weight as lipids (Beopoulos et al., 2009b).

Basidiomycetous yeasts strongly prevail among oleaginous yeasts, representing most of all the strains identified as lipid producers, even though some important oleaginous species have been identified among Ascomycota as well (e.g. *Yarrowia lipolytica*).

The most deeply investigated oleaginous yeasts belong to the genera *Yarrowia*, *Candida*, *Rhodotorula*, *Rhodosporodium*, *Cryptococcus*, and *Lypomyces* (Ageitos et al., 2011; Li et al., 2008; Rossi et al., 2009). *Yarrowia lipolytica*, previously referred to as *Candida lipolytica*, is a good candidate for single-cell oil production (Beopoulos et al., 2009a; Beopoulos et al., 2009b). *Yarrowia* are hemiascomycetous dimorphic fungi that belong to the order *Saccharomycetales*. They are able to degrade hydrophobic substrates such as n-paraffins and oils very efficiently and this physiological feature prompted the scientific community to explore several biotechnological applications (Bankar et al., 2009). The common habitats of these fungi are oil-polluted environments and foods such as cheese, yogurt, kefir, shoyu, meat, and poultry products. Despite *Y. Lipolytica* is distantly related to the conventional yeast *Saccharomyces cerevisiae,* the genome displays an expansion of protein families and genes involved in hydrophobic substrate (such as alkanes and lipids) utilization. Wild-type strains accumulate up to 38% of dry weight (DW) as lipids. Albeit the levels are lower than those of other oleaginous yeasts, it became a model organism because it can be subjected to genetic and metabolic engineering, having be developed a reliable and versatile system for disruption, cloning and expression of target genes.

Within the *Candida* genus, *Candida curvata* (Holdsworth & Ratledge, 1991) also referred as *Apiotrichum curvatum* and *Candida freyschussii* (Amaretti et al., 2011) synthetize and store significant amount of lipids. *Candida* comprises an extremely heterogeneous group of Ascomycota that can all grow with yeast morphology, classified in 150 heterogeneous species, among which only a minority have been implicated in human diseases, since approximately 65% of *Candida* species are unable to grow at 37°C, then they can not be successful pathogens or commensals of humans (Calderone, 2002). Therefore, most of the species can be exploited for biotechnological applications, despite of unwarranted negative public perceptions.

*Lipomyces* spp. present a great propensity to accumulate triacylglycerols. This genus belongs to the *Saccharomycetales* order and represents a unique branch in the evolution of the

value to plant and animal oils, but their production do not compete for food resources, in particular if it is based on inexpensive carbon sources, such as raw materials, by-products, and surplus. Furthermore, fungal SCO have a short process cycle, and their production is

The study of oleaginous yeasts has a long history: their ability to accumulate lipids has been known from the 70s, but only in the last years the attention has been focused on exploitation of SCO for biodiesel production. The yeasts represent a part of the microbiota in all natural ecosystems, such as soils, freshwaters and marine waters, from the ocean surface to the deep sea. Widely distributed in the natural environment, they colonize also more extreme environments, such as low temperatures, low oxygen availabilities, and oceanic waters (Butinar et al., 2007). Approximately 1500 species of yeasts belonging to over 100 genera have been described so far (Satyanarayana & Kunze, 2010). Although the vast majority of yeasts are beneficial to human life, only a few are opportunistic human pathogens. As a whole, they play a pivoltal role in the food chain, and in the carbon, nitrogen and sulphur cycles. Among the huge number of species that have been described, only 30 are able to accumulate more than 25% of their dry weight as lipids

Basidiomycetous yeasts strongly prevail among oleaginous yeasts, representing most of all the strains identified as lipid producers, even though some important oleaginous species

The most deeply investigated oleaginous yeasts belong to the genera *Yarrowia*, *Candida*, *Rhodotorula*, *Rhodosporodium*, *Cryptococcus*, and *Lypomyces* (Ageitos et al., 2011; Li et al., 2008; Rossi et al., 2009). *Yarrowia lipolytica*, previously referred to as *Candida lipolytica*, is a good candidate for single-cell oil production (Beopoulos et al., 2009a; Beopoulos et al., 2009b). *Yarrowia* are hemiascomycetous dimorphic fungi that belong to the order *Saccharomycetales*. They are able to degrade hydrophobic substrates such as n-paraffins and oils very efficiently and this physiological feature prompted the scientific community to explore several biotechnological applications (Bankar et al., 2009). The common habitats of these fungi are oil-polluted environments and foods such as cheese, yogurt, kefir, shoyu, meat, and poultry products. Despite *Y. Lipolytica* is distantly related to the conventional yeast *Saccharomyces cerevisiae,* the genome displays an expansion of protein families and genes involved in hydrophobic substrate (such as alkanes and lipids) utilization. Wild-type strains accumulate up to 38% of dry weight (DW) as lipids. Albeit the levels are lower than those of other oleaginous yeasts, it became a model organism because it can be subjected to genetic and metabolic engineering, having be developed a reliable and versatile system for disruption,

Within the *Candida* genus, *Candida curvata* (Holdsworth & Ratledge, 1991) also referred as *Apiotrichum curvatum* and *Candida freyschussii* (Amaretti et al., 2011) synthetize and store significant amount of lipids. *Candida* comprises an extremely heterogeneous group of Ascomycota that can all grow with yeast morphology, classified in 150 heterogeneous species, among which only a minority have been implicated in human diseases, since approximately 65% of *Candida* species are unable to grow at 37°C, then they can not be successful pathogens or commensals of humans (Calderone, 2002). Therefore, most of the species can be exploited for biotechnological applications, despite of unwarranted negative

*Lipomyces* spp. present a great propensity to accumulate triacylglycerols. This genus belongs to the *Saccharomycetales* order and represents a unique branch in the evolution of the

have been identified among Ascomycota as well (e.g. *Yarrowia lipolytica*).

not subjected to seasonal and cyclical weather variations.

(Beopoulos et al., 2009b).

cloning and expression of target genes.

public perceptions.

ascomycetes (van der Walt, 1992). *Lipomyces* are true soil inhabitants and have a worldwide distribution. The oleaginous species *Lipomyces starkeyi* has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions, and can produce lipid on xylose, ethanol, and L-arabinose, or using a mixture of glucose and xylose (Zhao et al., 2008), as well as other wastes (Angerbauer et al., 2008).

*Cryptococcus curvatus* is a yeast with industrial potential as single-cell oil because it can grow and accumulate lipid on a very broad range of substrates. It requires minimal nutrients for growth, accumulating up to 60% of its cellular dry weight (DW) as intracellular lipid (Meesters et al., 1996; Zhang et al., 2011). Yeasts of the *Cryptococcus* genus are widely distributed in nature and may be isolated from various substrates such as air, soil, bird excreta, water, animal surfaces and mucosae, leaves, flowers, and decomposing wood. Most species are considered as free-living (non-symbiotic) and only a few have medical importance being responsible for disease in man and animals (*C. neoformans* and *C. gattii*). *C. curvatus* is recognized as an opportunistic pathogen of animals, including humans (Findley et al., 2009).

Species belonging to the genus *Rhodosporidium*, and to its asexual counterpart *Rhodotorula*, have been claimed as oleaginous yeasts. They belong to one of the three main lineages of the *Basidiomycota*, the *Pucciniomycotina*. *Rhodotorula* is a common environmental inhabitant. The synthesis of different commercially important natural carotenoids by yeast species belonging to the genus *Rhodotorula* has led to consider these microorganisms as a potential pigment sources. Within this genus, the mesophilic red yeast *Rhodotorula glutinis* is able to synthetize and store lipids also growing on glycerol, whereas the psychrophilic species *Rhodotorula glacialis,* that are not red yeasts, accumulates lipids in a range of temperature between -3 and 20°C (Amaretti et al., 2010). The red yeast *Rhodosporidium toruloides* is an oleaginous mesophilic species. *Rhodosporidium* are able to carry out a number of diverse biochemical reactions such as biodegradation of epoxides, biphenyls and oxiranes (Smit, 2004), biosynthesis of carotenoids (de Miguel et al., 1997) and other types of biotransformations, but a major biotechnological exploitation is associated to their ability to convert glycerol and lignocellulosic biowastes into lipids (Hu et al., 2009; Yu et al., 2011). Among the oily yeasts, two novel species of the anamorphic basidiomycetous genus *Trichosporon* have been recently identified (*T. cacaoliposimilis* and *T. oleaginosus*) (Gujjari et al., 2011), despite lipid accumulation has not yet explored in the perspective of biodiesel production. *Trichosporon* are basidiomycetous yeasts widely distributed in nature, consisting of soil- and water-associated species, predominantly found in environmental substrates, such as decomposing wood. They present distinct morphological characteristics of budding yeast cells and true mycelia that disarticulate to form arthroconidia. Some species are causative agents of diseases in man and cattle. They can occasionally belong to the gastrointestinal microbiota of humans as well as transiently colonize the skin and respiratory tract.

Exploitation of oleaginous filamentous fungi for biodiesel production has a more recent history, which, with few exceptions, derives from studies focused to poly-unsaturated fatty acid production (PUFA), such as arachidonic acid and γ-linolenic acid. The most relevant example of this biotechnological application is represented by exploitation of *Mortierella alpina* to produce oils containing n-1, n-3, n-4, n-6, n-7, and n-9 PUFAs (Sakuradani et al., 2009). Among the major lipid producers there is *Mucor circinelloides,* a zygomycete fungus, which is emerging as opportunistic pathogen in immunocompromised patients (Li et al., 2011). *M. circinelloides* has been used for the first

Getting Lipids for Biodiesel Production from Oleaginous Fungi 75

Fig. 1. Reactions occurring sequentially in fatty acid synthetase: condensation of acyl-ACP and malonyl-ACP mediated by KS, NADPH-dependent reduction of the keto group to a hydroxyl group by means of KR, dehydration to create a double bond with DH and

The biosynthesis of FA requires the constant supply of acetyl-CoA as initial biosynthetic unit and of malonyl-CoA as the elongation unit, supplying two carbons at each step. Nonoleaginous yeasts receive acetyl-CoA mostly from glycolysis. In oleaginous yeasts, acetyl-CoA is mostly provided by the cleavage in the cytosol of citrate, which accumulated as a consequence of nitrogen limitation (Ratledge, 2002) (Fig. 2). In fact, lipid accumulation by

In oleaginous yeasts, nitrogen limitation activates AMP-deaminase (Ratledge & Wynn, 2002), which supply ammonium to the nitrogen-starved cell. As a consequence, mitochondrial AMP concentration decreases, causing isocitrate dehydrogenase activity to drop. The TCA cycle is then blocked at the level of isocitrate, which accumulates and equilibrates with citrate through aconitase. Excess of citrate from TCA cycle is exported out of the mitochondrion via the malate/citrate antiport. Cytosolic ATP-citrate lyase (ACL)

ACL represents one of the key enzymes that contribute to the oleaginous trait of yeasts, whereas its activity is negligible in non-oleaginous species. ACL is composed of two

Malonyl-CoA is produced from acetyl-CoA by acetyl-CoA carboxylase (ACC) that

ACC is also a key enzyme in *de novo* FA synthesis, since *ACC1* mutants became FA auxotrophs or maintain low levels of ACC activity (Tehlivets et al., 2007). ACC1 undergoes allosteric activation by citrate. Furthermore the transcription of *FAS1*, *FAS2*, and *ACC1* is

+ATP malonyl-CoA + ADP + Pi

subunits, encoded by *ACL1* and *ACL2* and is negatively regulated by exogenous FA.

reduction of the double bond by means of EAR. R = H, CH3(CH2)2n; nmax=7.

oleaginous fungi does not occur under balanced nutrient conditions.

cleaves citrate to give oxaloacetate and acetyl-CoA (Fig. 2).

Acetyl-CoA + HCO3-

coordinately regulated, being negatively regulated by FA.

condensate an acetyl-CoA unit with bicarbonate:

commercial production of microbial lipids (Ratledge, 2004). Lipid accumulation in *M. circinelloides* has been extensively studied (Wynn et al., 2001), and its TAG have been proposed as feedstock for producing biodiesel by direct transformation of its lipids (Vicente et al., 2009). *M. circinelloides* represents an outstanding model within the *Zygomycota* phylum, based on the availability of an efficient transformation procedure (Gutierrez et al., 2011) and on the whole sequence of genome (http://genome.jgipsf.org/Mucci2/Mucci2.home.html). Also the phylogenetically related *Umbelopsis isabellina* has emerging as a promising species to convert biomass residues to biodiesel precursors (Meeuwse et al., 2011a). To the best of our knowledge, limited are the attempts to get lipids with *Aspergillus oryzae* that, conversely, is extensively studied as lipase producer to carry out transesterification of TAG (Adachi et al., 2011).
