**1. Introduction**

Currently, carotenoids are valuable molecules due to their use in different industries such as chemical, pharmaceutical, poultry, food and cosmetics. These pigments have coloring and antioxidant properties that have attracted the attention of researchers and industries as they have useful applications on several fields [1]. Carotenoids are naturally occurring lipid-soluble

© 2016 The Author(s). Licensee InTech. 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. © 2018 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.

pigments, the majority being C40 terpenoids, which act as membrane-protective antioxidants scavenging O<sup>2</sup> and peroxyl radicals; their antioxidant ability is apparently attributed to their structure, which in some cases (for example: astaxanthin) contain two terminal rings joined by a double conjugated chain or polyene system. Carotenoids are classified in two major groups: i) carotenes, composed only by carbon and hydrogen, and ii) xanthophylls, which are oxygenated derivatives [2]. Approximately 600 different carotenoid structures have been described to date that can be found in most life forms and have various functions ranging from their original role as photosynthetic pigments to antioxidants, precursors of vitamin A or pigments involved in the visual attraction of pollinating animals [2].

initially metabolized by the glycolytic pathway, followed by alcoholic fermentation, even in the presence of oxygen. On the other hand, non-fermentable carbon sources, such as ethanol and succinate, are metabolized through acetyl-CoA oxidation entering directly to the citric acid cycle. In general, non-fermentable carbon sources as ethanol, enhance the synthesis of carotenoid pigments in *X. dendrorhous* [1, 16, 17] and a relation between carotenoid production and ergosterol and fatty acid synthesis have been reported, since these pathways use the

Microbiological Synthesis of Carotenoids: Pathways and Regulation

http://dx.doi.org/10.5772/intechopen.78343

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This chapter is focused on the microbiological synthesis of astaxanthin, taking as example the microorganisms *X. dendrorhous* and *H. pluvialis* since both are considered among the most promising for biotechnological production of this carotenoid. Also, the potential regulatory mechanisms that influence carotenogenesis is described, particularly, the influence of the carbon source and the regulation of carotenogenesis by catabolic repression in the yeast *X.* 

Astaxanthin (3,3′-dihydroxy-β, β′-carotene-4,4′-dione) belongs to the xanthophyll group and its structure consists of two terminal rings joined by a polyene chain (**Figure 1**). In addition to the existence of cis or trans geometric isomers, astaxanthin presents 3 configurational isomers: two enantiomers (*3R, 3′R* and *3S, 3′S*) and one meso form (*3R, 3′S*), due to the existence of two asymmetric carbons located at the 3, 3′ positions of the β-ionone ring with hydroxyl group (–OH) on both ends of the molecule [21, 22]. The stereoisomers (3*S*, *3′S*) and (3*R,* 3′*R*) are the most abundant in nature*. H. pluvialis* biosynthesizes the (3*S*, 3′*S*)-isomer whereas the yeast *X. dendrorhous* produces the (3*R*, 3′*R*)-isomer, while synthetic astaxanthin comprises isomers (3*S*,

Being a xanthophyll, astaxanthin possesses chemical properties and physiological characteristics of these compounds: it is highly lipophilic and shows an intense red color based on the light absorbed by its polyene system [22]. Due to its 3-hydroxyl and 4-keto functional groups in the terminal rings, astaxanthin has a higher polarity than other carotenoids and an antioxidant activity greater than that of β-carotene, lutein, zeaxanthin and canthaxanthin [5, 23, 24]. Due to its high antioxidant activity, there is an increasing number of studies referring to the astaxanthin beneficial properties for human health including several properties such as anti-inflammatory, anti-diabetic, antibacterial, immunostimulant, photoprotective, neuroprotective, anticancer and benefits to cardiovascular health, among others [2, 5, 22]. From an economical point of view, astaxanthin is the third most important carotenoid in the carotenoid global market after β-carotene and lutein. Astaxanthin market reached the 29% of total carotenoid sales with a global market size of \$225 million, estimating to increase to \$253 million by 2018, approximately [1]. Currently, the vast majority of the commercial offer corresponds to synthetic astaxanthin. However, the use of chemical compounds as food additives has been strictly regulated, favoring food free of them from both, the consumers and the

same substrates that derive from the mevalonic acid (MVA) pathway [18–20].

*dendrorhous*.

**2. Importance of astaxanthin**

3′*S*), (3*R*, 3′*S*) and (3*R*, 3′*R*) [22].

Commercial carotenoids are mainly obtained by extraction from plants and by chemical synthesis. However, some problems regarding seasonal and geographic variability that cannot be controlled arise in the case of production and marketing of several colorants of plant origin. On the other hand, the chemical synthesis may generate hazardous waste that can affect the environment. Unlike these traditional methods, the microbial production of carotenoids turns into a great opportunity of safe use. Microbial production has the advantage to use low-cost substrates, resulting in lower production costs. This explains the increasing interest in the production of microbial carotenoids as substitutes for synthetic carotenoids used as colorants in food and feed industries [1]. Among carotenoids, the xanthophyll astaxanthin stands out for its antioxidant and nutraceutical properties that are beneficial to human health, and for its use in the aquaculture industry as nutritional supplement of salmonid fish [3]. Many studies have pointed to search for a natural source of astaxanthin as an alternative production method that guarantees the beneficial properties of the mentioned compound. In nature, few astaxanthin-producing organisms are known, being the microalgae *Haematococcus pluvialis* and the yeast *Xanthophyllomyces dendrorhous* the most promising microbiological systems for the biotechnological production of this carotenoid. In this context, *X. dendrorhous* produces unmodified astaxanthin as the main carotenoid, does not require light for its growth and pigmentation and can use various carbon sources having a relatively rapid growth [2, 4]. In contrast, the production of astaxanthin by *H. pluvialis* requires specific conditions during its cultivation, because it changes its structure during the growth cycle. This means that the physical properties and nutrient requirements of the alga change during the culture process, altering the optimal conditions for growth and carotenoid production [1].

Despite the potential of *X. dendrorhous*, the specific production of astaxanthin by wild-type strains of this yeast is too low to be a commercially competitive source [5]. Due to the above, many researchers have tried to improve the production of astaxanthin using several methods as the optimization of culture conditions, classical random mutagenesis methods, genetic and metabolic engineering strategies [1, 4, 6–15]. These studies have led to an extensive knowledge about the biology of this yeast and the carotenoid synthesis pathway. However, in recent years, interest has been focused on elucidating the regulatory mechanisms operating on the production of carotenoids in this yeast. The carbon source is the most studied parameter that influences carotenogenesis in *X. dendrorhous*. Glucose and other fermentable sugars are initially metabolized by the glycolytic pathway, followed by alcoholic fermentation, even in the presence of oxygen. On the other hand, non-fermentable carbon sources, such as ethanol and succinate, are metabolized through acetyl-CoA oxidation entering directly to the citric acid cycle. In general, non-fermentable carbon sources as ethanol, enhance the synthesis of carotenoid pigments in *X. dendrorhous* [1, 16, 17] and a relation between carotenoid production and ergosterol and fatty acid synthesis have been reported, since these pathways use the same substrates that derive from the mevalonic acid (MVA) pathway [18–20].

This chapter is focused on the microbiological synthesis of astaxanthin, taking as example the microorganisms *X. dendrorhous* and *H. pluvialis* since both are considered among the most promising for biotechnological production of this carotenoid. Also, the potential regulatory mechanisms that influence carotenogenesis is described, particularly, the influence of the carbon source and the regulation of carotenogenesis by catabolic repression in the yeast *X. dendrorhous*.
