**2. Importance of astaxanthin**

pigments, the majority being C40 terpenoids, which act as membrane-protective antioxidants

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

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

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

or pigments involved in the visual attraction of pollinating animals [2].

and peroxyl radicals; their antioxidant ability is apparently attributed to their

scavenging O<sup>2</sup>

64 Progress in Carotenoid Research

carotenoid production [1].

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

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

**3. Microbiological synthesis of astaxanthin**

**3.1. Astaxanthin biosynthesis in** *X. dendrorhous*

[1, 20].

pene [1].

pathways in eukaryotic microorganisms involve three general steps [1]:

In general, the microbiological synthesis of carotenoids has conservative steps among the different species of carotenogenic microorganisms. The most conserved carotenoid biosynthesis

Microbiological Synthesis of Carotenoids: Pathways and Regulation

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

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**1.** The synthesis begins with the conversion of acetyl-CoA to 3-hydroxy-3-methyl glutaryl-CoA (HMG-CoA), catalyzed by HMG-CoA synthase. Then, HMG-CoA is converted into mevalonic acid (MVA), being the first precursor of the terpenoid biosynthetic pathway. The following steps include two sequential phosphorylation reactions performed by the enzymes mevalonate kinase and phosphomevalonate kinase, respectively, and a final decarboxylation step catalyzed by phosphomevalonate decarboxylase to produce IPP

**2.** IPP is isomerized to dimethylallyl pyrophosphate (DMAPP). The addition of three IPP molecules to DMAPP, catalyzed by prenyltransferases, produce geranylgeranyl pyrophosphate (GGPP). Then, the condensation of two molecules of GGPP produces phytoene (the first C40 carotene of the pathway), which is subsequently desaturated to produce lyco-

**3.** Lycopene undergoes many modification reactions and several cyclic carotenoids derived from lycopene such as β-carotene, γ-carotene, torulene, torularhodin and astaxanthin [1].

Despite the common steps in carotenogenesis, there are particular differences in the synthesis pathways between different microorganisms. In the following sections, the carotenogenic

It is currently believed that the main function of carotenoids in *X. dendrorhous* is to protect the yeast against damage caused by oxidative stress. This hypothesis is supported by the fact that strains that do not produce astaxanthin are more sensitive and grow less in the presence of ROS [23]. In addition, it has been reported that the presence of certain oxygen species increases the total carotenoid content [23, 24]. Other evidence points to the fact that *X. dendrorhous* has significantly lower catalase activity than other yeasts such as *Saccharomyces cerevisiae*, so carotenoids could compensate this low activity to help to preserve the viability of continuously growing yeasts [23]. On the other hand, *X. dendrorhous* does not have a cytosolic version of a superoxide dismutase, and carotenoids could also be compensating the lack of this enzyme [23]. *X. dendrorhous* is the only known yeast that produces astaxanthin *de novo* [22] and it has been approved by the FDA (Food and Drug Administration) for the commercial production of astaxanthin; therefore, it is a good candidate to allow the natural production of astaxanthin [27]. Knowledge about the biology of this yeast has increased and several methodologies to manipulate it have been developed, but still several aspects need to be improved for an eco-

The synthesis pathway preserves the basic steps of carotenoid synthesis in this yeast; however, some steps involve characteristic enzymes in fungi and other characteristic enzymes in

pathways in the microalgae *H. pluvialis* and in the yeast *X. dendrorhous* are described.

nomically competitive production of astaxanthin from *X. dendrorhous* [27].

**Figure 1.** Overview of *X. dendrorhous* carotenogenesis and related pathways. The steps for carotenoid biosynthesis from IPP, generated via the MVA pathway, are shown. Also, an abbreviated ergosterol synthesis pathway based on the route described in *S. cerevisiae* is schematized (in green), highlighting enzymes whose genes have been functionally characterized in *X. dendrorhous (*homologous to those present in *S. cerevisiae)* [19, 66]. MVA: mevalonate; IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl pyrophosphate; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate.

authorities [1, 2, 5]. In this context, the society nowadays is looking toward "green" solutions; thus, astaxanthin from natural sources, as from microorganisms, seems to be more favorable than its synthetic counterpart due to structure, function, application and security [25, 26].
