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Main Carotenoids Produced by Microorganisms

Carotenoids are the pigments present in plants, animals, and microorganisms which are responsible for a broad variety of colors found in nature. Their capacity as antioxidants mainly established their marketable success as health, food, and feed supplements, and cosmetics components. Currently, chemical synthesis dominates the worldwide market; however, due to the high biological value of natural carotenoids, the production scheme is moving towards microbial production as a profitable alternative.


Introduction
The simplest way to understand what carotenoids are is to look for colors in living organisms in a natural environment. Those reddish, orange, or yellowish pigments observed in living (micro)organisms are mainly carotenoids. They are one of the most widespread and ubiquitous lipid-soluble and nonnitrogenous pigments. Carotenoids form a subfamily of isoprenoids (also named terpenoids), which result in a diverse group of secondary metabolites. A prevalent example of this is the autumn colors and hues of trees and bushes. Other common examples of carotenoids include the orange-red condiment and food coloring annatto (derived from seeds of the achiote tree (Bixa Orellana)), petals, pollen (e.g., saffron), fruits (e.g., papaya, mandarins, oranges), vegetables (e.g., paprika, tomato), roots (e.g., carrots), animal tissues (e.g., salmonid and goldfish skin, flamingo and canary plumage, invertebrate exoskeletons), and animal products (e.g., egg yolks) (Figure 1) . Based on their oxygenation status, carotenoids can be divided in two main groups: (i) oxygenated molecules (oxycarotenoids) and (ii) non-oxygenated molecules. On the one hand, oxycarotenoids Carotenoids are the pigments present in plants, animals, and microorganisms which are responsible for a broad variety of colors found in nature. Their capacity as antioxidants mainly established their marketable success as health, food, and feed supplements, and cosmetics components. Currently, chemical synthesis dominates the worldwide market; however, due to the high biological value of natural carotenoids, the production scheme is moving towards microbial production as a profitable alternative.
[1] [2][3] [4] including carbonyl, carboxylic acid, ester, epoxy, hydroxy and methoxy groups are the xanthophylls (e.g., astaxanthin (C H O ), canthaxanthin (C H O ), lutein (C H O ) or zeaxanthin (C H O )). On the other hand, those strictly non-oxygenated molecules (hydrocarbons) are the carotenes (C H ) (e.g., lycopene, α-carotene, β-carotene) (Figure 2) . . They are naturally produced by photosynthetic species (plants, algae, and cyanobacteria), some groups of fungi and some non-photosynthetic bacteria . Although it is generally recognized that humans and animals are not able to produce their own carotenoids, the genome sequencing of the pea aphid (Acyrthosiphon pisum) detected carotenoid biosynthetic genes due to horizontal gene transfer from fungi .

Brief History of Carotenoids Discovery and Production
Carotenoids are taxonomically widespread and serve as pigments in plants and animals, and are the reason for the diverse and intense colors present in nature (Figure 1) . Because of this colorful characteristic, these compounds were one of the earliest studied phytochemicals , leading to the study of carotenoid pigments that began in the nineteenth century and spanning 200 years. This history can be divided into four periods, as Otto Isler indicated in his book entitled Carotenoids, in 1971 .
Beginning in the 19th century (first period), the core of carotenoid analysis was the isolation of the pigments and their characterization through their light absorption measurements, although their structures were still widely unknown . Henri Braconnot (1780-1855) was likely in charge of the first research on carotenoids in 1817, which was carried out in paprika (Capsicum annuum). More than a hundred years later (1927), a pigment from paprika was purified in its crystalline form under the name of capsanthin . In 1818, Aschoff isolated an apocarotenoid named crocin (from the word "crocos", which means saffron in German ). The apocarotenoids are carotenoid cleavage products as a result of the activity of specific carotenoid cleavage oxygenases . Crocin is the chemical ingredient primarily responsible for the saffron color (Crocus sativus) . A few years later, in 1823, the research on crab Following this first period (19th century) of carotenoid research, which focused on their isolation, the second period  was based on the determination of the empirical formula of carotenoids.
These tentative efforts to discover carotenoid's role in photosynthesis were also capital . The need for commercial production of natural pigments boosted intensive research on the microbial biosynthesis of carotenoids, which can be considered the fifth period of carotenoids research (1991 to present). β-carotene and astaxanthin, for example, are extensively studied and industrially produced by microbial fermentation . However, the screening of new producers of carotenoids (algae, bacteria, yeast and fungi), the development of fermentation processes or the use of genetic engineering or synthetic biology methodologies (e.g., search and use of genes involved in carotenoid biosynthesis, storage and catabolism) mark this fifth period .

What Are Carotenoids?
Based on their chemical characteristics, carotenoids are lipophilic compounds insoluble in water, which contain a chromophore. Corynebacterium glutamicum) and (iv) C (33 carotenoids; e.g., haloxanthin). In fungi (e.g., Xanthophyllomyces dendrorhous, Puccinia graminis ), the gene crtYB encodes a bifunctional enzyme that can perform as a phytoene synthase, as previously mentioned, and as a lycopene cyclase. The latter enzymatic activity is able to convert the acyclic ends of lycopene into βrings, and generates carotenoid diversity, by producing β-carotene and γ-carotene. Subsequently, ketolation and hydroxylation reactions occur in order to synthesize other carotenoids (Figure 3).

Biosynthetic Pathways of Carotenoids
Depending on the microorganism, some carotenoids can be synthesized by enzymes encoded by different genes .
β-carotene hydroxylase is an enzyme encoded by the crtZ gene, which is able to hydroxylate β-carotene to form β-cryptoxanthin, and further add another hydroxy group to finally synthesize zeaxanthin.
Canthaxanthin is produced by the transformation of two methylene groups located in the β-ionone rings of β-carotene into keto groups. This reaction is carried out by the enzyme β-carotene ketolase (CrtW), which initially adds a keto group to β-carotene, forming echinenone, and afterwards synthesizes canthaxanthin by adding an extra keto group .
Furthermore, astaxanthin is formed by the addition of 4-keto groups and 3-hydroxy groups to the molecule of β-carotene, by means of the enzyme astaxanthin synthetase (CrtS), helped by a cytochrome P450 reductase (CrtR). These enzymes are naturally present in some microorganisms, such as X.
dendrorhous . Moreover, the biosynthesis of astaxanthin in other microorganisms has been described by means of the enzymes β-carotene hydroxylase and β-carotene ketolase, encoded by the genes crtZ and crtW, respectively .
The lutein biosynthetic pathway is mainly present in plants. Lutein can be synthesized from lycopene through the action of two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C). The production of lutein has been observed in a strain of Synechocystis sp. transformed with the previously mentioned genes .  Barreiro and co-workers .

Carotenoids Market
As mentioned earlier, chemically-synthesized carotenoids dominate the market. Natural carotenoids obtained from plants, animals, and microorganisms are still limited. They can be synthetically produced using low-cost chemicals without living organisms involved in the process, which satisfy the majority of the worldwide market demand (>73% of the global market in 2013). This is particularly true in developing regions, such as the Asia-Pacific market. However, naturally extracted carotenoids are becoming increasingly accepted by the consumer market, resulting in a higher demand of natural products. This holds especially true in the European Union and North America, markets which are limiting the use of harmful additives in food and feed. Besides, these natural carotenoids are preferred by the health market because of their lower potential for toxic effects, in contrast with synthetic ones, and due to their higher biological properties (e.g., healthier, more favorable cis/trans isomer mixtures). The health-promoting properties of those chemically obtained are lower, which results in a less valuable, less desirable product.  [44] market value in 2020. Either way, it is clear that the global carotenoids market is on the brink of reaching a value of $2.0 billion, which is more than 2.5-fold higher than the $766 million estimated in 2011, and When a detailed analysis is done of the main carotenoids present in the market, the two that are the most common ones industrially produced are astaxanthin and β-carotene. Global market size of astaxanthin was estimated at $1.

Relevance of Carotenoids
Other than the stunning visual colors of carotenoids, what is their natural role of these compounds in nature?
Carotenoids avoid oxidative damage (antioxidant function) of cells and cellular organelles by quenching the scavenging singlet oxygen molecules, peroxy radicals, and other toxic oxygen species. This ability as quenchers of oxidative species explains their role in non-photosynthetic organisms as photoprotectors.
However, individual carotenoids additionally act through other mechanisms (e.g., β-carotene is pro- Astaxanthin (3, 3′-dihydroxy-β, β-carotene-4, 4′-dione) is a red-orange pigment with a hydroxyl and a ketone group at both ends of the molecule, which makes it a superior antioxidant to most of the other carotenoids (Figure 3).
Astaxanthin is the main carotenoid used as a feed additive for salmonids and crustaceans grown in captivity to provide the pink color that is characteristic of their flesh . Additionally, it is a valuable compound due to its powerful antioxidant properties, and it has also emerged as an interesting compound due to a variety of beneficial health effects. These include cardiovascular, anticancer, antidiabetic, anti-inflammatory, and immuno-stimulation, as well as cardiovascular, neuro-, ocular-, and skin-protective effects .
Roughly 95% of astaxanthin is produced by chemical synthesis. It can also be obtained from natural sources like plants or microorganisms. Chemical astaxanthin has a lower price in the market (around $1000/kg) compared to the natural one, and it is mostly used in aquaculture. Although synthetic astaxanthin is also approved for human use, there is increasing demand for astaxanthin from natural sources. Currently, large scale production of biological astaxanthin is carried out through cultivation of the microalgae Haematococcus pluvialis, and from the yeast X. dendrorhous (Phaffia rhodozyma) .
Astaxanthin can also be produced by fermentation of the marine bacteria Paracoccus carotinifaciens.
Β-Carotene is commercially used as a colorant in the food, feed, cosmetics, and pharmaceutical industries. It is also used as a pro-vitamin A supplement to achieve the recommended β-carotene dietary intake . It has an important role in human health, as a precursor of vitamin A, along with α-carotene and β-cryptoxanthin. This vitamin is important for many functions of the human body. It is particularly [51] [52] [
It is widely distributed in nature and commercialized in some regions for its coloring properties. Canthaxanthin is widely used in aquaculture and poultry as a feed colorant. In aquaculture, it is used for coloring rainbow trout and salmon flesh, administered frequently with astaxanthin. In poultry industry, it is used in the laying hen's diet for coloring egg yolks in combination with other carotenoids to obtain an optimal pigmentation, and in chicken to color the skin (Lucatin Red, BASF; Carophyll Red, DSM Nutritional Products). It is also used to feed ornamental birds and fishes to provide a brighter and healthier color to the feathers and the flesh.
In the food industry, canthaxanthin is approved as an additive in the EU (E161g) for coloring the "saucisse de Strasbourg", as well as coloring matter in feeding stuffs. In contrast to other carotenoids, canthaxanthin is not used as a nutraceutical .
Commercial canthaxanthin can be found in diluted or stabilized forms in order to prevent oxidation of the compound, including suspensions or solutions in edible fats or oils, emulsions, or water-dispersible powders . Nowadays, canthaxanthin is produced by chemical synthesis. Alternative ways of manufacturing this pigment are being explored in order to find a cost-efficient and more environmentally friendly process that meets market requirements. These new ways include natural sources such as plants, algae or bacteria, and also synthetic biology for its production in heterologous systems (Escherichia coli,
Lutein is best known for its role in visual health. Together with zeaxanthin and meso-zeaxanthin, these are the only carotenoids found in both the macula and lens of human eye. Several studies suggest that lutein and zeaxanthin are important for delaying and reducing age-related macular degeneration, and reduce the risk of cataracts. These carotenoids absorb light from the visible spectra, protecting the retina and the lens from potential damage caused by light exposure. Besides, these macular carotenoids protect the eye from oxidative stress and free radicals due to their antioxidant properties . Beneficial properties of lutein are not restricted to visual health; it is also a potent antioxidant that protects skin from photo-oxidative damage, since it filters near-UV high-energy blue light . It has also been described for having additional benefits for human health such as preventing cancer, cardiovascular disease, type 2 diabetes, or improving cognitive function , although some authors claim that additional studies should be carried out in order to generate enough evidence before deciding whether lutein should be clinically used or not .
In human nutrition, lutein is commercialized as a colorant, but it is mainly used due to its antioxidant Lutein is also used in poultry feed mainly to color eggs and to enhance chickens' skin with a visual vigorous color. It also brings health benefits to chickens supplemented with this carotenoid. It is generally administered together with other orange-red xanthophylls to obtain an intense yellow hue, generally canthaxanthin . Nowadays, lutein's presence in the egg yolk industry is shared with zeaxanthin and βapo-8′-carotenoic acid (apo-ester). The latter provides a similar yellow hue to egg yolks as lutein and zeaxanthin, but has a higher deposition rate than both of them, making it a more efficient pigment.

Lycopene
Lycopene (ψ, ψ-Carotene) is an open-polyene chain which lacks the β-ionone ring structures found in βcarotene (Figure 3). It is a red carotenoid synthesized naturally by some plants and microorganisms mostly in the isomer all-trans. The best known dietary intake of lycopene in humans is from tomatoes, but it can also be found at equivalent levels in other fruits such as persimmon, pink guava, or [ watermelon .
Lycopene is widely used as a pigment in the food industry. It is used, for example, in fruit juices and soft drinks, sport drinks, cereals, bread, dairy products, dietary products, and dietary supplements . In addition, there is growing interest in lycopene due to its health promoting properties, such as antioxidant activity that protects DNA, lipids, and proteins from oxidative damage, anti-cancer activity, or cardioprotective activity. Several mechanisms by which lycopene acts against cancer have been

Zeaxanthin
Zeaxanthin ((3R,3′R)-β, β-Carotene-3,3′-diol) is an isomer of lutein: the two alcohol groups of the molecule differ from each other by the shift of a single double bond (Figure 3). It is naturally found in fruits and vegetables such as papaya (14.1 µg/g fresh weight basis), corn (6 µg/g fresh weight basis), or spinach (15.1 µg/g dry weight basis) .
Zeaxanthin, together with lutein and meso-zeaxanthin, have been described as having a critical role in visual health. A higher dietary intake of these xanthophylls has been associated with reducing the risk of cataracts and age-related macular degeneration . These carotenoids absorb light from the visible spectra, protecting the retina and the lens from potential damage caused by light exposure. Besides, these macular carotenoids protect the eye from oxidative stress and free radicals due to their antioxidant properties .
Zeaxanthin is commercially used in the food, cosmetic, and pharmaceutical industries as pure formulation or in combination with other carotenoids for its coloring properties, and it is also used as a [55] [ supplement for its putative beneficial health effects (OPTISHARP , DSM Nutritional Products) .
In addition to its use as a human food supplement, zeaxanthin, together with lutein and β-apo-8′carotenoic acid (apo-ester), is used in poultry feed, mainly to provide a yellow color to egg yolk, and a visually healthy color to the skin of chickens. It is generally administered together with other orange-red xanthophylls to obtain an intense yellow hue, generally canthaxanthin. Nowadays, the preferred carotenoid for providing a yellow hue to yolk is the apo-ester, due to the fact that it has a higher deposition rate than lutein and zeaxanthin, thus making it a more efficient pigment

Biotechnological Production of Carotenoids
At present, the growing market of carotenoids is mainly met through chemical synthesis, although this has several disadvantages. First, higher production costs make chemical processes unprofitable in some cases, such as lutein or lycopene. Additionally, the chemical synthesis of carotenoids requires a very high level of control and can generate by-products that have undesirable side effects, and could cause food safety problems. Finally, these processes will become unsustainable in the near future, since they use non-renewable resources (generally from petrochemical origins, as in the case of canthaxanthin).
Therefore, in light of the global economic value of carotenoids and the growing awareness of consumers, the production of carotenoids from natural or renewable sources has become an area of intense research .

Astaxanthin
Astaxanthin is a xanthophyll carotenoid which can be found in numerous natural sources from algae and yeasts, to animals such as salmon, trout, krill, shrimp, and crayfish . However, like most carotenoids, commercial astaxanthin is mainly produced by chemical synthesis (ca. 95%), which is an expensive and unsustainable process, and natural sources such as fermentative production or crustacean byproducts are gaining importance . Four natural sources are a realistic alternative to synthetic astaxanthin: (i) counterpart. Thus, several important commercial companies, such as Cyanotech Corporation, Mera Pharmaceuticals Inc., AIgatechnologies or Fuji Chemical Industry Co. Ltd. are involved in large scale production of astaxanthin from H. pluvialis .
Nonetheless, natural astaxanthin is not able to compete with the synthetic alternative at lower valueadded markets currently, mainly due to the production costs (in the best case scenario, 1536€/kg vs. 880€/kg, respectively) . However, due to the increasing demand for natural astaxanthin and its high price, there is constant growth of astaxanthin production from natural sources over the last few years.
Thus, the improvement of astaxanthin titer by microbial fermentation is required to be competitive against its synthetic manufacturing . However, astaxanthin production by natural microalgae requires large open areas and long production times. This has led to numerous studies in recent years about improvement of the culture conditions, or even the genetic modifications of bacteria and yeasts for the production of astaxanthin, such as E. coli and S. cerevisiae.
A promising natural source of astaxanthin at the industrial scale is the heterobasidiomycetous yeast X.
dendrorhous, due to its ability to accumulate large amounts of this carotenoid pigment, with productivities of 350 mg/L astaxanthin in 800 L fermenters . Diverse studies on astaxanthin production yields by optimization of fermentation conditions revealed that its production is mainly influenced by the Far and away, the most promising strain is the well-known biotechnological chassis E. coli. This bacterium, which cannot naturally synthesize carotenoids, was genetically engineered by the introduction of carotenogenic genes. Therefore, recent studies show that the heterologous expression of β-carotene ketolase (CrtW) from Brevundimonas sp. and β-carotene hydroxylase (CrtZ) from Pantoea agglomerans, along with the genetic modification of the groES and groEL chaperones, allows a production of 1.18 g/L of astaxanthin after 60 h of fermentation in shake flasks ( Table 1) .

β-Carotene
Although the oldest way to obtain carotenoids is extraction by physicochemical processes from plant material (the richest sources of β-carotene are dehydrated peppers, carrots and grape leaves), chemical synthesis from β-ionone has been the primary source of β-carotene production since it was developed in 1950 by Milas . Nevertheless, among all the known methods of β-carotene production, again, microbial biosynthesis is gaining major interest. Natural β-carotene can be obtained from microalgae (D.
salina), vegetables (like carrots or pumpkins), or by fermentation of microorganisms (mainly fungus as B. trispora and yeasts such as X. dendrorhous or Y. lipolytica) .
Currently, as with astaxanthin, the main natural source of β-carotene are algae, whose production account for about 35% of total revenue. Much attention is drawn to the green microalgae D. salina, which is the main commercial source of microbial origin . However, other well-known microalgae, such as C. zofingiensis, are gaining interest, and recent studies have reported that a mutant of the algae C.
zofingiensis is able to accumulate high amounts of β-carotene (34.6 mg/L) when it is induced by glucose with high light irradiation .
Other of the main sources of production of β-carotene is the fungus B. trispora. β-carotene production by B. trispora was reported in 1963, but this achievement was abandoned because the profitability of the process was not competitive with chemical biosynthesis. However, after some decades, microbial synthesis of carotenoids has attracted researcher's attention, and great efforts have been dedicated to optimizing culture and growth conditions aimed to improve β-carotene biosynthesis from B. trispora, [92] [108] [109] [110] reaching a production of 78.0 mg/g DCW at lab scales in 2012 . B. trispora produces β-carotene through a complex process, where mycelia of the (+) and (−) sexual mating types of this fungi need to be co-cultivated .
Conversely, because yeasts are unicellular organisms with a relatively high growth rate in low-cost fermentation media, they have an advantage over algae and fungi . Thus, it has been reported that there is an even more promising source of natural β-carotene: the yeast Y. lipolytica. Despite it being best known for the industrial production of astaxanthin, it was reported that engineered Y. lipolytica strains can produce 797.1 mg/L β-carotene as an astaxanthin intermediary. Thus, the expression of phytoene desaturase (crtI) and lycopene cyclase (crtYB) obtained from X. dendrorhous, along with the up-regulation of geranyl-geranyl diphosphate synthase (GGS1/crtE) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG1) and the down-regulation of the competing enzyme squalene synthase (SQS1), could increase the β-carotene biosynthesis in Y. lipolytica ( Table 1) .

Canthaxanthin
Canthaxanthin was initially extracted from an edible mushroom (C. cinnabarinus) in 1950 , and, since then, it has been detected in several organisms as plants, trout, birds (flamingo), crayfish, fungi (A. Since canthaxanthin is the precursor of other carotenoids, such as astaxanthin, just a few papers have reported canthaxanthin production as the main goal ( Table 1).

Lutein
The xanthophyll lutein generally appears in nature, along with its stereoisomer zeaxanthin, and it occurs in abundance in green leafy vegetables such as kale, collard, and spinach. Other important natural sources of lutein and zeaxanthin are egg yolk, chicken (broilers), and cheese .
Lutein is one of the unique carotenoids that is obtained mainly from natural sources, since its commercial production through chemical synthesis has not been viable due to its poor overall yield . Currently, the flower petals of yellow Marigold (T. erecta L.) is the main source in the industrial extraction of lutein.
However, marigold presents several drawbacks: (i) the flowers must be periodically harvested, and the petals have to be separated prior to extraction; (ii) lutein content in marigold petals is variable; and (iii) slow growth and low yield of biomass production . Thus, there is a huge opportunity for natural lutein production from microalgae, which could become the best alternative source. Lutein biosynthesis has been reported within members of Chlorophyta (such as Scenedesmus obliquus, Chlorella minutissima, D. salina, H. pluvialis, and C. vulgaris), Chlorarachniophyta, Cryptophyta, Euglenophyta, and Rhodophyta algal species . Two main factors make microalga a good lutein source: (i) its lutein content; and (ii) its biomass productivity. Nowadays, only Chlorella sorokiniana, Murielopsis sp., and Scenedesmus almeriensis are considered for mass production in largescale systems .
One of the greatest promises in the future production of lutein is the microalgae Chlamydomonas reinhardtii, which is used as host for the heterologous expression of some carotenoid biosynthesis genes. [114] [116] Thus, the expression of the phytoene synthase gene (psy) from D. salina results in the production of 2.6fold greater amounts of lutein compared to the wild type strain. Similar results were reported due to the overexpression of the phytoene desaturase (pds) or the expression of the phytoene-β-carotene synthase gene (pbs) from the red yeast X. dendrorhous, increasing, in this case, the lutein production by 60% under low light conditions .
Another strategy to achieve better yields in lutein production in microalgae is the fine-tuning of culture conditions (mainly light intensity, CO content, color and time of exposure, temperature, and nitrogen source) of the well-known producer organisms . Furthermore, 11-fold higher lutein productivity (4.96 mg L d ) has been reported in the green microalgae S. obliquus and 290 mg lutein m d in a 4000 L photobioreactor for S. almeriensis . Similarly, growth and lutein production processes have been optimized for the algae Chlorella sorokiniana and Chlorella protothecoides, although all these assays are still being carried out at the bench or pilot scales (Table 1) .
Finally, lutein producing bacteria has yet been found in nature, however the genetic modification of the cyanobacteria Synechocystis sp. PCC to express β-and ε-cyclase, and β-and ε-hydroxylase from Arabidopsis, results in a lutein producing strain .

Lycopene
Lycopene is naturally present in fruits and vegetables such as papayas, red peppers and, especially, tomatoes (about 80% of their total carotenoid content) , which is one of the main sources of lycopene nowadays . However, increasing demand for lycopene has led to a search for new sources.
Thus, although lycopene can be synthesized chemically, its production process is extremely complex and costly, highlighting microbial synthesis as a promising method in recent years .
As in most cases, the model microorganism E. coli was one of the first developed chassis for lycopene production, reaching titers of 2.7 g/L. Nevertheless, the release of some endotoxins during the fermentation process has led other microorganisms, especially well-known industrial yeasts such as S.
cerevisiae, P. pastoris or Y. lipolytica, to take the lead in the development of new lycopene production processes. The production of lycopene by the fungus B. trispora was previously reported, but numerous studies have focused on the use of yeast because B. trispora requires the addition of cyclase inhibitors such as imidazole or pyridine derivatives, in order to avoid formation of β-carotene. Besides, yeasts usually possess endogenous carotenoid metabolic pathways, so they can easily be genetically modified to drive metabolic flux toward lycopene synthesis from the common precursors isopentyl diphosphate (IPP) and dimethyl diphosphate (DMAPP).
Some species are able to produce natural lycopene, such as X. denrorhous (P. rhodozyma). However, due to the lack of genetic manipulation tools, the product concentration by using this natural yeast is not usually high, and they are not reported as industrial producers. Thus, they just provide gene elements occurring in lycopene heterologous synthesis pathways . Three key crt genes are necessary for a heterologous lycopene pathway expression: (i) crtE, GGPP synthase, catalyzes the first step of downstream lycopene biosynthesis; (ii) crtB, phytoene synthase, leads to the formation of the colorless phytoene; and (iii) crtI, phytoene desaturase, catalyzes the conversion of phytoene to lycopene. Thus, the heterologous expression of two copies of crtE from Pantoea ananatis, two copies of crtI from B. trispora, and one copy of crtB from P. agglomerans in S. cerevisiae, along with the knocking out of endogenous bypass genes, culminates in the production of 3.28 g/L lycopene in 7-L fermenters ( Table 1).
Finally, through a combination of the modification of the native isopentenol utilization pathway (IUP) in the oleaginous yeast Y. lipolytica, with further genetic and fermentation optimizations, a final lycopene titer of 4.2 g/L was achieved in 3-L bioreactors ( To date, the xanthophyll zeaxanthin has been mainly produced by extraction from natural sources like green vegetables and yellow corn, although it can also be found in oranges, tangerines, spinach, lettuce, squash, and chicken egg yolk. Zeaxanthin could be chemically synthesized, but the process is complex and expensive. Therefore, natural alternatives lead the sources of industrial production, even when the processes net low extraction rates and produce large amounts of waste. As a result, the idea of using new biological technology has attracted more and more attention in the last years, although, so far, all these studies continue on a bench scale. Compared with other carotenoids, zeaxanthin production rates are extremely low and the use of metabolically engineered microorganisms as a source is sought. Many bacteria are able to naturally accumulate zeaxanthin; the best-known among them are those of the genera Mesoflavibacter and Muricauda. For example, it has been reported that Muricauda lutaonensis is able to produce 3.12 mg L of zeaxanthin when cultured in 2-L biorreactors , and Mesoflavibacter zeaxanthinifaciens showed a production yield of 910 µg/g DCW at the lab scale ( Table 1) .
To date, there are two realistic industrial sources of zeaxanthin: bacteria and algae. By far the most promising algae is C. zofingiensis, which after random mutagenesis with N-methyl-N′-nitro-Nnitrosoguanidine (MNNG), was able to accumulate 36.79 mg/L zeaxanthin when induced by glucose and growing with nitrogen deficiency and high light irradiation; an especially remarkable fact since no algae have been found to have profitable potential for zeaxanthin production . On the other hand, gene editing using CRISPR-Cas9 was conducted in C. reinhardtii aimed to eliminate the synthesis of lutein and enhance the production of high purity zeaxanthin, resulting in a mutant able to produce 6.84 mg/L zeaxanthin .
Finally, among the most promising bacteria it is worth highlighting the model host E. coli. Thus, it has been reported that the heterologous expression of the ctrY and ctrZ genes from P. ananatis resulted in the production of 11.95 mg/g DCW of zeaxanthin , levels much higher than those discussed initially.
Unfortunately, these levels do not reach those shown by the well-known P. putida, which produces 51.3 mg/L of zeaxanthin after expression of the crt genes from P. ananatis and optimization of the fermentation parameters ( Table 1) .

Future Outlook
Currently, the entirety of the carotenoids market is dominated by chemical synthesis, but natural carotenoids obtained from plants, animals, and microorganisms, due to their higher biological and health properties, are taking the attention of those markets that demand natural products. The microbiological production of these industrially-relevant compounds appears to be a suitable alternative, which still needs future development on strain selection, omics methodology applications as data sources for synthetic biology developments, and culture condition improvements. However, consumer priorities are driving the transition from chemical to biological production, where fungi, bacteria, and algae are the main players.
The nutritional (food and feed), health, and cosmetics applications of these colored compounds have profitable benefits today, which will be increased due to food and beverage supplementation, intensive aquiculture as substitute for fishery depletion, and health properties.
Carotenoid discovery, research and development has a long story, which is as highly active and industrially attractive now more than ever.