**3.2. Biosynthesis of xanthophylls**

of another IPP into FPP to yield GGPP (C20). Finally, phytoene synthase (*crtB*) catalyzes the first committed step of carotenoid biosynthesis, the formation of one molecule of phytoene (C40) from two molecules of GGPP (**Figure 2**). Phytoene is a colorless acyclic carotene with only three conjugated double bonds. All the C40 carotenoids are derived from phytoene, which accounts for over 90% of total carotenoids to date [22]. Based on molecular structures, carotenoids are classified into two groups: carotenes and xanthophylls. Carotenes are hydrocarbon carotenoids with only carbon and hydrogen atoms (e.g., lycopene and β-carotene), whereas xanthophylls are oxygenated carotenoids by hydroxylation, ketolation and epoxidation (e.g., astaxanthin, lutein, **Figure 2**) [31]. In plants, algae, fungi and bacteria, apocarotenoids are derived from the oxidation of carotenoids or other apocarotenoids with carotenoid cleavage enzymes (such as carotenoid cleavage dioxygenases or CCDs and apocarotenoid cleavage

oxygenases or ACOs) [32]. Some apocarotenoid examples are shown in **Figure 2**.

As a colorless carotene, phytoene is the common precursor to all the C40 carotenoids (**Figure 2**). It exhibits excellent anti-UV activity [33] and is clinically proved to have activities of skin whitening and wrinkle reduction [34]. Hence, there are increasing cosmetic products developed based on phytoene. Phytoene is an intermediate carotenoid in plants and exists only as a minor product; hence, it is expensive to extract phytoene from plant materials. Consequently, it is promising to engineer microbes to produce higher concentrations of phytoene and more importantly, to produce it at high purity without other carotenoids. By deleting the *crtI* gene, encoding phytoene desaturase (see below), from an engineered lycopene-producing strain of *Escherichia coli* previously developed in our laboratory [19], it was relatively simple to generate strains of producing more than 50 mg/L of high-purity phytoene in simple low-cell density shake flasks [35]. Although this carotene with a high-potential market in cosmetics could be relatively simple to transfer to the industry, this is only just the beginning to attract interest, as witnessed by a French company Deinove (www.deinove.com) [36]. Despite certainly being more efficient than the use of the tomato strain developed to this end, there could still be considerable progress made by optimizing the engineered strains such as that used in our study and coupling this to high cell density fermentation processes to achieve a more

Lycopene, a red color pigment most commonly associated with tomatoes, belongs to one of the top six commercial carotenoids. It is produced from the dehydrogenation of phytoene catalyzed by different types of phytoene desaturases (*crtI*, *PDS* or *ZDS,* **Figure 2**). Lycopene has been used as animal feed, food coloring and nutritional products. Some clinical studies have suggested that lycopene functions in reducing the risk of prostate cancers [37, 38]. In recent years, multiple research groups reported relatively high concentrations of lycopene produced in *E. coli* and yeasts. In *E. coli*, Kim et al. have used a mixture of carbon sources containing glucose, glycerol and arabinose to produce lycopene at 1.35 g/L [39]. Our laboratory initially optimized the MEP pathway which enabled the *E. coli* strain to produce at 20 mg/g

**3. Production of carotenoids in engineered microbes**

**3.1. Biosynthesis of carotenes**

90 Progress in Carotenoid Research

cost-effective process.

The modification of carotenes by enzymes such as hydroxylases and ketolases leads to the synthesis of xanthophylls (**Figure 2**). Due to the polarity introduced by oxygen, xanthophylls have different physical properties and physiological activities. For example, unlike carotenes, most xanthophylls do not possess provitamin A activity but do have higher anti-oxidant activities. The reason is that, in addition to the polyene structure, the functional groups of xanthophylls such as keto groups in the β-ionone rings can also quench singlet oxygen resides [31].

Among various xanthophylls, astaxanthin is the most important commercial product. Astaxanthin is a red pigment with numerous health benefits. As a potent anti-oxidant, astaxanthin protects the tissue against UV-light damage [51–53] and exhibits anti-cancer activity [54, 55] and anti-inflammatory properties [56]. In double-blind, randomized controlled trials, astaxanthin lowered oxidative stress in obese subjects and improved cognition and promoted proliferation of nerve stem cells [57]. Astaxanthin also improves integrated immune response [58], reveals anti-aging effects by protecting red blood cells in both aging and young people


Due to the wide application of astaxanthin, many researchers have been working hard to engineer microbes to produce high titer and yield of astaxanthin. It is not trivial to optimize the biotransformation of β-carotene to astaxanthin as the biosynthetic pathway is rather complex with many intermediates and a complex network of enzymatic reactions [67]. By screening different β-carotene hydroxylases and ketolases, there has been success to improve astaxanthin production from sub-milligram to milligram per gram DCW [67, 68]. Further optimization of the metabolic pathway leading to astaxanthin synthesis has led to improved yields which are now promising for commercialization. For example, Zhou et al. developed a *S. cerevisiae* strain that overproduced astaxanthin at 47.2 mg/L and 8.1 mg/g DCW, where they used a direct evolution approach to generate a triple mutant of beta-carotene ketolase with higher activity [69]. Lin et al. integrated a multicopy of the key biosynthetic genes of astaxanthin (*hpchyb* and

Biosynthesis of Carotenoids and Apocarotenoids by Microorganisms and Their Industrial Potential

**(mg/L)**

**Content (mg/g DCW)**

Astaxanthin 1.6 0.29 3 days in flasks [77]

**Culture conditions**

http://dx.doi.org/10.5772/intechopen.79061

flasks

bioreactors

bioreactors

bioreactors

bioreactors

bioreactors

flasks

plates

**References**

93

[72]

[79]

[69]

[70]

[66]

[71]

[81]

[82]

**No. Hosts Carotenoids Titer** 

9 *Xanthophyllomyces dendrorhous, previously as Phaffia rhodozyma*

1 *Saccharomyces cerevisiae* Astaxanthin / 4.7 3–4 days in

 *Saccharomyces cerevisiae* Astaxanthin / 0.029 5 days in flasks [73] *Escherichia coli* Astaxanthin / 2.64 2 days in flasks [74] *Escherichia coli* Astaxanthin / 0.31 2 days in flasks [68] *Escherichia coli* Astaxanthin 2.1 1.41 2 days in flasks [75] *Escherichia coli* Astaxanthin 2.9 1.99 2 days in flasks [67] *Corynebacterium glutamicum* Astaxanthin / 1.6 2 days in flasks [76]

10 *Xanthophyllomyces dendrorhous* Astaxanthin / 9.0 8 days in flasks [78]

11 *Xanthophyllomyces dendrorhous* Astaxanthin 561 5.0 4–5 days in

12 *Saccharomyces cerevisiae* Astaxanthin 47.2 8.1 3–4 days in

13 *Kluyveromyces marxianus* Astaxanthin / 9.90 3 days in

14 *Yarrowia lipolytica* Astaxanthin 54.6 3.5 3–4 days in

15 *Escherichia coli* Astaxanthin 320 15.0 2 days in

17 *Escherichia coli* Zeaxanthin / 11.9 2 days in

18 *Escherichia coli* Zeaxanthin 722 23.2 2.5 days in

**Table 2.** Microbial production of astaxanthin and zeaxanthin in literature.

16 *Xanthophyllomyces dendrorhous* Zeaxanthin 10.8 0.5 7.5 days in flasks [80]

**Table 1.** Microbial production of carotenes in literature.

[59, 60] and relieves eye fatigue especially beneficial for persons spending too much time on the computer and smartphones [61]. In addition, astaxanthin supplement can prevent atherosclerotic cardiovascular disease [62, 63] and diabetes [64, 65]. More importantly, besides all these benefits, astaxanthin is clinically proven to be safe for human and animals. Therefore, astaxanthin has been widely used in fish feeding, food, nutritional, medicinal and cosmetic industries. The current global annual market of astaxanthin is around 250 tons worth \$447 million [66], and it is growing rapidly. Synthetic astaxanthin, like β-carotene, is less popular with consumers and yields a mixture of three isomers, RR, RS and SS, at the ratio of 1:2:1 and appears to be less available for assimilation than the natural forms. Astaxanthin produced by the microalga *Haematococcus pluvialis* has a higher cost and lower purity than synthetic astaxanthin so additional work is required before good natural astaxanthin can be marketed effectively. Furthermore, astaxanthin in microalgae, shrimp and fish exists as an ester form rather than in the free form, which limits its nutraceutical applications.

Due to the wide application of astaxanthin, many researchers have been working hard to engineer microbes to produce high titer and yield of astaxanthin. It is not trivial to optimize the biotransformation of β-carotene to astaxanthin as the biosynthetic pathway is rather complex with many intermediates and a complex network of enzymatic reactions [67]. By screening different β-carotene hydroxylases and ketolases, there has been success to improve astaxanthin production from sub-milligram to milligram per gram DCW [67, 68]. Further optimization of the metabolic pathway leading to astaxanthin synthesis has led to improved yields which are now promising for commercialization. For example, Zhou et al. developed a *S. cerevisiae* strain that overproduced astaxanthin at 47.2 mg/L and 8.1 mg/g DCW, where they used a direct evolution approach to generate a triple mutant of beta-carotene ketolase with higher activity [69]. Lin et al. integrated a multicopy of the key biosynthetic genes of astaxanthin (*hpchyb* and


**Table 2.** Microbial production of astaxanthin and zeaxanthin in literature.

[59, 60] and relieves eye fatigue especially beneficial for persons spending too much time on the computer and smartphones [61]. In addition, astaxanthin supplement can prevent atherosclerotic cardiovascular disease [62, 63] and diabetes [64, 65]. More importantly, besides all these benefits, astaxanthin is clinically proven to be safe for human and animals. Therefore, astaxanthin has been widely used in fish feeding, food, nutritional, medicinal and cosmetic industries. The current global annual market of astaxanthin is around 250 tons worth \$447 million [66], and it is growing rapidly. Synthetic astaxanthin, like β-carotene, is less popular with consumers and yields a mixture of three isomers, RR, RS and SS, at the ratio of 1:2:1 and appears to be less available for assimilation than the natural forms. Astaxanthin produced by the microalga *Haematococcus pluvialis* has a higher cost and lower purity than synthetic astaxanthin so additional work is required before good natural astaxanthin can be marketed effectively. Furthermore, astaxanthin in microalgae, shrimp and fish exists as an ester form

rather than in the free form, which limits its nutraceutical applications.

**No. Hosts Carotenes Titer (mg/L) Content** 

92 Progress in Carotenoid Research

1 *Escherichia coli* Phytoene 50 35 1–2 days, in flasks [35] 2 *Escherichia coli* Lycopene 224 34.5 1–2 days, in flasks [48] 3 *Escherichia coli* Lycopene / 20 1–2 days, in flasks [40]

4 *Escherichia coli* Lycopene 1500 35 2 days, in

5 *Escherichia coli* Lycopene 1350 32 2 days, in

6 *Saccharomyces cerevisiae* Lycopene 1610 24.4 5–6 days, in

7 *Saccharomyces cerevisiae* Lycopene 1650 54.6 5–6 days, in

9 *Yarrowia lipolytica* Lycopene 213 21.1 10 days, in

10 *Blakeslea trispora* β-Carotene 5600 / 7 days, in

11 *Escherichia coli* β-Carotene 2100 60 3–4 days, in

12 *Escherichia coli* β-Carotene 3200 / 2–3 days, in

13 *Yarrowia lipolytica* β-Carotene 6500 90 5–6 days, in

14 *Yarrowia lipolytica* β-Carotene 4000 50 10–11 days, in

**Table 1.** Microbial production of carotenes in literature.

8 *Yarrowia lipolytica* Lycopene / 16 7–8 days, in flasks [42]

**(mg/g DCW)** **Culture conditions References**

[19]

[39]

[41]

[49]

[43]

[50]

[45]

[44]

[47]

[46]

bioreactors

bioreactors

bioreactors

bioreactors

bioreactors

bioreactors

bioreactors

bioreactors

bioreactors

bioreactors

*bkt*) into the *Yarrowia lipolytica*. As a result, they were able to achieve about 9.97 mg/g DCW of astaxanthin [70]. By developing and using an efficient multidimensional heuristic process and colorimetric medium screening approach, our laboratory has achieved one of the best results of astaxanthin using *E. coli*, 320 mg/L and 15 mg/g DCW [71]. As summarized in **Table 2**, the engineered *S. cerevisiae* [69], *Y. lipolytica* [66], *Kluyveromyces marxianus* [70] and *E. coli* [71] have produced promisingly high titers and yields of astaxanthin. The recently achieved titers and yields [66, 70, 71] are from 10-fold to 100-fold higher than those previously reported in *S. cerevisiae* [72, 73]*, E. coli* [74, 75]*, Corynebacterium glutamicum* [76] and *Xanthophyllomyces dendrorhous,* previously as *Phaffia rhodozyma* [77–79].

Consequently, overexpression of the YBBO enzyme improved the final yield (76 mg/L) and purity (88%) of retinol in the final products [86]. Based on our lycopene chassis strain, we developed a 'plug-n-play' system that could easily adapt our *E. coli* strain into different apocarotenoids, such as α-, β-ionones and retinol [19] with promising results obtained (**Figure 3**).

Biosynthesis of Carotenoids and Apocarotenoids by Microorganisms and Their Industrial Potential

http://dx.doi.org/10.5772/intechopen.79061

95

Both α-ionone and β-ionone have exceptional aroma activities as their odor threshold is at the sub-ppb range [7, 87]. Hence, they have been widely used as fragrance molecules in cosmetics and perfumes. As consumers prefer natural ingredients, the market demand for natural ionone is increasing dramatically. In addition, there is a chiral center for α-ionone. Natural α-ionone from plants (such as raspberry) is (R)-(+)-(E)-alpha-ionone. In contrast, synthetic α-ionone has two isomers (R and S). The R-enantiomer has a unique and strong floral flavor and aroma, described as a violet-like, fruit-like or raspberry-like flavor, while the S-enantiomer is woody or β-ionone like. Lashbrooke et al. did a proof-of-principle production of α-ionone at about 300 ng/L [88]. By coupling the modular metabolic engineering approach and enzyme engineering methods (N-terminal truncation and protein fusion), we developed an *E. coli* strain to produce 'natural identical' α-ionone at almost 500 mg/L, about 1400 times higher than that previously reported [19] (**Table 3**). Similarly, Phytowelt (www.phytowelt. com), a German company, has also developed an *E. coli*-based process to produce α-ionone,

**Figure 3.** A 'plug-n-play' platform for biosynthesis of apocarotenoids. Adapted from author' s paper [19]. crtY—lycopene beta-cyclase; CCD1—carotenoid cleavage dioxygenase; BCDO (or blh)—β-carotene dioxygenase; ybbO—NADP+-

**4.2. Biosynthesis of α- and β-ionone**

dependent aldehyde reductase.

demonstrating that it has attracted more commercial interest.

Zeaxanthin is another important xanthophyll with high commercial values. Lutein, an isomer of zeaxanthin, is typically found in plants (such as corn), whereas zeaxanthin is present in cyanobacteria and some non-photosynthetic bacteria [2]. Although both lutein and zeaxanthin are used as colorants and potentially in pharmaceutical and nutraceutical industries, the demand for alternative sources of zeaxanthin is more urgent than lutein. Till now, *X. dendrorhous* has been engineered to produce 10 mg/L of zeaxanthin [80]. The first attempt to produce zeaxanthin in *E. coli* achieved 11.9 mg/ g DCW in bioreactors [81]. A few years later, the same group applied a dynamic control system to *E. coli* in which 720 mg/L of zeaxanthin was produced [82] (**Table 2**).
