**4.1. Biosynthesis of retinol or vitamin A**

As an important nutritional compound and an active cosmetic ingredient, retinol market size is estimated at 1.6 billion dollars [85]. Jang et al. pioneered retinol production in metabolically engineered *E. coli* [85]. Unlike carotenoids that are stored intracellularly in the lipid structures of microbes, apocarotenoids are smaller and thus can pass the cell membrane into the culture media. Consequently, a two-phase culture system was applied to capture extracellular retinol and improve its production by minimizing its degradation [85]. The same group later identified a gene (*ybbo*) that has retinal reductase activity that converts retinal into retinol. 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**).

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

*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* 

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

As shown in **Figure 2**, carotenoids can be further converted into apocarotenoids by CCDs or other oxygenases. Here, three apocarotenoids of high commercial values are highlighted here. Retinol, or vitamin A, is one of the most important apocarotenoids to humans. Retinol exhibits an essential function in vision, bone development and also promotes skin health as an anti-oxidant [83]. The other two are aromatic molecules, α-ionone, which naturally exists in raspberry, and β-ionone, which is found in many flowers, for example, rose, osmanthus and violet [84]. The chemical synthesis of these three molecules is not very difficult and contributes significantly to the current market share. However, consumers prefer natural derivatives and are willing to pay higher prices for natural ingredients [19]. As mentioned in the introduction, the extremely low concentrations in natural plant materials make their extraction an extremely expensive process. Consequently, the fermentation of engineered microbes is a

As an important nutritional compound and an active cosmetic ingredient, retinol market size is estimated at 1.6 billion dollars [85]. Jang et al. pioneered retinol production in metabolically engineered *E. coli* [85]. Unlike carotenoids that are stored intracellularly in the lipid structures of microbes, apocarotenoids are smaller and thus can pass the cell membrane into the culture media. Consequently, a two-phase culture system was applied to capture extracellular retinol and improve its production by minimizing its degradation [85]. The same group later identified a gene (*ybbo*) that has retinal reductase activity that converts retinal into retinol.

*dendrorhous,* previously as *Phaffia rhodozyma* [77–79].

**4. Production of apocarotenoids in engineered microbes**

was produced [82] (**Table 2**).

94 Progress in Carotenoid Research

promising alternative route.

**4.1. Biosynthesis of retinol or vitamin A**

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, demonstrating that it has attracted more commercial interest.

**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+ dependent aldehyde reductase.


TRY figures [46, 47, 71] higher than existing processes. It is not surprising that some of them will be translated into more cost-effective industrial processes. More importantly, scientists and engineers are working together to continue improving microbial strains and fermentation processes. Breakthrough by innovation and collective knowledge will markedly reduce product cost and make it more competitive. In addition, the recent trend of consumers' preference to 'natural' or 'bio-based' ingredients will make microbial-derived carotenoids and

Biosynthesis of Carotenoids and Apocarotenoids by Microorganisms and Their Industrial Potential

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

97

Amid diverse natural products, carotenoids and apocarotenoids are particularly interesting. This is not only due to their bright color and pleasant fragrances but also their light-harvesting capability, the electron/energy transferring ability, the potent anti-oxidant properties, the hormone function, vitamin A activity and numerous other health benefits to both human and other life forms on the Earth. Increasingly, clinical studies have supported the concept that the regular uptake of carotenoids can prevent many serious diseases. The list of benefits and applications keeps growing and with the market for commercial exploitation it can be confidently expected to increase. In light of this and the extremely low levels found in plant materials, it is urgent to find solutions enabling these valuable molecules to be supplied in a sustainable and cost-effective manner. In the past decade, the metabolic engineering of microorganisms has progressed remarkably for the production of carotenoids and apocarotenoids. Some of these processes are being commercialized already but the scope to further extend this family of molecules is high, adding an increasingly solicited pipeline of natural

This work was supported by the research funding of Biotransformation Innovation Platform (BioTrans), Agency for Science, Technology and Research (A\*STAR), Singapore. The author appreciates Dr. Nic Lindley, Ms. Sudha Devi Manbahal Shukal and Ms. Chin Chin Lim in

Biotransformation Innovation Platform (BioTrans), Agency for Science, Technology and

apocarotenoids more appealing.

products to compete with chemical synthesis.

Address all correspondence to: zcqsimon@outlook.com

**Acknowledgements**

BioTrans for invaluable advice.

Research (A\*STAR), Singapore

**Author details**

Congqiang Zhang

**6. Conclusion**

**Table 3.** Microbial production of retinol, α- and β-ionones in literature.

Although several groups have attempted to produce β-ionone using yeast or *E. coli*, their yields are relatively low. Simkin et al. firstly engineered *E. coli* cells to synthesize β-ionone but with only detectable trace amounts being reported [89]. Beekwilder et al. engineered *Saccharomyces cerevisiae* for the production of β-ionone; however, the titer achieved was only 0.22 mg/L [87]. López et al. inserted extra copies of geranylgeranyl diphosphate synthase gene and CCD1 gene from the plant *Petunia hybrid*, which enabled their *S. cerevisiae* strain to produce about 6 mg/L of β-ionone when grown in a bioreactor [90]. To date, the best-reported β-ionone strain was from our laboratory, where the engineered *E. coli* strain produced 500 mg/L of β-ionone [19] (**Table 3**).
