**2. Biosynthesis of carotenoids and apocarotenoids in nature**

example, the loveliness of yellow marigolds comes mainly from lutein, a yellow carotene; the redness of watermelons and tomatoes is because they are rich in lycopene, a red carotene; and the scarlet plumage of flamingos stems from another red carotenoid, astaxanthin. The beautiful colors of plants are also responsible for attracting insects and animals for their pollination and seed dispersal [1]. The carotenoid color originates in the structure of multiple conjugated double bonds. This unique structure enables two essential features of carotenoids: the lightharvesting capability and a powerful anti-oxidant effect by the quenching of free radicals, singlet oxygen and reactive oxygen species. In photosynthetic organisms, carotenoids are indispensable for photosynthesis and photo-protection [2]. In non-photosynthetic organisms including animals, the anti-oxidant activity not only protects cells from oxidative damages (e.g., oxidative DNA damage [3]) but can provide additional benefits for humans such as antiinflammatory and anti-cancer effect [4]. In addition, carotenoids play an important health role as pro-vitamin A compounds. About 30–50 carotenoids are believed to have vitamin A

activity including two well-known compounds: β-carotene and α-carotene [2].

symbiotic association between plants and fungi [9, 10].

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Vitamin A includes retinol, retinal and retinoic acid, which are all apocarotenoids. Apocarotenoids are a group of oxidative products of carotenoids. While carotenoids contribute to the visual beauty of flowers and fruits, apocarotenoids are famous for the pleasant aromas and give rise to fragrance and palatable flavors of many flowers and fruits (such as rose, violet, tomato and raspberry) [5–7]. These apocarotenoid aromas, in a similar manner to the colored carotenoids, attract pollinators and promote plant-insect interactions [8]. In addition, some apocarotenoids act as hormones. For example, the plant growth hormone, abscisic acid, has multiple functions in plant development processes including bud dormancy and response to environmental stress and plant pathogens [5]. Strigolactones are another important subclass of apocarotenoids, functioning as shoot-branching inhibitors and promoting the formation of

Due to the color, aroma, remarkable nutrition and health benefits, carotenoids and apocarotenoids have been widely used in food, feed, nutritional, pharmaceutical and personal care industries. The market demands for carotenoids and apocarotenoids are rising rapidly as increasing clinical research studies report various health and pharmaceutical benefits [11–13]. The global carotenoid market is projected to reach 1.53 billion USD by 2021 [14]. The regular uptake of food with a high content of carotenoids (e.g., β-carotene) or retinoids is vital to alleviate vitamin A deficiency. Vitamin A deficiency can lead to severe aftermath including blindness, decreased immune function and even death [15]. Lutein and zeaxanthin are critical for eye health by preventing age-related macular degeneration [16]. Astaxanthin has even more benefits such as potent anti-oxidant activities, promoting immune response, reducing eye fatigue, enhancing muscle performance and so on [11]. Because of low exceptional fragrance property, α-ionone and β-ionone are widely used in cosmetics such as perfumes [17]. Crocin is another valuable apocarotenoid and is responsible for the red pigmentation of saffron, a

high-value spice with retail prices ranging between 2000 and 7000 euros/kg [18].

Despite carotenoids and apocarotenoids being widely distributed in nature, their cellular contents are extremely low. For example, 100 tons of raspberries, or 20 hectares of agricultural area, could only yield 1 g of α-ionone [19]. Similarly, it requires the manual harvest of stigmas from as many as 110,000–170,000 flowers to obtain 1 kg of saffron [20], justifying the high cost of To understand how carotenoids and apocarotenoids can be produced in microbes, it is essential to elucidate the biosynthetic enzymes which constitute these metabolic pathways.

Carotenoids are a subclass of terpenoids (or isoprenoids); thus, as other terpenoids, they share the common C5 building blocks, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). In nature, there exist two independent biosynthetic pathways to produce IPP/DMAPP: the mevalonate (MVA) pathway [23] and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, also referred to as the 1-deoxy-D-xylulose 5-phosphate (DXP) or the non-MVA pathway [24].

The MEP pathway starts from the condensation of pyruvate and glyceraldehyde-3-phosphate, which are catalyzed by DXP synthase (*dxs*), to produce DXP, which is subsequently reduced into MEP by DXP reductase (*dxr*). MEP is converted into 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME) by CDPME synthase (*ispD*). CDPME is subsequently transformed into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP) through two intermediates, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDPMEP) and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC) by CDPME kinase (*ispE*), MEC synthase (*ispF*) and HMBPP synthase (*ispG*), respectively. Finally, HMBPP reductase catalyzes HMBPP into a 5-6:1 ratio of IPP and DMAPP, while IPP isomerase (*idi*) inter-converts IPP and DMAPP to adjust the ratio according to the cellular requirements (**Figure 1**).

In the MVA pathway, two molecules of acetyl-CoA are condensed into one molecule of acetoacetyl-CoA by acetyl-CoA acetyltransferase (*atoB*). Acetoacetyl-CoA is converted into mevalonate via an intermediate (S)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase and HMG-CoA reductase, respectively. IPP is produced from mevalonate by another three enzymes, mevalonate kinase (*mk*), phosphomevalonate kinase (*pmk*) and

phosphomevalonate decarboxylase (*pmd*). Thus, while the MEP pathway produces mixtures of DMAPP and IPP, the MVA pathway produces only IPP and requires *idi* to generate

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Most bacteria including cyanobacteria use exclusively the MEP pathway, whereas most eukaryotes and archaea possess only the MVA pathway. Interestingly, plants have both pathways: the MVA pathway located in the plant cytoplasm and the MEP pathway located in plastids. This is consistent with the hypothesis that chloroplasts originate from cyanobacteria endosymbionts [25]. Both pathways have been engineered to produce terpenoids including carotenoids. The MEP pathway has a higher theoretical yield than the MVA pathway [26] due to its adoption of a variety of cofactors (ATP, NADPH, CTP and flavodoxin, etc.) whereas the MVA pathway mainly uses ATP. However, in practice, it is easier to manipulate the MVA pathway and its theoretical yield has been achieved for certain products [27–30]. In contrast, the practical yield of the MEP pathway is often limited by the low activity of ispG and ispH enzymes and their special requirement of iron-sulfur cofactors. To the best of my knowledge, the highest reported yields of terpenoids synthesized by the MEP pathway in literature are

The two pathway metabolites IPP and DMAPP are condensed to give geranyl diphosphate (GPP, C10) or farnesyl diphosphate (FPP, C15), catalyzed by GPP synthase (*gpps*) or FPP synthase (*fpps*), respectively. Geranylgeranyl diphosphate (GGPP) synthase catalyzes the addition

DMAPP (**Figure 1**).

less than 20% of its theoretical yield [30].

**Figure 2.** Biochemical pathway of carotenoids and apocarotenoids.

**Figure 1.** Biosynthetic pathway of terpenoid precursors. Carotenoids are a subclass of terpenoids. In nature, two major biosynthetic pathways of terpenoids exist, one is the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and the other is the mevalonate (MVA) pathway. Abbreviations: *dxs* (DXP synthase), *dxr* (DXP reductase), *ispD* (4-diphosphocytidyl-2-C-methyl-D-erythritol, or CDPME synthase), *ispE* (CDPME kinase), *ispF* (2-C-methyl-D-erythritol-2,4-diphosphate synthase), *ispG* (1-hydroxy-2-methyl-2-(*E*)-butenyl-4-diphosphate synthase), *ispH* (1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase), *atoB* (acetoacetyl-CoA thiolase), *hmgs* (hydroxymethylglutaryl-CoA, or HMG-CoA synthase), *hmgr* (HMG-CoA reductase), *mk* (mevalonate kinase), *pmk* (phosphomevalonate kinase), *pmd* (phosphomevalonate decarboxylase), and *idi* (IPP isomerase).

phosphomevalonate decarboxylase (*pmd*). Thus, while the MEP pathway produces mixtures of DMAPP and IPP, the MVA pathway produces only IPP and requires *idi* to generate DMAPP (**Figure 1**).

Most bacteria including cyanobacteria use exclusively the MEP pathway, whereas most eukaryotes and archaea possess only the MVA pathway. Interestingly, plants have both pathways: the MVA pathway located in the plant cytoplasm and the MEP pathway located in plastids. This is consistent with the hypothesis that chloroplasts originate from cyanobacteria endosymbionts [25]. Both pathways have been engineered to produce terpenoids including carotenoids. The MEP pathway has a higher theoretical yield than the MVA pathway [26] due to its adoption of a variety of cofactors (ATP, NADPH, CTP and flavodoxin, etc.) whereas the MVA pathway mainly uses ATP. However, in practice, it is easier to manipulate the MVA pathway and its theoretical yield has been achieved for certain products [27–30]. In contrast, the practical yield of the MEP pathway is often limited by the low activity of ispG and ispH enzymes and their special requirement of iron-sulfur cofactors. To the best of my knowledge, the highest reported yields of terpenoids synthesized by the MEP pathway in literature are less than 20% of its theoretical yield [30].

The two pathway metabolites IPP and DMAPP are condensed to give geranyl diphosphate (GPP, C10) or farnesyl diphosphate (FPP, C15), catalyzed by GPP synthase (*gpps*) or FPP synthase (*fpps*), respectively. Geranylgeranyl diphosphate (GGPP) synthase catalyzes the addition

**Figure 2.** Biochemical pathway of carotenoids and apocarotenoids.

**Figure 1.** Biosynthetic pathway of terpenoid precursors. Carotenoids are a subclass of terpenoids. In nature, two major biosynthetic pathways of terpenoids exist, one is the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and the other is the mevalonate (MVA) pathway. Abbreviations: *dxs* (DXP synthase), *dxr* (DXP reductase), *ispD* (4-diphosphocytidyl-2-C-methyl-D-erythritol, or CDPME synthase), *ispE* (CDPME kinase), *ispF* (2-C-methyl-D-erythritol-2,4-diphosphate synthase), *ispG* (1-hydroxy-2-methyl-2-(*E*)-butenyl-4-diphosphate synthase), *ispH* (1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase), *atoB* (acetoacetyl-CoA thiolase), *hmgs* (hydroxymethylglutaryl-CoA, or HMG-CoA synthase), *hmgr* (HMG-CoA reductase), *mk* (mevalonate kinase), *pmk* (phosphomevalonate kinase), *pmd* (phosphomevalonate decarboxylase), and *idi* (IPP isomerase).

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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**.

dry cell weight (DCW) of lycopene [40] and more recently reconstituted the MVA pathway in *E. coli* to produce lycopene in a glucose-defined medium, reaching 1.5 g/L in a simple nonoptimized fed-batch process [19]. Xie et al. evolved the bifunctional enzyme crtYB to acquire only the phytoene synthase function. By applying this mutated enzyme and optimizing the copy number of crt genes, the engineered *Saccharomyces cerevisiae* strain produced 1.61 g/L of lycopene [41]. In addition, *Yarrowia lipolytica*, the oleaginous yeast, has also been engineered

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Moving further along the carotenoid biosynthetic pathway, lycopene is usually cyclized into β-carotene or α-carotene by a lycopene cyclase (**Figure 2**). Both α- and β-carotenes are yellow pigments; β-carotene is more commonly marketed, being one of the most important commercial carotenoids. As mentioned earlier, β-carotene is a direct precursor of vitamin A (**Figure 2**). It has been widely used as a colorant, nutritional supplement, animal feed and in pharmaceutical and personal care industries. Chemically synthesized β-carotene is less popular among consumers than that extracted from natural sources or so-called 'bio-based' sources. At the same time, naturally derived β-carotene has gradually taken over the market. Currently, β-carotene is produced mainly in the microalga *Dunaliella* [21] and the fungus *Blakeslea trispora* [2]. As summarized in **Table 1**, many groups have engineered fast-growing microorganisms and achieved high titers of β-carotene. Yang et al. have applied a hybrid MVA pathway in *E. coli* to overproduce β-carotene at 3.2 g/L [44]. Zhao et al. have engineered the central metabolic pathway to increase cofactor supply in an *E. coli* strain, which enabled the strain to produce at 2.1 g/L of β-carotene [45]. *Y. lipolytica* has shown potential as a better host for producing β-carotene; 4 g/L of β-carotene was achieved in an *Y. lipolytica* strain by integrating multiple copies of key enzymes (hmgr in **Figure 1** and the bi-functional enzymes phytoene synthase/ lycopene cyclase carRP) [46]. Recently, based on an engineered lipid overproducing strain of *Y. lipolytica*, Larroude et al. have rewired it to produce at 6.5 g/L and 90 mg/g DCW of β-carotene [47]. These results are relatively better than those previously achieved [48–50]. It would not be surprising if some of these examples would lead to the successful commercial-

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

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

such as keto groups in the β-ionone rings can also quench singlet oxygen resides [31].

to produce lycopene but at slightly lower yields [42, 43].

ization notably of novel β-carotene sources in the near future.

**3.2. Biosynthesis of xanthophylls**
