What Are the Sources of Carotenoids?

Carotenoids, due to their antioxidant and anticancer properties, have been widely applied in the pharmaceutical, healthcare, food, and cosmetics industries [1]. Their primary sources include microalgae, higher plants, microorganisms, and certain animals. Since the types of carotenoids present in each type of algae or plant vary significantly, different extraction methods must be employed to isolate various carotenoids from different algae or plants. Astaxanthin and β-carotene extracted from microalgae have already been commercialised. In recent years, the demand for carotenoids has significantly increased. According to statistics, the compound annual growth rate (CAGR) of the carotenoid market value from 2016 to 2021 was 3.5%; by 2021, the global production value could reach 1.52 billion USD [2].

Economic development has improved people's living standards, leading to greater emphasis on nutrition and health, and increased demand for carotenoids. Additionally, compared to chemically synthesised carotenoids, naturally synthesised carotenoids are more favoured. However, due to the low carotenoid content in algae and plants, even with the continuous development of extraction technologies, it is still difficult to meet the growing demand for natural carotenoids. The continuous advancement of synthetic biology technologies has significantly promoted the synthesis of carotenoids and de-auxiliary carotenoids in microbial host cells [3]. The strong hydrophobic nature of carotenoids makes them easily embedded in phospholipid bilayer membranes, which poses significant challenges for host cells and, to some extent, limits the selection of suitable host cells for carotenoid synthesis [4]. With the further elucidation of the carotenoid synthesis pathway, most of the enzymes and regulatory genes involved in the catalytic reactions have been identified. This provides a theoretical basis for the construction of carotenoid-producing cell factories and also provides scientific support for their widespread application in industries such as medicine, cosmetics, and food.

1 Sources and biological functions of carotenoids

Carotenoids are diverse and widely distributed, with carotenoids found in the leaves and petals of higher plants, microorganisms, and algae [5]. Most carotenoid molecules have a C40 conjugated double bond polyene chain and a terminal carbon ring in their molecular skeleton, and the characteristics of each carotenoid are determined by the types of aromatic rings and oxygen-containing functional groups [6]. Based on chemical structural characteristics and the presence of functional groups, carotenoids can be further classified into carotenes and xanthophylls. Carotenoids not only regulate plant growth and development but also have close relationships with human nutrition and health [7]. They are natural antioxidants with preventive and disease-alleviating functions [8] and serve as precursors for the biosynthesis of vitamin A (retinol).

1.1 Sources of carotenoids

Natural carotenoids are primarily obtained from algae, plants, and microbial fermentation. In algae, Haematococcus pluvialis and Dunaliella salina can synthesise astaxanthin and β-carotene, respectively; Haematococcus pluvialis has the strongest ability to synthesise astaxanthin, with astaxanthin accounting for approximately 90% of the total carotenoids in the cell, and its weight can reach about 7% of the dry cell weight [9]. Bacteria and fungi can also synthesise carotenoids, such as Erwinia and Red Fife yeast [10]. The roots, stems, leaves, and petals of higher plants can also synthesise various carotenoids. Due to the natural carotenoid synthesis pathways in algae, microorganisms, and plant systems, they are ideal candidates for carotenoid synthesis cell factories.

Astaxanthin belongs to the ketocarotenoid class and is widely found in algae, fungi, and bacteria. It has three optical isomers: levo-astaxanthin, dextro-astaxanthin, and all-trans-astaxanthin. Different isomers exhibit variations in antioxidant activity [11]. β-carotene is widely distributed in nature, with two β-carotenoid rings at its terminal end [12]. It primarily exists in four isomeric forms, and the main difference between naturally synthesised and chemically synthesised β-carotene lies in the proportion of all-trans and cis isomers [13]. Lutein is primarily found in the leaves and flowers of green plants [14]. Its chemical molecular structure includes two ketone rings and three chiral centres, with eight isomers coexisting in nature. It primarily participates in capturing light energy, regulating plant growth and development, etc.[15]. Most algae contain lutein, such as Chlorella, Chlorella vulgaris, and Chlorella vulgaris. Among these, Chlorella has the highest content and is an advantageous algal strain for lutein production [16].

1.2 Biological Functions of Carotenoids

Carotenoids, due to their strong antioxidant properties, play a very important role in antioxidant defence (Table 1). Studies have shown that adding 250 mg/kg body weight of lutein not only effectively reduces radiation-induced oxidative damage in albino mice but also helps maintain the stability of their antioxidant system [17]. Additionally, different carotenoids exhibit significant variations in antioxidant potency, and when combined in specific concentration ratios, they exhibit synergistic effects in antioxidant activity. For example, when the concentration ratio of astaxanthin to β-carotene is 1:1, their synergistic antioxidant effect is strongest [18]; when the mass ratio of zeaxanthin to lutein is 2:1, their synergistic antioxidant effect is strongest [19].

Lutein and zeaxanthin are important components of the macular pigment in the human cornea, protecting the retina from blue light damage and enhancing visual acuity [20]. Therefore, lutein is commonly used in eye health supplements to prevent and alleviate age-related macular degeneration, cataracts, and retinal neural diseases [21]. When the intake of lutein and zeaxanthin is insufficient, the risk of macular degeneration increases [22]. Among carotenoids, astaxanthin and canthaxanthin exhibit better anti-cancer effects. Studies have shown that astaxanthin significantly reduces cancer incidence, inhibits malignant proliferation and metastasis of cancer cells, and reduces tumour weight and size [23]. Astaxanthin exhibits even higher anticancer activity. Studies indicate that astaxanthin-induced cell apoptosis is associated with reactive oxygen species (ROS), and ROS-induced cellular toxicity leads to the catalytic cleavage of caspase-3 and -9 [24]. Studies on prostate cancer have found that fucoxanthin and its metabolite fucoxanthinol can inhibit cell growth, induce apoptosis in prostate cancer PC-3 cells, and activate caspase-3 [25]. Fucoxanthin and fucoxanthinol can also induce tumour cell cycle arrest by regulating the expression of various molecules and signal transduction pathways [26].

2. Biosynthesis of carotenoids

2.1  Carotenoid biosynthesis

The synthesis pathways of carotenoids in plants have been studied the most extensively. In recent years, scientists have conducted in-depth analyses of the synthesis pathways in microorganisms and algae, revealing that different organisms have distinct synthesis pathways, resulting in varying types and yields of carotenoids [28]. The enzymes required for carotenoid synthesis differ between plants and microorganisms, with more specialized functions in plants. For example, the enzymes responsible for catalysing the synthesis of octahydrolycopene and the cyclisation of lycopene are carried out by two separate enzymes in plants, whereas in yeast and moulds, this process is completed by a single enzyme [29-30].

Although microalgae are classified as lower plants, they possess characteristics of both higher plants and microorganisms, enabling them to synthesise a wide variety of carotenoids. They can synthesise α-carotene and lutein, which are unique to higher plants, as well as carotenoids such as astaxanthin and canthaxanthin, which are commonly found in microorganisms. Therefore, microalgae have unique advantages for use as host cells for carotenoid synthesis[31]. The elucidation of the carotenoid synthesis pathway provides a theoretical basis for the construction of carotenoid synthesis cell factories. The synthesis begins with the precursor compound geranylgeranyl pyrophosphate (GGPP). The following section briefly describes the carotenoid synthesis process using higher plants as an example (Figure 2).

2.1.1 The biosynthetic pathway of GGPP

The biosynthesis of GGPP is a crucial step in carotenoid synthesis, and its synthesis process can be simply divided into two major steps: the synthesis of the precursor isopentenyl diphosphate (IPP) and the synthesis of dimethylallyl diphosphate (DMAPP) from IPP. Depending on the location of synthesis, the synthesis pathway of IPP can be further divided into the mevalonate pathway and the DMAPP pathway. DMAPP); and the synthesis of GGPP from the precursor IPP and DMAPP. Depending on the location where synthesis occurs, the IPP synthesis pathway is further divided into the mevalonic acid (MVA) pathway [32] and the methyl erythritol phosphate (MEP) pathway [33], both of which are compartmentalised. Among these, the MVA pathway is primarily found in the cytoplasmic matrix and endoplasmic reticulum of most mammals and yeast cells, with acetyl coenzyme A as the starting material; the MEP pathway is generally present in the protoplasts of higher plants, some bacteria, and algae [34], with 3-phosphoglycerate (GA-3-P) and pyruvate as the starting materials [35]. After the formation of IPP and DMAPP, the catalytic steps of the MVA and MEP pathways are largely identical.

2.1.2 Synthesis of Carotenoids from GGPP

Starting from GGPP, enzymes involved in the synthesis of various carotenoids include oxidoreductases (EC1) such as PDS (phytoene desaturase) and ZDS (ζ-carotene desaturase), transferase enzymes (EC2) such as PSY (phytoene synthase), and isomerase enzymes (EC5) such as LCYe (lycopene ε-cyclase) and LCYb (lycopene β-cyclase), among others.

The main process is as follows: first, GGPP is catalysed by PSY to synthesise phytoene, and other carotenoids are further derived from phytoene through dehydrogenation and cyclisation. PSY is the key rate-limiting enzyme in this pathway, with its encoding genes being CrtB in bacteria and PSY in eukaryotes. Modulating its expression level or activity can regulate the metabolic pathway flux [36]. For example, in oilseed rape and potato-derived callus tissues, overexpression of constitutive PSY increases total carotenoid content in cells, and β-carotene synthesis is also significantly enhanced [37].

Since PSY is a single-copy gene in most plants, it is an ideal target for improving carotenoid content in plants using genetic engineering techniques [38]. Secondly, octahydrolycopene is converted into ζ-carotenoid under the catalysis of PDS, and ζ-carotenoid is further converted into lycopene under the catalysis of ZDS. Gao et al. [39] found that white light can inhibit the expression of CpPDS and CpZDS in grapefruit (Citrus paradisi) callus, thereby reducing lycopene synthesis. Qin et al. [40] found that after mutation of the AtPDS3 gene in the carotenoid synthesis pathway of Arabidopsis, the expression levels of genes such as AtPSY and AtZDS significantly decreased, leading to impaired carotenoid synthesis and inhibition of the chlorophyll and gibberellin synthesis pathways.

Lycopene can be converted into different carotenoids under the catalysis of different enzymes: under the catalysis of CrtE, it can be cyclised to form δ-carotene, which is further converted into ε-carotene; under the catalysis of CrtY, it can be converted into γ-carotene, which is further converted into β-carotene. Additionally, CrtB can catalyse the conversion of δ-carotene into α-carotene. The carotenoid types synthesised from GGPP are extremely diverse, constituting an important component of the natural carotenoid synthesis pathway. A thorough understanding of this pathway will provide theoretical foundations for the design, modification, and application of carotenoid biosynthesis pathways.

2.2 Synthesis from carotenoids to xanthophylls

The metabolic pathway for synthesising xanthophyll pigments from carotenoids requires five types of oxidoreductases, including LUT1 (carotenoid ε-hydroxylase), CrtZ (β-carotene 3-hydroxylase), LUT5 (β-ring hydroxylase), ZEP (zeaxanthin epoxidase), and VDE (violaxanthin deepoxidase). After undergoing consecutive hydroxylation reactions, β-carotene first forms β-cryptoxanthin, which is then converted into zeaxanthin.

Among these, the process by which zeaxanthin undergoes ring-opening to form flavoquinone, which is then further converted into violaxanthin, is reversible; the enzymes catalysing the forward two-step reaction (i.e., the cyclisation reaction) are all ZEP, and the reaction occurs under weak light or dark conditions. In Arabidopsis, the gene encoding this enzyme is AtABA1; the enzymes catalysing the reverse two-step reaction (i.e., the decyclisation reaction) are all ZEP, and the reaction occurs under strong light conditions; in Arabidopsis, the gene encoding this enzyme is AtNPQ1, and the entire cycle is referred to as the lutein cycle (Lutein cycle) [41]. Currently, the catalytic enzymes involved in each reaction step have been identified, especially in the higher plant Arabidopsis (Table 2). The study of the carotenoid to lutein synthesis pathway can be used for directed evolution or stress response methods to synthesise specific types of carotenoids.

3 Construction of carotenoid synthesis cell factories and synthetic biology strategies

The biosynthesis pathway of carotenoids can be divided into upstream and downstream pathways, with the most basic IPP/DMAPP as the node. The upstream pathway involves the synthesis of IPP and DMAPP, which can be achieved through two pathways: MEP and MVA. The downstream pathway starts from IPP and DMAPP, undergoes multiple reactions and modifications, and ultimately synthesises various carotenoids and their derivatives.

The construction of a carotenoid synthesis cell factory is a complex process involving the assembly and adaptation of multiple modules. This not only requires the selection of appropriate catalytic components based on the target product but also, in some cases, the enhancement of NADPH and ATP synthesis, increased supply of GGPP precursors, or the introduction of an exogenous MVA pathway to alleviate feedback inhibition effects of metabolic intermediates [42]. The catalytic components required for carotenoid synthesis pathways include various enzymes that catalyse the chemical reactions of the pathway, such as synthases, dehydrogenases, cyclases, hydroxylases, and ketolases. To increase carotenoid yield, it is necessary to maximise the metabolic flux from substrates to target products in the host cells while minimising the production of non-essential by-products or metabolic intermediates. Therefore, it is necessary to select optimal host cells and catalytic components and combine them optimally from multiple dimensions, including catalytic properties, expression levels, and host adaptability.

3.1 Selection and modification of carotenoid synthesis host cells

The continuous development of synthetic biology technologies has significantly advanced the efficient synthesis of carotenoids and their derivatives in chassis cells such as Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica (Table 3). Most carotenoids exhibit strong hydrophobicity, leading to significant damage to cell membrane structures and impairing normal cellular physiological functions after synthesis within cells [44]. Additionally, the limited membrane structures in microbial chassis cells also restrict the potential for increasing carotenoid yields. Furthermore, the strong reducing properties of carotenoids can trigger stress responses in chassis cells, leading to a significant increase in intracellular reactive oxygen species (ROS) levels and feedback inhibition of cell growth [45].

Therefore, employing inducible promoters to decouple the growth and production of production strains [46], creating engineered transporters and membrane vesicle transport systems, can promote carotenoid efflux, alleviate membrane system stress [47], and reduce the feedback inhibition effect on carotenoid synthesis. The complex internal environment of chassis cells determines that the synthesis of target products is inevitably influenced by various intracellular factors. In particular, endogenous non-essential genes significantly influence carotenoid synthesis capacity [48]. Regulating, designing, and modifying non-essential genes in the host cell can enhance the compatibility between exogenous expression modules and their internal environment, improve cellular tolerance, and strengthen metabolic flux in the target pathway.

However, considering the limited number of non-essential genes that can be rationally designed and their limited impact on the internal environment, non-rational design strategies such as random mutagenesis are needed to increase genetic and phenotypic diversity, thereby accelerating the laboratory evolution of strains [49].

Plant chassis systems, which are more closely aligned with the natural hosts of products in terms of protein expression, post-translational modification, and catalytic environment, have garnered increasing attention from researchers in recent years. Currently, researchers can use tobacco, tomato, and rice as chassis cells to produce carotenoids such as lycopene [50]. For example, Professor Liu Yaoguang's team introduced the carotenoid synthesis pathway into rice endosperm, resulting in a new rice variety rich in various carotenoids [51]. Additionally, Chlamydomonas reinhardtii and Synechocystis, which possess natural carotenoid synthesis pathways, are also ideal plant chassis cells [52].

3.2 Modular assembly and adaptation of the carotenoid synthesis pathway

The construction of carotenoid cell factories involves the assembly of multiple modules, as well as the combination and adaptation of factors such as catalytic performance and expression levels between pathway modules. The ultimate goal is to maximise the metabolic flux from substrate to target product while minimising the accumulation of non-essential by-products and metabolic intermediates [53]. The rate-limiting enzymes in carotenoid synthesis include CrtE, CrtI, CrtZ, and CrtW, which exhibit relatively broad substrate specificity and can catalyse multiple consecutive reactions. However, rate-limiting enzymes from different sources may require different numbers of reaction steps when catalysing consecutive reactions, significantly affecting the proportion of the target compound in total carotenoid content [54]. Additionally, differences in substrate selectivity among catalytic components can affect the conversion rates of metabolic intermediates [55]. Therefore, screening and combining catalytic components from different sources is an effective strategy to enhance carotenoid synthesis flux and reduce the accumulation of metabolic intermediates [56].

Furthermore, adjusting module expression levels can also enhance overall metabolic flux and weaken rate-limiting steps [57]. When modulating module expression intensity, factors such as promoter strength, copy number, and the integration position of the module on the chromosome can be altered. Typically, modules can be cloned into different plasmids for expression, facilitating the rapid establishment of expression libraries with diverse expression intensities and enabling the adjustment of expression levels for different modules. Furthermore, by combining different promoter strengths and adjusting the replication origin of the plasmid, the diversity of the library can be increased, and the dynamic range of module expression intensity can be expanded [58]. To achieve stable expression of carotenoid synthesis pathway gene modules, the chassis genome integration approach can be adopted. The insertion position and copy number of expression modules on the chromosome significantly influence the overall expression level of the modules and the flux of the carotenoid synthesis pathway.

In the construction of carotenoid cell factories, to achieve optimal compatibility between modules, it is necessary to screen various factors such as the catalytic performance of the catalytic elements, gene copy number, expression levels, and the integration position and arrangement order of the elements on the chromosome. This requires the construction of a sufficiently large library to meet the required coverage. Modular metabolic engineering (MME) can cluster and group catalytic units involved in metabolic pathways, treating each group of catalytic units as a module [59]. This method only involves balancing the expression levels between modules, which greatly reduces the complexity of carotenoid cell factory construction.

4 Summary and Outlook

Carotenoids, with their vibrant colours and important biological functions, are widely used in the pharmaceutical, food, and health industries and have high commercial value. In recent years, the demand for carotenoids has been steadily increasing. Currently, the chemical total synthesis technology for carotenoids is mature and serves as the primary production source; however, its edible safety remains uncertain. Therefore, constructing carotenoid-synthesising cell factories to produce related products has garnered increasing attention. To maximise the production capacity of carotenoid synthetic cell factories, it is necessary to optimise their design and regulation. To effectively address issues such as metabolic imbalance and intermediate accumulation, it is essential to construct regulatory elements, design gene circuits to precisely regulate material and energy flows, and leverage high-throughput screening, enzyme design, computer simulation, model analysis, and coupled gene control elements.

The continuous development of synthetic biology technologies has brought new opportunities for the construction of carotenoid cell factories. This not only enables the modularisation of carotenoid synthesis-related components in engineering but also endows them with highly favourable biological characteristics. This provides more possibilities for integrating relevant functional components to construct biological systems with specific biological functions and achieve large-scale design, development, modification, and application. The carotenoid synthesis metabolic pathways obtained in this way not only exhibit better predictability but also simplify the modification process and enhance the efficiency of traditional metabolic engineering. Additionally, computer-aided design and deep learning can accelerate and optimise metabolic pathway design and process construction. By leveraging a continuous design, construction, testing, and learning model, it is expected to achieve the desired effects of the target process in advance, which will facilitate the development of more efficient and stable artificial synthetic cell factories. The interdisciplinary integration of multiple fields will undoubtedly drive the construction of carotenoid synthesis cell factories toward high-throughput, intelligent, and efficient directions.

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