How to Synthesise Beta Carotene?

Carotenoids are orange-coloured carotenoids primarily found in fruits, vegetables, and algae. β-carotene, as a member of the tetrapterene family, possesses significant biological value, serving as an antioxidant to enhance human immunity and exhibiting anti-cancer properties [1]. It is also a precursor to vitamin A and has numerous applications in pharmaceuticals, nutritional supplements, cosmetics, and food [2]. With the continuous improvement of people's health awareness, the market value of nutritional supplements is gradually increasing, and the market prospects are promising. Carotenoids have garnered widespread attention, particularly β-carotene, astaxanthin, lycopene, and lutein. According to forecasts, the global carotenoid market size is expected to grow from 2 billion US dollars in 2022 to 27 billion US dollars by 2027, with a compound annual growth rate (CAGR) of 5.7%.

Currently, the production of β-carotene primarily relies on natural extraction, chemical synthesis, and microbial synthesis [3]. Natural extraction generally involves extracting carotenoids from plants, vegetables, and algae, with challenging purification processes and low yields. Chemical synthesis faces issues such as multi-step reactions, environmentally unfriendly processes, and the production of by-products and harmful substances. Microbial synthesis offers advantages such as high product yield, no by-product formation, low production costs, mild production conditions, reduced labour requirements, and environmentally friendly processes. Therefore, the construction of microbial cell factories for the heterologous synthesis of β-carotene has increasingly attracted the attention of researchers.

Due to its numerous biological functions, the market demand for β-carotene is growing rapidly, necessitating the development of new biotechnological production platforms. Research on synthesising β-carotene using synthetic biology has made significant progress, such as innovative metabolic engineering strategies, optimisation of fermentation conditions, and diversification of chassis cell selection, which have significantly increased the yield of β-carotene. However, due to limitations in biotechnology, there is still a considerable gap before industrial-scale production can be achieved. Therefore, this paper reviews the physicochemical properties, biosynthetic pathways, and current research status of β-carotene, systematically summarises metabolic engineering strategies for synthesising β-carotene, and identifies the challenges and future research directions for producing β-carotene using synthetic biology technologies. This study aims to provide a reference for constructing microbial cell factories for β-carotene and other natural products.

1 Physical and Chemical Properties and Functions

β-carotene is a isoprenoid compound, a liposoluble carotenoid found in plants and microorganisms, with the chemical formula C40H56, with a molecular weight of 536.88 and a melting point of approximately 178°C. β-carotene is a tetrapterone compound composed of eight isoprene units and two β-carotenoid rings, with its molecule consisting entirely of carbon and hydrogen atoms, and the core structure containing 40 carbon atoms.

In nature, most β-carotene exists in the all-trans form, with a small proportion in the cis structure, as shown in Figure 1. β-Carotene exhibits lipophilicity and high hydrophobicity due to its conjugated double bonds and central symmetry [4]. β-Carotene has different solubilities in various solvents, being easily soluble in organic solvents such as chloroform and acetone, but insoluble in water. This substance is unstable to light and heat, prone to decomposition, and requires storage at low temperatures away from light [5]. During the extraction of β-carotene, antioxidants such as vitamin C or 2,6-di-tert-butyl-p-cresol are often added to prevent oxidation and decomposition, thereby improving stability.

β-carotene has multiple preventive and therapeutic effects on diseases and is beneficial to human health. Firstly, β-carotene has a preventive effect against cancer. Research has shown that there is a significant association between β-carotene intake and the risk of lung cancer, meaning that higher intake of β-carotene helps reduce the risk of lung cancer [6]. Secondly, β-carotene also has the function of preventing cardiovascular diseases by inhibiting the ability of macrophages to oxidatively modify low-density lipoproteins, thereby reducing the risk of atherosclerosis and decreasing the incidence of cardiovascular diseases and related deaths [7].

Worth noting, beta-carotene, as a potent antioxidant, can eliminate oxygen free radicals in the human body and possesses highly efficient singlet oxygen quenching ability. Additionally, as a provitamin A compound, β-carotene is an important source of vitamin A, playing a crucial role in cell differentiation, embryonic development, and the prevention of dry eye disease. It also helps strengthen the immune system and enhance resistance to infections [8]. The potential health benefits of β-carotene continue to be explored, and the development and utilisation of functional foods rich in β-carotene are increasingly prevalent [9]. Therefore, the development of microbial cell factories for the efficient synthesis of β-carotene through biotechnological methods holds significant market application value.

2 β-Carotene Biosynthetic Pathway

Carotenoids are tetrapteroid compounds with an isopentenyl diphosphate (IPP) skeleton. The biosynthesis of β-carotene is part of the carotenoid biosynthetic pathway. IPP and dimethylallyl diphosphate (DMAPP) are the initial structural units for the synthesis of lycopene, β-carotene, and other carotenoids [10]. The synthesis of IPP and DMAPP primarily occurs through two pathways: the isoprenylation pathway and the isoprenyl-diphosphate pathway (IDP).  DMAPP) are the initial structural units for the synthesis of lycopene, β-carotene, and other carotenoids [10]. The synthesis of IPP and DMAPP primarily originates from two pathways: the mevalonic acid (MVA) pathway in the cytoplasm and the methyl erythritol phosphate (MEP) pathway in the plastids. The biosynthetic pathway of β-carotene can be broadly divided into upstream and downstream components. The upstream biosynthetic pathway involves the utilisation of the MVA and MEP pathways to obtain the five-carbon precursor IPP, forming the IPP biosynthetic module. The downstream pathway involves the conversion of the five-carbon precursor into β-carotene, forming the β-carotene biosynthetic module (Figure 2).

2.1 IPP Biosynthetic Module

The MVA pathway is present in most eukaryotes, archaea, and higher plants. It uses acetyl CoA produced by glycolysis as the initial substrate. Two molecules of acetyl CoA are converted into acetyl-CoA (acetoacetyl-CoA) by acetyl-CoA thiolase (AACT), and then acetyl-CoA is reduced to AACoA by acetyl-CoA reductase (ACAR). AACoA is then oxidised to acetyl-CoA by acetyl-CoA oxidase (ACO), and the final product is MVA.  AACoA), which is then condensed by hydroxymethylglutaryl-CoA synthase (HMGS) to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Finally, HMG-CoA is reduced to MVA by hydroxymethylglutaryl-CoA reductase (HMGR), an irreversible reaction that constitutes the main rate-limiting step of the MVA pathway [11]. Subsequently, under the sequential catalysis of multiple enzymes, MVA is converted into IPP through phosphorylation and decarboxylation. IPP can be isomerised into DMAPP by isopentenyl diphosphate isomerase (IDI).

The MEP pathway, present in many bacteria, algae, and plants, uses pyruvate and glycerol-3-phosphate as substrates. Under the catalysis of 1-deoxy-D-xylulose-5-phosphate synthase (DXS), DXS) to form DXP. Then, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) converts DXP into MEP, which is further converted into IPP and DMAPP by multiple enzymes. DXS and IDI are considered the rate-limiting enzymes in the isopentenylation pathway. enhancing the activity of these enzymes can increase β-carotene yield [12]. In addition to carotenoid synthesis, IPP and DMAPP are also precursors for the synthesis of many important drugs, such as artemisinin, oleanolic acid, and squalene.

2.2 β-Carotene Biosynthesis Module

In the β-carotene biosynthesis module, IPP undergoes condensation and cyclisation under the biocatalysis of a series of enzymes to form β-carotene. The synthesis of β-carotene primarily occurs in the chloroplasts of algae or the cytoplasm of microorganisms. Specifically, IPP and DMAPP molecules produced by the MVA and MEP pathways undergo consecutive enzymatic reactions to sequentially condense and form geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP).  FPP), and geranylgeranyl pyrophosphate (GGPP).

Two molecules of GGPP are first converted into phytoene by phytoene synthase (CrtB), and then further converted into lycopene by phytoene desaturase (CrtI). The formed lycopene can be cyclised into β-carotene under the catalysis of lycopene β-cyclase (CrtY), or serve as a precursor for the synthesis of other carotenoids. In studies, CrtYB was found to be a bifunctional enzyme capable of both converting GGPP into octahydrolycopene and cyclising lycopene into β-carotene [13]. CarRP from Mucor circinelloides is also a protein with two distinct enzymatic activities, simultaneously exhibiting the catalytic activity of octahydrolycopene synthase and lycopene cyclase [14]. Additionally, IWASAKA discovered a trifunctional enzyme, CrtIBY, in Aurantiochytrium sp. Strain KH105, which can directly convert GGPP into β-carotene [15].

3 Recent Advances in Microbial Synthesis of β-Carotene

Due to the potential commercial value of β-carotene, the construction of microbial cell factories for β-carotene production has garnered increasing attention. Using molecular biological techniques, multiple key genes of the β-carotene synthesis pathway have been overexpressed, significantly increasing β-carotene accumulation in bacteria, yeast, and algae. Currently, the main chassis microorganisms used for β-carotene production include Escherichia coli, Saccharomyces cerevisiae, and Lipolytikus brevis, among others, with some achieving considerable yields. Specific modification strategies and yields are listed in Table 1.

Escherichia coli is a widely used model microorganism. Due to its clear genetic background, simple genetic manipulation, and rapid growth rate, Escherichia coli has gradually become one of the most commonly used chassis cells for constructing microbial cell factories, making it an ideal host cell. Dai et al. utilized an RBS library to regulate the expression of key genes dxs, idi, and crt in the β-carotene synthesis pathway, thereby increasing β-carotene production. The combined regulation of the crt operon, dxs, and idi genes resulted in a 35% increase in β-carotene production [16]. The pentose precursor IPP and cofactors are two important factors in the production of terpenoids. Therefore, by overexpressing the rate-limiting enzyme DXS in the MEP pathway in Escherichia coli to increase the carbon flux of IPP, the yield of carotenoids increased by 3.5-fold [17].

Zhao et al. integrated β-carotene synthesis genes from Pantoea agglomerans into the Escherichia coli genome, then designed central metabolic modules to increase the supply of precursors (IPP and DMAPP) and cofactors (ATP and NADPH), thereby enhancing β-carotene production, followed by fed-batch fermentation, resulting in β-carotene production of 2.1 g/L [18]. The MVA pathway holds significant potential for the synthesis of isoprenoid compounds. The heterologous expression of the complete MVA pathway and β-carotene synthesis genes in engineered Escherichia coli increased β-carotene production to 465 mg/L [19]. In engineered Escherichia coli, Yang simultaneously overexpressed the MEP and MVA pathways to increase intracellular IPP and GPP concentrations, resulting in a significant increase in β-carotene production, with a final fermentation yield of 3.2 g/L [20].

Baker's yeast is widely recognised as a safe yeast strain due to its simplicity in genetic manipulation and tolerance to production conditions, making it widely used in industrial production as a host for synthesising various high-value-added compounds. Although Saccharomyces cerevisiae does not naturally produce β-carotene, it does synthesise the important precursor compound carotene-1,2,3,4,5,6,7,8,8,9,10-decahydro-1,10-dihydroxy-10-methoxy-9,10-dihydro-1,10-dihydroxy-1,10-dihydroxy-1,10-dihydroxy-1,10-dihydroxy-1,10-dihydroxy-1,10-dihydroxy-1,10-dihydroxy-1,10-dihydroxy-1,10-dihydroxy-1,10-d

YAMANO et al. first reported the heterologous production of β-carotene in Saccharomyces cerevisiae. They achieved the production of lycopene and β-carotene by expressing the crtE, crtB, crtI, and crtY genes from Pantoea ananatis; however, the yield was very low, at only 103 μg/g [21]. To further increase β-carotene yield, VERWAAL et al. regulated the expression of key genes and preliminarily established a β-carotene cell factory in Saccharomyces cerevisiae. They overexpressed GGPP synthase and tHMG1, and the recombinant Saccharomyces cerevisiae ultimately produced 5.9 mg/g of β-carotene [22]. Maintaining metabolic flux balance and controllable gene expression during biosynthesis are critical for the efficient production of high-value-added chemicals. Xie et al. took FPP, a key metabolic intermediate in the β-carotene biosynthetic pathway, as a node for ordered regulation, adjusting downstream flux, enhancing precursor supply, and inhibiting competitive pathways, achieving β-carotene production of 376 mg/L through fermentation [23].

Fan et al. combined multiple metabolic strategies to optimise the carbon metabolism pathway, enhance the supply of precursor acetyl CoA, weaken the ergosterol synthesis pathway, and increase β-carotene storage capacity. They also optimised the copper ion-induced GAL system to enhance the biomass of Saccharomyces cerevisiae. After multiple strategy modifications, the β-carotene titer reached approximately 166 mg/L, which is five times higher than that of the parental strain before modification [24].

Lipolytically modified yeast is a promising non-model fungus that cannot naturally synthesise β-carotene but can produce large amounts of acetyl CoA precursors and accumulate significant lipid content. Since β-carotene is a lipophilic compound, lipolytically modified yeast may serve as an excellent host for the overproduction of β-carotene. Currently, various genetic manipulation tools have been developed and utilised, with an increasing number of researchers conducting engineering modifications. Jing et al. first overexpressed exogenous genes in Lipolytococcus yeast, constructed a β-carotene biosynthetic pathway, and eliminated the rate-limiting steps in the MVA pathway through metabolic engineering. Additionally, they further enhanced the lipid synthesis capacity of the engineered strain and increased the copy number of key genes in the biosynthetic pathway, obtaining an engineered strain capable of efficiently synthesising β-carotene. Fed-batch fermentation yielded 2.7 g/L of β-carotene. The results indicate that increased intracellular lipids promote β-carotene synthesis, and the expression of multiple copies of key genes is an important strategy for enhancing metabolic flux [25].

Gao used a strong promoter and overexpressed the β-carotene biosynthetic pathway, resulting in β-carotene yields over 100 times higher than the wild-type strain. Using an optimised medium for fed-batch fermentation, 4 g/L of β-carotene was produced. This study indicates that Lipolysichlorella is an ideal host for heterologous synthesis of β-carotene [26]. LARROUDE combined traditional metabolic engineering strategies with novel synthetic biology tools to enhance lipid synthesis and gene copy number, and used optimal promoters to increase metabolic flux toward the MVA pathway, significantly improving β-carotene yield. Batch fed-batch fermentation yielded a total β-carotene production of 6.5 g/L [27]. The integration of metabolic engineering strategies with fed-batch fermentation processes provides a new approach for the synthesis of carotenoids and other terpenoids using lipase-degrading yeast.

4 Optimisation Strategies

With the continuous innovation and development of biotechnology, β-carotene can be rapidly and conveniently heterologously synthesised in microorganisms such as Lipolytikus yeasts and Escherichia coli through metabolic engineering and synthetic biology technologies, leading to continuous breakthroughs in β-carotene production. To further explore methods to enhance β-carotene production and achieve higher economic benefits, innovative metabolic engineering strategies are still required. Currently, several strategies have been identified to enhance β-carotene production, primarily focusing on NADPH and ATP supply, acetyl CoA precursor supply, suppression of competitive pathways, cellular compartmentalization, and enhanced lipid synthesis (Figure 3).

4.1 NADPH and ATP Supply

ATP and NADPH are two important cofactors for the production of terpenoids. The levels of these cofactors within cells determine metabolic flux, as they participate in enzymatic reactions and regulate chemical equilibrium states. Therefore, regulating the levels of intracellular cofactors can increase the metabolic flux toward β-carotene synthesis. NADPH is the primary provider of reducing equivalents required for biosynthetic metabolism and an essential reducing factor protecting cells from oxidative stress. Additionally, NADPH has been reported as the primary rate-limiting factor for lipid synthesis in lipid-degrading yeast [38].

ATP plays a crucial role in biosynthesis, metabolic regulation, and maintaining cell growth. Regulating ATP supply within cells can effectively regulate cellular metabolism [39]. The MVA and MEP pathways require over 17 enzymatic reactions and the regeneration of coenzymes. Synthesising one molecule of IPP via the MVA pathway consumes two molecules of NADPH and three molecules of ATP, while the MEP pathway requires three molecules of NADPH and two molecules of ATP [40]. For example, in yeast, the reduction of HMG-CoA to methyleneglycolate by HMGR requires NADPH as a cofactor [41].

Therefore, some researchers have attempted to improve β-carotene yield by increasing NADPH supply. Zhao et al. constructed a β-carotene synthesis pathway in E. coli and designed a central metabolic module to increase ATP and NADPH supply, to enhance β-carotene production. After regulating genes involved in ATP synthesis, the pentose phosphate pathway, and the tricarboxylic acid cycle, β-carotene production increased by 21%, 17%, and 39%, respectively. The combined optimisation of the TCA and PPP modules exhibited a synergistic effect on β-carotene production, resulting in a 64% increase in β-carotene yield [18].

Liu et al. investigated the potential sources of NADPH in Saccharomyces cerevisiae. In addition to the pentose phosphate pathway, which provides NADPH to the cytoplasm, the mannitol cycle, malic acidase, aldehyde dehydrogenase, and glutamate dehydrogenase all participate in the generation of cytoplasmic NADPH in Saccharomyces cerevisiae, which is used for lipid biosynthesis [42]. In the carotenoid biosynthetic pathway, the precursor of β-carotene is lycopene. In Sun et al.'s study, by regulating the expression of genes encoding α-ketoglutarate dehydrogenase, succinate dehydrogenase, and aldolase B in the central metabolic module, NADPH and ATP supply were increased, resulting in a 76% increase in lycopene production, which could provide insights for the biosynthesis of β-carotene [43].

4.2 Increasing the supply of precursor acetyl CoA

Lipolysable yeast is an ideal host for carotenoid synthesis, with high intracellular acetyl CoA flux, which serves as a raw material for the MVA pathway. Adequate acetyl CoA supply is crucial for terpenoid compound synthesis. Increasing intracellular acetyl CoA supply facilitates enhanced β-carotene synthesis. Most acetyl-CoA in the cytoplasm is produced by ATP citrate lyase (ACL) from ATP, which is then cleaved by citrate from the mitochondria into oxaloacetate and acetyl-CoA [44].

To maintain high levels of acetyl-CoA in the cytoplasm, citrate must continuously flow out of the mitochondria for hydrolysis. Zhang et al. overexpressed AMPD in Saccharomyces cerevisiae, inhibiting the activity of isocitrate dehydrogenase, thereby increasing citrate and acetyl-CoA levels [45]. Fan et al. investigated the effect of intracellular acetyl-CoA supply on β-carotene production by constructing a heterologous PK/PTA pathway, enhancing the pentose phosphate pathway, and inhibiting the endogenous glycolytic pathway in Saccharomyces cerevisiae to increase acetyl-CoA supply, ultimately achieving a β-carotene yield of 105.94 mg/L in the engineered strain, representing a 56% increase compared to the control strain [24]. Jin adopted a different strategy to improve acetyl-CoA entry into the MVA pathway by overexpressing TGL3, PXA1, MFE1, POT1, and PEX10 to enhance fatty acid β-oxidation, weakening the lipid synthesis pathway to recover acetyl CoA from lipids, thereby increasing the carbon flux in the β-carotene synthesis pathway [46].

4.3 Increasing lipid accumulation

β-carotene is a lipophilic compound primarily stored in cell membranes and lipid droplets. WU modified the cell membrane in E. coli through membrane engineering to enhance its ability to store β-carotene, while modifying membrane morphology and lipid synthesis pathways showed synergistic effects, resulting in a 2.9-fold increase in β-carotene yield [28]. ZHAO et al. aimed to increase β-carotene accumulation in Saccharomyces cerevisiae by using metabolic engineering to enhance lipid content, designing different lipid metabolism pathways in β-carotene-producing strains, where the overexpression of sterol acyltransferases ARE1 and ARE2 increased β-carotene production by 1.5-fold, and the deletion of phospholipase PAH1, DPP1, and LPP1 doubled β-carotene production. Combining these two strategies resulted in a 2.4-fold increase in β-carotene production compared to the original strain [33].

Lipolytic yeast is a natural oil-producing yeast, making it more suitable than brewer's yeast for producing hydrophobic β-carotene. However, since both lipid synthesis and β-carotene synthesis require acetyl CoA as a precursor, it is necessary to investigate how to balance the flux distribution between these two synthetic pathways to achieve an optimal state for further increasing β-carotene production. LARROUDE et al. constructed a lipid-producing Saccharomyces yeast strain capable of high lipid and β-carotene production. Compared to the control strain, lipid accumulation increased by 3.6-fold, with β-carotene production of 8.9 mg/g DCW and 35.7 mg/L, which were 2.61-fold and 1.93-fold higher than the control, respectively [27].

4.4 Downregulation of competitive pathways

In the β-carotene biosynthetic pathway, IPP and DMAPP serve as major metabolic intermediates, which sequentially condense to produce GPP, FPP, and GGPP. Under the catalysis of various enzymes, these intermediates further generate monoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpenes [47]. To redirect metabolic flux toward β-carotene synthesis, it is often necessary to inhibit competitive pathways, suppress side reactions, and enhance target product yield. The ergosterol synthesis pathway is a competitive pathway for β-carotene synthesis, but ergosterol is a component of cell membranes, and its absence leads to severe growth defects [48].

To maintain normal cell growth and increase β-carotene metabolic flux, KILDEGAARD et al. downregulated ergosterol biosynthesis in lipase-defective yeast by truncating the natural promoter or using a weak promoter. The β-carotene titer of the resulting strain increased by 2–2.5-fold, with the highest titer observed when the promoter was shortened to 50 bp, reaching 797.1 mg/L [49]. Fan et al. introduced a PEST sequence at the N-terminus of squalene synthase to reduce protein stability and weaken ergosterol synthesis. The results showed that the introduction of the PEST sequence redirected metabolic flux from the ergosterol synthesis pathway to the β-carotene synthesis pathway, thereby increasing β-carotene yield [24]. Cao adopted a similar strategy to weaken ergosterol biosynthesis by replacing its native promoter with a weak HXT1 promoter to downregulate ERG9 expression [44].

4.5 Compartmentalisation strategies

Compartmentalisation strategies can also inhibit the transfer of metabolic flux to competitive pathways, serving as an effective regulatory strategy to enhance the production efficiency of microbial cell factories [50]. Compartmentalisation refers to the division of different functional regions within a cell into distinct compartments, primarily including mitochondria, peroxisomes, the endoplasmic reticulum, and the Golgi apparatus [51]. Each cellular compartment possesses a unique physicochemical environment with distinct metabolites, enzymes, and cofactors. Utilising cellular compartmentalisation can promote the synthesis of terpenoid compounds [52]. Intracellular assembly pathways can reduce interference between endogenous and exogenous pathways, increase the concentration of substrates and enzymes in specific spaces, thereby enhancing reaction rates and production efficiency. Additionally, they can restrict key metabolic intermediates within compartments, inhibiting their transfer to competitive pathways. Mitochondria are semi-autonomous organelles. MATSUMOTO attempted to localise the carotenoid synthesis pathway to mitochondria to improve carotenoid production in Saccharomyces cerevisiae. Compared to strains expressing the carotenoid synthesis pathway in the cytoplasm, carotenoid production increased by 13.82-fold using compartmentalised synthesis [53].

Isoprene is a precursor for β-carotene synthesis. Lv combined mitochondrial engineering with cytoplasmic engineering to utilise acetyl CoA comprehensively. Compared to recombinant strains utilising mitochondrial or cytoplasmic engineering alone, isoprene levels increased by 2.1-fold and 1.6-fold, respectively. This strategy provides an effective method for improving isoprene levels in yeast, and may also be applicable to β-carotene biosynthesis [54]. β-Carotene and astaxanthin are both carotenoids. Ma localised the astaxanthin synthesis pathway to liposomes, the endoplasmic reticulum, and the peroxisome, respectively. Compared to the cytosolic pathway, localising the synthesis pathway to subcellular organelles significantly increased yield, not only accelerating the conversion of β-carotene to astaxanthin but also significantly reducing the accumulation of intermediates. Additionally, by simultaneously localising the astaxanthin synthesis pathway to the endoplasmic reticulum, peroxisomes, and the endoplasmic reticulum, the highest astaxanthin yield of 858 mg/L was achieved [55].

5 Summary and Outlook

β-carotene has been widely applied in the food, nutritional supplements, pharmaceutical, and cosmetics industries. In recent years, its market demand has continued to expand, making the establishment of an efficient, environmentally friendly, and sustainable production method for β-carotene critically important. With the rapid development of metabolic engineering and synthetic biology, as well as the continuous in-depth research into its biosynthetic pathways, constructing microbial cell factories for β-carotene production has become one of the most promising production methods. This paper provides an overview of the method of synthesising β-carotene using microorganisms, focusing on the latest research progress and summarising commonly used metabolic engineering strategies. Currently, strategies to increase β-carotene production primarily include supplying precursor acetyl CoA, providing cofactors such as ATP and NADPH, enhancing lipid accumulation, downregulating competitive pathways, and compartmentalization strategies. Optimisation of the MVA and MEP pathways is a common method to increase IPP flux. In addition to the aforementioned metabolic engineering strategies, introducing non-native pathways to enhance precursor supply may significantly impact β-carotene production. For example, by introducing an artificial isopentenol utilisation pathway (IUP) into lipase-depleted yeast, the levels of IPP and DMAPP were increased by 15.7-fold, leading to a significant increase in carotenoid production [40]. Therefore, the use of microorganisms for β-carotene production holds great potential for future development.

Although significant research progress has been made in the biosynthesis of β-carotene, the construction of microbial cell factories remains a complex and multifactorial challenge. As a result, the biosynthesis of β-carotene still faces numerous issues, and few engineered microorganisms can achieve industrial-scale production. Further reforms and innovations in biotechnological methods are necessary. For instance, the genetic characteristics of microorganisms limit their application in production. When constructing biosynthetic pathways, while some microbial processes focus on genomic integration, most microorganisms still rely on plasmid expression, which often requires extensive use of antibiotics and increases metabolic burden. Additionally, plasmid expression is prone to genetic instability. Therefore, more efficient and faster gene manipulation tools are needed to address this issue, such as the CRISPR/Cas9 gene editing system. SCHWARTZ developed a CRISPR/Cas9-based tool for targeting and label-free integration of target genes into the genome of Lipolytococcus lactis [56].

Enzymes play a crucial role in the construction of cell factories. Enzymes from different sources may exhibit varying expression levels when heterologously expressed in the host, necessitating careful selection of enzyme sources or targeted enzyme modification. Kang utilized RIAD-RIDD assembly to construct an Idi-CrtE multi-enzyme complex, linking metabolic pathways, significantly increasing the flux of carotenoids [57]. β-carotene is an intracellular product that requires cell lysis and organic solvent extraction during extraction. Additionally, β-carotene is extremely unstable and sensitive to light and heat, prone to oxidative degradation. Therefore, the choice of cell disruption method significantly influences β-carotene extraction efficiency, necessitating careful selection of appropriate extraction methods to ensure its stability. Further research into β-carotene extraction processes is required. Addressing these challenges may accelerate the industrial application of β-carotene and provide insights for the production of other carotenoids.

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