Review
Research Progress of Nervonic Acid Biosynthesis
2023 Volume 72 Issue 10 Pages 889-900
Details
2023 Volume 72 Issue 10 Pages 889-900
Nervonic acid (NA) is a very-long-chain monounsaturated fatty acid with great application values. It plays a vital role in the development of brain nervous system and the treatment of neurological diseases, so it has attracted much attention from all walks of life. Although NA has a wide range of sources, its current acquisition methods are still mainly relied on chemical synthesis and plant extraction, which are challenging to meet the market and green industry demands, limiting its development and application. In recent years, with the rapid development of synthetic biology technology, NA biosynthesis has become an alternative production strategy. In this study, we summarize the physicochemical properties, pharmacological activities, resources, biosynthetic pathways and heterologous biosynthesis of NA, and discuss the challenges and prospects of NA biosynthesis. The application prospects of cell-free systems and retrobiosynthesis in NA synthesis were also reviewed.
Nervonic acid (NA) is a very-long-chain monounsaturated fatty acid, which was first found in the white matter of human and bovine brains 1) . Since then, NA has shown great potential for various health benefits, such as recovering damaged nerve fibers 2) , promoting brain development 3) , improving memory 4) , treating Alzheimer’s disease 5) , and preventing obesity-related metabolic diseases 6) . Therefore, the research on its source, extraction and preparation method, and pharmacological function has gradually increased.
Originally, the primary source of NA was derived from shark brains, but due to the decline of shark resources and the awareness of animal protection, this source has been abandoned. Some endemic plants in China, such as Malania oleifera and Acer truncatum Bunge, can also produce NA. However, the plant growth cycle is long, and the extraction process is inefficient and polluting, which is conflicts with the concept of sustainable green development. Therefore, NA biosynthesis has become an alternative strategy to meet the market. In this review, we aim to provide a comprehensive overview of the current knowledge and progress on NA, and highlight the challenges and opportunities for its future development.
NA (24:1, n-9) is an omega-9 monounsaturated long-chain fatty acid, also known as cis-15-docosatetraenoic acid due to its cis-unsaturated bond at the C15 position (Fig. 1). It is a monounsaturated analog of lignoceric acid (24:0) and exists as an extension product of oleic acid (18:1, n-9). Its immediate precursor is erucic acid (22:1, n-9). The molecular formula of NA is C24H46O2, the relative molecular mass is 366.621, the melting point is 42-43°C, and the density is 0.9 g/cm3. It is non-polar but irritating. NA is insoluble in water but soluble in alcohol and forms flake white crystals at room temperature 7) .
Chemical structure of nervonic acid (A) and its biosynthetic pathway in plants (B). Accase: acetyl CoA carboxylase; MAT: malonyl CoA-ACP transacylase; FAS: fatty acid synthase; KAS: ketoacyl-ACP synthase; Fat A/B: fatty acyl-ACP thioesterase; LACS: long chain acyl coenzyme A synthase; KCS: 3-ketoacyl coenzyme A synthase; KCR: 3-ketoacyl coenzyme A reductase; HCD: 3-hydroxyacyl coenzyme A dehydratase; ECR: trans-2,3-enoyl coenzyme A reductase; G3P: glycerol-3-phosphate; GPAT: 3-phosphoglycerol acyltransferase; LPA: lysophosphatidic acid; LPAAT: lysophosphatidic acid acyltransferase; PA: phosphatidic acid; PAP: phosphatidic acid phosphatase; DAG: diacylglycerol; DGAT: diacylglycerol transferase; TAG: triacylglycerol.
NA can be found in various natural sources, such as microorganisms, algae, plants and animals (Table 1). Some microorganisms, such as Francisella tularensis 8) , Neurospora crassa 9) , Phycomyces blakesleanus 9) and Mortierella isabellina 10) , can produce NA. However, most of them are highly pathogenic to humans and animals, and are not suitable for NA production. The most commonly used microorganisms are oleaginous yeasts, which are genetically engineered to produce NA. The metabolic pathways, synthetic pathways, and culture conditions are continuously optimized to increase its yield. Algae, such as Nitzshia cylindrus 11) , Mychonastes afer 12) , Nannoloris sp. QUCCCM31 13) , can also produce NA, but they are not widely used due to the high cultivation cost. Plants have the highest NA content, especially M. oleifera, which has 55.7%?67% NA in its seeds 14) . However, plants have many disadvantages, such as a long growth cycle, environmental and climatic sensitivity and variable seed yield. These disadvantages can be alleviated by the application of plant tissue culture technology and cell suspension culture system for NA production. Suspension cells have many advantages, such as fast growth, high proliferation, high cell viability, and vigorous metabolism, which can significantly shorten the growth time and increase the NA biosynthesis. Animal lipids also contain NA, which was first discovered in shark oil; therefore, NA is also known as shark oleic acid. However, due to the low extraction rate and the limited resources, NA is rarely obtained from animal sources.
Natural sources of NA.
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The main methods for obtaining NA from plant extracts are mechanical pressing, leaching, enzymatic hydrolysis, and ultrasonic-assisted extraction 19) . Some studies have compared the efficiency of these methods. Hu et al. extracted NA from the seed oil of Acer truncatum Bunge using soxhlet extraction, mechanical pressing, and ultrasonic-assisted extraction, and found that the ultrasonic-assisted extraction method was the best, with a NA content of 37.02% after purification 19) . Zhao et al. obtained the seed essential oil of Acer truncatum Bunge’s through liquid oil pressing and CO2 supercritical extraction technology, and purified NA by high-speed countercurrent chromatography with a content of 18.1% 20) . Xu et al. used aqueous enzymatic extraction to extract the seed oil of Acer truncatum Bunge and obtained fatty acid with good quality, with a NA content of 7.2% 21) . However, problems such as seed breakage during collection and seed oil loss during extraction remain to be solved.
The main methods for purifying NA are metal salt precipitation, solvent crystallization, column chromatography, molecular distillation, etc. Lin et al. compared the metal salt precipitation method and crystallization method for purifying NA from Vitex negundo L’s seed oil. The initial crude had a NA content of 3.5%. After purification, the NA content increased to 16.5% and 5.6%, respectively. The metal salt precipitation method had a higher purification efficiency than the crystallization method 22) . Fu et al. crystallized the mixed fatty acids extracted from M. oleifera twice with absolute ethanol and removed them once with a material-to-liquid ratio of 1:100 (g/mL), and achieved a NA content of 13.9% 23) . Liu et al. used a urea-loaded chromatography column to separate the mixed fatty acid by ultrasonic, reflux, and soxhlet extraction methods, and concentrated it to obtain NA with a content of 84.7% 24) . Xu et al. trans-esterified the mixed fatty acids extracted from A. truncatum Bunge with anhydrous ethanol and an alkaline catalyst, followed by dehydration, and vacuum molecular distillation to obtain more than 50.0% of ethyl nervate and biodiesel 25) .
NA have been well studied for its pharmacological effects. NA can significantly improve human memory. Wang patented the application of NA in breastfed infant formula and found that the experimental group had significantly better association, IQ values, comprehension, and recognition abilities than the control group 26) . Moreover, a recent study showed that supplementation of Acer truncatum Bunge seed oil in rats stimulated pathways related to memory and cognition improvement, such as neurotrophic factor signaling, glycerophospholipid metabolism, and sphingolipid metabolism pathways by KEGG enrichment analysis 4) . NA is also associated with psychiatric disorders such as depression and Alzheimer’s disease. Kageyama et al. measured NA in the cerebrospinal fluid of patients with psychiatric disorders and found that its levels were significantly negatively correlated with depression and positively correlated with mania, suggesting that NA could be a potential biomarker for mood symptoms 27) . Pamplona et al. found that higher NA concentration in the blood reduced the risk of Alzheimer’s disease, but other studies reported opposite results 4) . Astarita et al. found a significant increase of NA in the brains of Alzheimer’s disease patients 28) . Therefore, the role of NA in Alzheimer’s disease is unclear. Oda et al. found that NA was significantly negatively correlated with body mass index (BMI), leptin, triacylglycerol, cholesterol, and fasting glucose, suggesting that NA may have a preventive effect on obesity and other related metabolic diseases 6) , 29) .
When NA is ingested, it forms sphingolipids by binding to sphingosine via amide bonds to form 2) . Sphingolipids are the major components of white matter and the myelin sheath of nerve fibers 30) , 31) . Studies have shown that NA intake increases the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARα), peroxisome proliferator-activated receptor alpha (RER), and Sirtuin 1 (SIRT1) in mice, enhancing fatty acid metabolism 6) . Therefore, NA is mainly added to health products and milk powder to promote and improve brain development and fatty acids metabolism. NA has some effect on mice with Parkinson’s disease, and the molecular mechanism has been elucidated 32) . It up-regulates tyrosine hydroxylase, down-regulates alpha-synuclein, partially inhibits oxidative stress, and does not affect striatal DA, serotonin, and metabolites in mice administered NA. Umemoto et al. found that levels of malondialdehyde, a marker of lipid peroxidation, were significantly reduced in NA-treated mouse cells. NA pretreatment can activate the cellular antioxidant system and significantly increase the expression levels of superoxide dismutase (Mn-SOD and Cu/Zn SOD) and γ-glutamyl cysteine synthase (GCLC), which are responsible for the synthesis of glutathione. Therefore, NA can act as neuroprotective mediator in the brain, which may be related to its therapeutic effects on Alzheimer’s and other brain diseases 33) .
The NA biosynthesis pathway has been elucidated and its synthesis can be divided into three stages (Fig. 1) : (1) synthesis of some essential fatty acids; (2) synthesis of oleic acid; (3) extension of the chain length of oleic acid or other monounsaturated fatty acids. In plants, the first stage takes place in the plastid matrix, while stages two and three occur in the endoplasmic reticulum (ER). In animals and yeast, however, stage one occurs in the cytoplasm, and other two stages are also carried out in ER 34) . NA biosynthesis begins with acetyl-CoA. First, acetyl-CoA and bicarbonate are converted to malonyl-CoA by acetyl-CoA carboxylase (Accase), and then malonyl-CoA is converted to malonyl-ACP by malonyl-CoA-ACP transacylase (MAT). Then C16:0-ACP is synthesized by fatty acid synthase (FAS) and ketoacyl-ACP synthase I/III (KAS I/III). C16:0-ACP is extended to C18:0-ACP by ketoacyl-ACP synthase II (KAS II) and then desaturated to form C18:1-ACP. Or C16:0-ACP is desaturated to C16:1-ACP and then extended to C18:1-ACP. In stage two, C18:1-ACP, also known as oleic acid-ACP, is released from fatty acyl-CoA by fatty acyl-ACP thioesterase (Fat A/B) 35) , 36) , and the released fatty acids are rapidly esterified by long-chain acyl-coenzyme A synthase (LACS) to prevent their efflux out of the cell. After transporting the esterified fatty acids to the cytoplasm, the fatty acids are converted to acyl-CoAs by LACS 37) . In the last stage, very long-chain fatty acids (VLCFAs, 22-26 carbons), including NA, are synthesized as acyl-CoAs by the fatty acid enzyme extension complex, which includes 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxacyl-CoA dehydratase (HCD) and trans-2,3-enoyl-CoA reductase (ECR) 38) . The fatty acid elongation enzymes in animals and yeast differ from those in plants. In humans, enzymes ELOVL2 and ELOVL4 are used to extend and synthesize VLCFAs 39) . While, in Saccharomyces cerevisiae, ELOVL2 is responsible for synthesizing saturated and monounsaturated fatty acids with a carbon chain length of 24 40) . As shown in Fig. 1, in plants, malonyl coenzyme A and long-chain acyl-coenzyme A are condensed by KCS, then reduced by KCR to produce 3-hydroxyacyl coenzyme A, and then dehydrated by HCD to produce 2-enoyl coenzyme A. Finally, 2-enoyl coenzyme A is reduced to long-chain acyl-coenzyme A by ECR. Each fatty acid extension cycle begins at 18:1 with two carbons contributed by malonyl coenzyme A to the acyl chain, and after three fatty acid extensions, oleic acid (18:1) is extended to NA (24:1). The synthesized NA is in the form of CoA, and then the nervonoyl-CoA is acylated in the sn-1 and sn-3 positions of the glycerol-3-phosphoglycerol backbone by 3-phosphoglycerol acyltransferase and diacylglycerol acyltransferase 41) and stored in triacylglycerol.
7.2.1 Classification of KCS
KCS is an essential rate-limiting enzyme in the biosynthetic pathway of NA 42) . Studies on KCS have increased in recent years. In 1995, James et al. first discovered that the fae1 gene was involved in the synthesis of VLCFAs in Arabidopsis thaliana. The amino acid sequence of FAE1 was similar to other condensing enzymes such as chalcone synthase (CHS), astragalus synthase (STS), and β-ketoacyl carrier protein synthase III (KASIII) 43) . Later, Costaglioli et al. divided the 21 genes (KCS1-KCS21) in A. thaliana KCS gene family into four types: KCS1-like, FAE1-like, FAH-like, and CER6 (2005). Joubes et al. further classified them into eight classes: α, β, γ, δ, ε, ζ, η, and θ, based on their genetic relationship, protein topology, and 3D modeling structure 44) .
7.2.2 Substrate specificity of KCS
Studies have shown that KCS from different species or the same species exhibit different substrate specificities. For example, KCS of Cardamine graeca showed good catalytic activity for C20:1 and C22:1, which increased the NA production of transgenic plants dozens of times. However, Mo KCS11 in the NA-rich M. oleifera had low catalytic efficiency, and only increased the NA yield of transgenic plant by several folds. The catalytic properties of some other plant KCSs are summarized in Table 2.
Catalytic properties of some KCS enzymes from plants.
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7.2.3 Analysis of key catalytic residues in KCS
In 2001, Ghanevati and Jaworski examined the effect of six conserved cysteines (C84, C223, C270, C312, C389, C460) and four conserved histidine residues (His302, 387, 391, 420) in the active site of FAE1 KCS on its activity by using a site-directed mutagenesis and expressing them in S. cerevisiae 51) . They found that mutating any of these four conserved histidine residues or C223 resulted in a complete loss of KCS activity. Histidine also plays an important role in other condensing enzymes similar to KCS. The crystal structures of the large intestine fatty acid synthases KASI, II, III and, chalcone synthase reveal the presence of histidine residues near the active site cysteine, and mutational analysis of chalcone synthase 52) and Escherichia coli KASIII 53) showed that the active site histidine plays a key role in the decarboxylation of malonyl substrates. Later, to investigate domains/residues associated with substrate specificity, Blacklock and Jaworski studied the catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and Brassica napus FAE1 KCS, which have distinct substrate specificities, by using domain swapping and site-directed mutagenesis 54) . The results showed that the N-terminal region excluding the transmembrane domain and the K92 residue were responsible for substrate specificity of FAE1 KCS. In 2013, Sun et al. found that KCS has four conserved histidine and six conserved cysteine residues in cruciferous plants with high and low erucic acid content. They concluded that the conservation or variation of the active sites did not affect the high level of erucic acid production 55) . Therefore, it is necessary to characterize more KCS enzymes with different substrate specificity and catalytic activity to reveal the underlying rules.
7.3.1 Enzymes in the lipoyl coenzyme A synthesis pathway
In addition to the key rate-limiting enzyme KCS, other important enzymes in the NA biosynthetic pathway have also been extensively studied. LACS, an AMP-binding protein, plays vital roles in various fatty acid-derived pathways, including lipid metabolism, β-oxidation, intracellular fatty acid homeostasis, and fatty acid transport in different cells 56) . LACS rapidly converts monounsaturated fatty acids to acyl-CoAs through a two-step reaction, resulting in the production of 12-20 carbon fatty acyl-CoA 57) . The elongation of acyl-coenzyme A occurs through KCS, KCR, ECR, and HCD, except for KCS, which have broad substrate specificities and correlated expressions. Mutations in the Bn-fae1 gene can impact the transcription of Bn-kcr1 and the translation of 3-ketoacyl coenzyme A reductase, suggesting a relationship between the expression of KCR and KCS 58) . KCR enhances the activity of the fatty acid elongase ELOVL6, potentially by inducing conformational changes or facilitating product removal. ECR affects the enzymatic activity of HCD and may be the second rate-limiting step in fatty acid elongation 59) .
7.3.2 Enzymes in the TAG pathway
In the synthesis of triacylglycerol (TAG), the long-chain acyl-coenzyme A generated by GPAT, LPPAT, PAP, and DGAT is sequentially incorporated into the sn-1 and sn-3 positions. The rate-determining enzyme for TAG synthesis is DGAT, which has distinct substrate preferences in different plants. Plants with higher NA content prefer long-chain fatty acid DAG as a substrate. For example, in the castor oil plant, DAG with specific fatty acids in both sn-1 and sn-2 positions is preferred by DGAT 60) . Safflower seeds utilize sn-1,2-DAG and sn-2,3-DAG as substrates for DGAT 61) .
Due to the limited natural sources and low yield, NA can not meet the demands of market and sustainable development. The synthetic pathway of NA has been fully elucidated, so heterologous synthesis of NA has become an important alternative strategy to replace natural sources. Heterologous synthesis mainly includes chemical synthesis and biosynthesis.
The first report of the chemical synthesis of NA 62) was by Hale et al., who reduced ethyl mustard to erucyl alcohol, then converting it to bromides and catalyzing it with malonic acid. Recently, Gao et al. patented a method of forming NA by ring-opening epoxidation of erucic acid with Grignard’s reagent, followed by oxidation of iodobenzene acetate and 2,2,6,6-tetramethylpiperidine oxide (TEMPO), with a yield of up to 95% 63) . This method has the advantages of cheap substrate and simple operation process, but it still suffers from the high pollution and required reagents of chemical synthesis.
Plants are considered to be good candidates for NA synthesis in bioreactors because of their high oil content. In the early 20th century, seeds from genetically modified plants were used to produce NA. Later, with the discovery of enzymes with better activity and the rapid development of plant transgenic technology, the NA production in plants gradually increased. Microalgae and microorganisms also have benefits, such as easy cultivation, rapid propagation and large-scale fermentation. Some oleaginous yeasts have been genetically modified for NA synthesis 7) , 18) . Comparing to chemical synthesis, biosynthesis is newer and more environmentally friendly.
Several studies have reported the successful production of NA in transgenic oil plants by introducing elongase genes from different sources. For example, in 1997, Cargill increased NA yield of rapeseed by dozens of times through inserting the elongase gene from L. annua seeds 64) . Guo et al. enhanced the NA production of A. thaliana by 30-40 times by transferring the KCS gene of L. annua with Napin promoter 50) . Taylor et al. isolated a KCS gene from C. graeca and introduced it into A. thaliana, Brassica carinata, and B. napus with Napin promoter, and increased their NA production 18) . Li et al. demonstrated the high elongation activity of the Mo KCS11 gene from the NA-rich plant M. oleifera by expressing it into A. thaliana, B. napus, and Camelina sativa, which proved to have good elongation activity 45) . The biosynthesis progress of NA by transgenic plants is summarized in Table 3.
Progress in biosynthesis of NA transgenic plants.
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Transgenic oil plants have the advantage of high fatty acid content, which can significantly increase NA yield. However, they also have the limitations of traditional plants, which can be overcome by tissue culture and suspension cell culture system.
Taylor et al. reported that overexpression of the KCS gene in S. cerevisiae enabled the synthesis of NA 18) . Qiao et al. genetically modified Yarrowia lipolytica and obtained an engineered strain that could produce NA, accounting for 1.5% of the total oil content 7) . Meng et al. from the Qingdao Institute of Bioenergy and Processes, Chinese Academy of Sciences, patented a “recombinant yeast strain for NA production and its application" 66) . In this patent, they overexpressed the Cg KCS gene from C. graeca, the Ma LCE1 gene for C16 and C18 fatty acid elongation from Mortierella Alpina, the DGAT 1 gene, and Δ9 desaturase gene in Y. lipolytica to achieve NA production. After optimizing the culture conditions, NA reached 1.44 g/L, which is the highest yield in Y. lipolytica. Fillet et al. integrated the codon-optimized KCS gene from Crambe abyssinica and C. graeca into the genome of Rhodosporidium toruloides and constructed the NA-producing engineered strains CRA-01 and NER-01, respectively 67) . Among them, strain CRA-01 had a high erucic acid yield, and strain NER-01 had the highest NA yield, reaching 8 g/L after culture optimization. This is also the highest NA production in microorganisms reported so far.
However, microbial production of NA faces challenges due to the low tolerance of the product and the host’s need to synthesize large amounts of other essential substances to maintain life. The toxicity of the synthesized products to the cells can be reduced by optimizing the fermentation system, by rapidly excreting them from the system, or by optimizing the chassis to divert more “energy" to NA production. Moreover, for large-scale fermentation of engineered bacteria for NA production, it is necessary to continue designing and optimizing the composition of the fermentation medium and the control parameters of the fermenter to achieve higher yields 68) .
Microalgae are rich in compounds such as carbohydrates, proteins, minerals, oils, fats, and polyunsaturated fatty acids, as well as biologically active compounds such as polyphenols, terpenoids, carotenoids, and tocopherols. These compounds have antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory, and antitumor properties. Algae extracts obtained with little or no solvents are safe for plants, animals, and humans. They have been used in modern agriculture as biostimulants, bioregulators, fertilizers, feed additives, food, pharmaceuticals and cosmetics 69) .
In 2011, Li et al. from Guangdong Ocean University introduced the KCS gene from M. oleifera into the host cells of oil-rich Neochloris oleoabundans and Chlorella sp. They obtained transgenic microalgae that could produce large amounts of NA, which accounted for 40% of the total fatty acids 70) . Ashtiani et al. increased NA synthesis by mixing cultures of Rhodotorula glutinis and Chlorella vulgaris 71) . They set up several cultural protocols to explore the optimal cultural conditions. They co-cultured yeast cells or media with different ratios of microalgae. When co-cultured microalgae with yeast in a ratio of 2:1, NA production was 7-fold higher than that of microalgae cultured alone. When the cell-free medium with yeast removed was co-cultured with microalgae in a 2:1 ratio, the NA yield was 9-fold higher than microalgae cultured alone. As mentioned earlier, the microalgae M. afer has a promising application, and several studies have been conducted to explore its ability to produce more NA. Hu et al. used a two-step method to culture the microalgae M. afer HSO-3-1 72) . The first stage used a high nitrogen source to make the microalgae accumulate biomass; the second stage used a low nitrogen source and high light to induce the microalgae to produce lipids. The yield of NA and linolenic acid reached 8.5% and 10.1% of total fatty acids, respectively, after 12-14 days of incubation. Qiu et al. investigated the effects of plant growth regulators and antioxidants on NA production by M. afer HSO-3-1 and found that naphthalene acetic acid (NAA) significantly increased its NA production; when the concentration of NAA was 0.3 mg/L, the yield of NA was 73% higher than that of control, accounting for 6.3% of total fatty acids content 73) .
Microalgae have the advantages of fast growth rate, high lipid content, and strong adaptability to the environment. However, large-scale cultivation of microalgae is costly and easy cause certain damage to the environment. Therefore, it is necessary to find or genetically engineer a green, easy-to-cultivate microalga as a factory for NA production.
Currently, NA is primarily biosynthesized by microorganisms, algae, and plants, and extensive research has been conducted on this topic. However, there are still some challenges that need to be addressed. Algae cultivation is expensive and can cause rapid ecological damage. While microorganism, such as R. toruloides can produce NA at high yields (8 g/L), the product only accounts for 20% of the total fatty acid due to host limitation, this yield is considerably far lower than both chemical synthesis and biosynthesis through transgenic plants.
We have proposed several strategies to increase the production of NA. One of them is to target the substrate specificity of KCS, which is a key rate-limiting enzyme in NA biosynthesis. However, studies on this aspect are currently lacking. A significant research breakthrough would be to identify the critical residues that determine the acyl chain length specificity of KCS by performing site-directed mutagenesis on specific functional motifs, such as the putative acyl-CoA binding site. Additionally, KCS proteins can be engineered to favor certain substrates. Another strategy is to modify the chassis to provide sufficient substrate. Acetyl-CoA is the precursor of NA, and its conversion from glucose and other substances can be enhanced, furthermore, by modifying the acetyl-CoA condensate and malonyl-CoA/ACP transacylase to convert acetyl-CoA to malonyl-ACP as much as possible, the NA yield can be improved. Moreover, by reducing the activity of desaturases, oleic acid can be directed to enter the fatty acid elongation pathway instead of being desaturated to produce polyunsaturated fatty acids. A third strategy is to engineer acyltransferases to integrate NA into more TAG (triacylglycerol) sites. Currently, NA can only be integrated into the sn-1 and sn-3 positions of TAG, but not the sn-2 position. To increase the NA content of the final product, NA can be integrated into the sn-1, sn-2, and sn-3 positions. This can be achieved by modifying the substrate specificity of LPAAT to catalyze NA at the sn-2 site of TAG.
Although transgenic plants have been shown to produce high yields of NA, the extended growth cycle and environmental factors such as climate, soil, geographic location, and irrigation can complicate production. Therefore, relying solely on transgenic plants is not a foolproof strategy. Additionally, the complex biosynthetic pathway of NA, involving multiple enzymes and dozens of steps from acetyl-CoA to the final product, decreases biosynthetic efficiency. Thus, there is an urgent need for alternative biosynthetic strategies that are more efficient, economical, and environmentally friendly.
Recently, cell-free systems have been successfully applied to produce high-value natural products, such as nonribosomal peptides (NRPs) 74) . In vitro synthesis of NRPs can avoid cytotoxicity and metabolic burden caused by heterologous expression in vivo. For instance, valinomycin, a complex NRPs compound, was entirely synthesized in a cell-free protein synthesis (CFPS) system, with a maximum yield of 30 mg/L, which is more than twice the yield (13 mg/L) in vivo 75) . Similarly, nisin, a ribosomally synthesized and post-translationally modified peptide (RIPP) compound, has also been synthesized through a cell-free system. In addition to improving the production of RIPPs, in vitro synthesis systems can also help determine the rate-limiting step or assess the tolerance of enzymes to substrates to guide in vivo production 76) , 77) . For example, Feng et al. constructed a cell-free system to synthesize L-theanine and used it to efficiently screen isoenzymes that produce L-theanine and converted toxic ethylamine into L-theanine to the maximum extent possible 78) .
Therefore, cell-free synthesis system, with the advantages of a short reaction cycle, high tolerance to the products, and less pollution to the environment, is a promising option for NA production. However, the inherent biosynthesis pathways of natural products are generally long and not conducive to heterologous biosynthesis. One solution is to use the reverse biosynthesis with the help of artificial intelligence (AI) to construct a new and shorter biosynthetic pathway that can be realized in vitro. For example, the Korea Advanced Institute of Science and Technology (KAIST) recently constructed a biosynthetic pathway for short-chain primary amines through reverse synthesis 79) . They identified the direct precursors for 15 short-chain primary amines and achieved one-step synthesis of these compounds. They also developed a metabolically engineered E. coli strain for the production of representative SCPAs from glucose. With the development of artificial intelligence technology, reverse biosynthesis approach holds promise for developing new biosynthetic pathways for NA and other complex natural products.
The authors declare that there are no conflicts of interest.
C. Ling, F. Li, and J-Y. Zhao collected and analyzed the data. X-L. Han and M-L. Wen conceived and supervised the project. C. Ling, M-L. Wen, and X-L. Han wrote the paper with contributions from all authors.
This work was supported by Yunnan Provincial Education Department Research Fund Project (Grant No. 2023J0004).
