Melatonin Biosynthesis Pathways in Engineered Microorganisms (2022) PDF

Synthetic and Systems Biotechnology 7 (2022) 544–553 Contents lists available at ScienceDirect Synthetic and Systems Biotechnology...

Synthetic and Systems Biotechnology 7 (2022) 544–553 Contents lists available at ScienceDirect Synthetic and Systems Biotechnology journal homepage: www.keaipublishing.com/en/journals/synthetic-and-systems-biotechnology Review Article Melatonin biosynthesis pathways in nature and its production in engineered microorganisms Xiaotong Xie a, Dongqin Ding b, c, Danyang Bai b, c, Yaru Zhu b, c, Wei Sun d, Yumei Sun a, *, Dawei Zhang b, c, ** a Dalian Polytechnic University, Dalian, 116000, PR China b Tianjin Institutes of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China c Biodesign Center, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China d Tianjin University of science and technology, Tianjin, 300308, PR China A R T I C L E I N F O A B S T R A C T Keywords: Melatonin is a biogenic amine that can be found in plants, animals and microorganism. The metabolic pathway of Melatonin melatonin is different in various organisms, and biosynthetic endogenous melatonin acts as a molecular signal Biosynthesis and antioxidant protection against external stress. Microbial synthesis pathways of melatonin are similar to those Plants of animals but different from those of plants. At present, the method of using microorganism fermentation to Animals Microorganisms produce melatonin is gradually prevailing, and exploring the biosynthetic pathway of melatonin to modify Metabolic modification microorganism is becoming the mainstream, which has more advantages than traditional chemical synthesis. Enzyme catalysis mechanism Here, we review recent advances in the synthesis, optimization of melatonin pathway. L-tryptophan is one of the two crucial precursors for the synthesis of melatonin, which can be produced through a four-step reaction. Enzymes involved in melatonin synthesis have low specificity and catalytic efficiency. Site-directed mutation, directed evolution or promotion of cofactor synthesis can enhance enzyme activity and increase the metabolic flow to promote microbial melatonin production. On the whole, the status and bottleneck of melatonin biosynthesis can be improved to a higher level, providing an effective reference for future microbial modification. 1. Introduction (Fig. 1). Nowadays, it is estimated that 50 to 70 million Ameri­ cans chronically suffer from a sleep or circadian disorder, and the global Melatonin (N-acetyl-5-methoxytryptamine) is an important indole­ market of melatonin was estimated at USD 1 billion in 2020, and it is amine derivative. In 1967, Sagan speculated that melatonin was syn­ expected to reach 3.4 billion USD by 2026 (https://marketresearchexper thesized by mitochondria and chloroplasts, which at the time were tz.com/report/global-melatonin-market-92220). Recently, it was re­ considered to have evolved from precursors similar to Rhodospirillum ported that more than 300 million people in China suffer from sleep rubrum and cyanobacteria [1,2]. Melatonin has anti-inflammatory, disorders. However, sales of melatonin products in the global market antioxidant and autophagic properties [3,4]. The level of melatonin in have soared, and demand exceeds production, so that it is necessary to plants affects their response to biotic and abiotic stress. In animals, further improve industrial approaches for melatonin synthesis. Com­ melatonin can regulate sleep and body temperature and improve mercial melatonin is mainly produced by chemical synthesis, which is dairy production. In humans, it was demonstrated to protect lym­ highly polluting. The raw material supply from animal and plant phocytes against DNA damage induced by ionizing radiation , regu­ extraction is small, and the extraction rate is low. In recent years, the late the circadian rhythm , protect the skin , and ameliorate rapid development of microbial fermentation methods has offered hope diabetes. Very recently, melatonin was also investigated as a po­ for more economic and environmentally friendly production. It is a tential adjuvant treatment for slowing down the effects of COVID-19 promising method to design, optimize and introduce melatonin Peer review under responsibility of KeAi Communications Co., Ltd. * Corresponding author. ** Corresponding author. Tianjin Institutes of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China. E-mail addresses: [email protected] (Y. Sun), [email protected] (D. Zhang). https://doi.org/10.1016/j.synbio.2021.12.011 Received 9 November 2021; Received in revised form 14 December 2021; Accepted 24 December 2021 Available online 12 January 2022 2405-805X/© 2022 The Authors. Published by KeAi Communications Co. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). X. Xie et al. Synthetic and Systems Biotechnology 7 (2022) 544–553 2. Natural melatonin synthesis pathways 2.1. Melatonin synthesis pathway in plants Melatonin was first reported in plants in 2000 and was found to be distributed in various plant tissues. Mitochondria and chloroplasts are the main sites of melatonin synthesis in plants. From an evolutionary point of view, the precursor of mitochondria probably was similar to a purple nonsulfur bacterium, and chloroplasts are probably the de­ scendants of cyanobacteria [29,30]. Studies have shown that L-trypto­ phan can be converted into melatonin via two pathways in plants, one in healthy plants and the other under senescence or biological stress [28, 31]. Under normal plant growth, L-tryptophan is fist carboxylated by L- tryptophan decarboxylase (TDC) into tryptamine in cytoplasm. The second committed enzyme trptamine-5-hydroxylase (T5H), which cat­ alyzes tryptamine into serotonin and requires acetyl-coenzyme A (Ac- CoA) on the endoplasmic reticulum. Usually in fresh plants, the third step is the acetylation of serotonin into N-acetylserotonin by serotonin N-acetyltransferase (SNAT) in the chloroplast. N-Acetylserotonin methyltransferase (ASMT) is believed to be the last enzyme, it catalyzes N-acetyl-serotonin into melatonin by methylation reaction in the cyto­ Fig. 1. The application value of melatonin. plasm. When plants under senescence and cadmium stress, the serotoin maybe accumulated so that the melatonin synthesis would favor use synthesis pathways into microorganisms. ASMT catalyzes serotonin into 5-methexytryptamine, and then synthe­ Due to the complex metabolic network and numerous secondary size melatonin by SNAT. During the last step, the conversion of metabolites of microorganisms, the synthesis pathway of melatonin has acetyl-5-hydroxytryptamine to melatonin is accompanied by the not been investigated in many species. Only a few strains have been co-conversion of S-adenosyl-L-methionine (SAM) to S-adenosyl-L-ho­ found to produce melatonin by themselves, including yeast and pseu­ mocysteine (SAH). Overall, when plants are subjected to biological domonads, but the synthesis efficiency is limited [13–16]. Pseudomonas stress and senescence, the same as the normal growth conditions of fluorescens can synthesize melatonin by itself as a growth signal mole­ plants is that tryptophan is carboxylated and hydroxylated to produce cule or as a protective agent against ROS in the medium to promote early serotonin. The difference is that serotonin is first methylated to produce adaptation. However, the melatonin concentration reached a maximal 5-methyltryptamine, and then 5-methyltryptamine is acetylated to value of only 1.32 μg/L. Back et al. speculated that the melatonin produce melatonin. In plants, the existence of two melatonin synthesis pathway of yeast is similar to that of cyanobacteria or plants metabolic pathways may be determined by the strict regulatory mech­. Melatonin is a secondary metabolite of yeast which can interact anisms of plants themselves (Fig. 2). However, the deeper regulation of with glycolytic proteins and has a positive effect on fermentative enzymes that plants biosynthesize melatonin under different conditions metabolism by increasing resistance to oxidative stress and UV radiation needs to be explored. [18–20]. Yeast follows the Ehrlich pathway during early alcohol fermentation and will use amino acids such as L-tryptophan as the source of nitrogen. The melatonin production of Saccharomyces cerevisiae QA23 and Levucell SC20 (from animal nutrition) reached 1.35 ± 1.69 and 93.14 ± 85.86 ng/mL during alcoholic fermentation, respectively [14,22]. Heterologous expression systems can be widely employed to screen various putative melatonin synthesis genes from animals and plants to overproduce melatonin in various microorganisms. There are few reports on the ectopic overproduction of melatonin in E. coli because of either the low or insoluble expression of melatonin biosynthesis genes [23–25]. Zhang et al. combined the physostigmine biosynthetic genes from Streptomyces albulus, a gene encoding phenylalanine 4-hydroxylase from Xanthomonas campestris and caffeic acid 3-O-methyltransferase (COMT) from Oryza sativa in E. coli, which resulted in a melatonin yield of 136.17 ± 1.33 mg/L in a shake flask and 0.65 ± 0.11 g/L in a 2L-bio­ reacorr. When a melatonin-producing strain was assembled using the three plasmids ddc, aanat and asmt, encoding the cofactor pter­ in-4α-carbinolamine dehydratase and using glucose as the sole carbon source for tryptophan supply, the final melatonin yield reached a record level of over 2 g/L. However, the synthesis of melatonin in microorganisms is complex and the yield is generally low. The metabolic engineering approaches are not systematic and there is a lack of research on related pathways in Fig. 2. Synthesis pathway of melatonin in plants the prior art. Therefore, we need to explore metabolic engineering TDC, tryptophan decarboxylase; T5H, tryptamine-5-hydroxylase; ASMT, N strategies to facilitate the large-scale production of melatonin by -acetylserotonin methyltransferase; COMT, acetylserotonin O microorganisms. -methyltransferase; SNAT, serotonin N -acetyltransferase. 545 X. Xie et al. Synthetic and Systems Biotechnology 7 (2022) 544–553 2.2. Melatonin synthesis pathway in animals main rate-limiting enzymes that have a major impact on L-tryptophan synthesis are anthranilate synthase encoded by trpE and DHAP synthase Animals have a single source of melatonin, mitochondria. Moreover, encoded by aroG. These enzymes are subject to feedback inhibition by animals cannot synthesize L-tryptophan themselves, and it must be tryptophan and phenylalanine, respectively. The transcriptional regu­ ingested externally. Therefore, animals have lower melatonin meta­ lation of the synthesis and transport is mainly achieved by the repressor bolism than plants. In animals, the synthesis of melatonin proceeds via a proteins encoded trpR. Weakening the TrpR repressor protein can relieve four-step pathway. First, tryptophan hydroxylase produces 5-hydroxy­ its inhibitory effect on the enzymes in the L-tryptophan synthesis tryptophan. This reaction requires the cofactor BH4 and oxygen. Next, pathway and increase the production of L-tryptophan [36,37]. tryptophan carboxylase converts 5-hydroxytryptophan into serotonin, Restraining the expression of tnaA (L-tryptophanase) was necessary to which is the main precursor of melatonin synthesis. This reaction is prevent the formation of serotonin and improve hydroxylation for in­ accompanied by the release of carbon dioxide. Subsequently, aralkyl­ crease the production of melatonin. amine N-acetyltransferase produces N-acetyl serotonin at the expense of Both E. coli and yeast can synthesize L-tryptophan, which can be used acetyl-CoA is. Finally, N-acetyl-serotonin methyltransferase generates to synthesize melatonin. At present, most strains with high tryptophan the final product melatonin accompanied by the conversion of the yield were constructed from E. coli, indicating that it may be a better cofactor SAM into SAH [17,31] (Fig. 3). chassis for the production of melatonin. Zhu et al. expressed heterolo­ gous glutamine synthetase in the engineered E. coli strain KW001, fol­ 3. Synthesis and modification pathways of melatonin in lowed by the overexpression of icd and gdhA, as well as the expression of microorganisms mutated serA and thrA variants. Finally, sthA and pntAB, encoding transhydrogenase, were overexpressed to maintain cofactor balance. In addition to plants and animals, many microbes are also capable of The engineered strain could produce 1.71 g/L L-tryptophan in shake- melatonin synthesis [16,18,19,33]. The application of engineered mi­ flask fermentation, which was 2.76-times higher than the titer of the croorganisms to synthesize melatonin is a potential industrial produc­ parental strain. Zeng and Chen rationally engineered wild-type tion method. The pathway through which microorganisms synthesize E. coli W3110 by knocking out tryptophanase (tnaA), 5-methyltetrahy­ melatonin is similar to that of animals. It requires encompasses drofolate-homocysteine methyltransferase (MTR), and 3-deoxy-7-phos­ consecutive enzymatic reactions with the involvement of coenzymes or phoheptanonic acid synthase (aroFGH), as well as overexpressing cofactors. However, there are few literature reports on the improvement D-3-phosphoglycerate dehydrogenase (serA), which resulted in a final of microbial melatonin production through metabolic engineering. Ac­ L-tryptophan titer of 30–34 g/L in a 1.5 L bioreactor. Xiong et al. cording to the basic approaches of metabolic engineering, the produc­ first introduced phosphoketolase from Bifidobacterium adolescentis to tion of melatonin can be improved by increasing the supply of strengthen E4P formation, after which the phosphotransferase system precursors, optimizing the expression of pathway enzymes, or was substituted with PEP-independent glucose transport, meditated by a improving the utilization of cofactors and coenzymes. glucose facilitator from Zymomonas mobilis and native glucokinase. Finally, they rewired the PEP-pyruvate-oxaloacetate node. Fed-batch fermentation in a 5-L bioreactor produced 41.7 g/L L-tryptophan, 3.1. L-tryptophan synthesis which is the highest yield reported to date. L-tryptophan is a precursor for the synthesis of melatonin. Normally, glucose is transported through the phosphotransferase system 3.2. Serotonin synthesis to produce glucose hexaphosphate, which then enters the glycolytic pathway and the pentose phosphate pathway to produce phosphoenol­ Serotonin is formed by a two-step conversion of L-tryptophan. First, L- pyruvate pyruvic acid (PEP) and erythrose 4-phosphate (E4P), respec­ tryptophan is hydroxylated to 5-hydroxyserotonin by TPH, which in tively. Then, 3-deoxy-D-arabinoheptulose (DAHP) is produced by the turn is carboxylated by TDC to yield serotonin. Serotonin is mainly condensation of PEP and E4P, and passes through the shikimic acid known for its role as a major neurotransmitter and mood regulator, pathway to generate chorismate. The biosynthetic pathway of L-trypto­ leading to the epithet ‘hormone of happiness’ based on the relationship phan is summarized in Fig. 4. In E. coli, L-tryptophan is synthesized by a between low serotonin levels and depression. Serotonin is synthe­ long pathway of enzymes encoded by the trpEDCBA operon. The sized in the central nervous system and gastrointestinal tract of Fig. 3. The biosynthetic pathways of melatonin in all known living organisms. 546 X. Xie et al. Synthetic and Systems Biotechnology 7 (2022) 544–553 Fig. 4. The biosynthetic pathway of L-tryptophan. Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; 6PGNL 6-phosphoglucono-lactone, G3P, glyceraldehyde-3- phosphate; 3 PG glycerate-3-phosphate; 3-Hpyr, 3-phosphonoox­ ypruvate; PEP, phosphoenolpyruvate; E4P, erythrose- 4-phosphate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; PRPP, phosphoribosyl pyr- ophosphate; PYR, pyruvate; AKG; α-ketoglutarate; Glu, glutamate; Gln, glutamine; P-Ser, 3-phosphoser­ ine; L-Ser, serine;DAHP, 3-deoxy-d-arabi-noheptulos­ onate-7-phosphate; SHIK, shikimate; CHA, chorismate; ANT, anthranilate; PRANT, N-(5′ -phos­ phoribosyl)-anthranilate; Trp, tryptophan. mammals. The presence of serotonin is strictly related to the syn­ Kino et al. found that L101F and W180F changes in the active center of thesis of melatonin in plants and animals. It is a key intermediate in the Pseudomonas aeruginosa have an effect on substrate specificity and hy­ melatonin synthesis pathway. The biosynthetic pathway of serotonin droxylase activity. The catalytic rate constant Kcat increased from 0.4 to from L-tryptophan is summarized in Fig. 5. 2.08 after mutation at the two sites [47,48]. Rosetta modeling is a useful In microbial production, serotonin is mainly synthesized from tool for studying the effects of mutations on catalytic function in more externally added L-tryptophan. Germann et al. used truncated H. sapiens detail. Generally, high-throughput screening based on biosensors can be HsTPH146-460, lacking both the N- and C-terminal regulatory regions in used to identify effective mutations. Maxel et al. applied this selection order to increase heterologous expression and enhance protein stability, platform to screen a NADPH-dependent-hydroxybenzoic acid hydroxy­ along with H. sapiens DDC, Bos taurus AANAT and H. sapiens ASMT to lase that uses 3,4-dihydroxyphenyl acid as a substrate, and obtained produce 2.4 mg/L of serotonin. Tryptophan hydroxylase has been variants with roughly 8-fold improved apparent catalytic efficiency expressed in E. coli with low enzymatic activity, as the solubility and (Kcat/Km) for 3,4-DHBA, compared to the wild type [49,50]. stability of the enzyme seem to be affected in this host. A Rational design of enzymes is becoming the dominant approach. serotonin-producing strain of Saccharomyces cerevisiae was constructed Therefore, it is necessary to elucidate the structure of the enzyme and by introducing L-tryptophan hydroxylase from Cupriavidus taiwanensis modify it to enhance the specificity of substrate binding by library together with a cofactor reconstitution pathway, and the yield of sero­ construction and rational in silico design. Windahl et al. solved the tonin reached 154.3 ± 14.3 mg/L. Therefore, introducing heter­ first crystal structure of an aromatic amino acid hydroxylase, TPH. The ologous enzymes is helpful for the improvement of 5HT production. The substrate specificity of the TPH is controlled through interactions with production of serotonin can also be improved by regulating the the side chain of tryptophan. The side chain of the tryptophan is bound expression levels of relevant enzymes. Directed evolution of enzymes in a hydrophobic pocket lined by residues Tyr236, Thr266, Pro267, can increase their affinity for the substrate and reduce side-reactions. Glu268, Pro269, His273, Phe314, Phe319, and Ile367. Human TPH Improving the supply of cofactors and oxygen are also effective ways can be expressed in E. coli and human embryonic kidney cells (HEK293). to improve the titer of serotonin. With L-tryptophan as the substrate, the Km value of wild-type TPH is 41 μM, while the Km value of the TPH2 mutant R441H was 33 μM. 3.2.1. L-tryptophan hydroxylase (Table 1 and Table 2). Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O2, and 3.2.2. L-tryptophan decarboxylase tetrahydrobiopterin (BH4) to produce 5-hydroxytryptophan and 4a- L-tryptophan decarboxylase (TDC) is a cytosolic type-II aromatic L- hydroxytetrahydrobiopterin. The role of TPH is to replace the H amino acid decarboxylase [53–57]. It catalyzes the final step in the at the C5 position on the benzene ring of tryptophan with an OH radical, microbial pathway of serotonin synthesis. TDC catalyzes two reactions, which as the first step in the synthesis of serotonin. The hydroxylation one of which produces tryptamine from L-tryptophan and the other se­ reaction is the first and rate-limiting step in the synthesis of serotonin rotonin from 5-hydroxytryptophan. Because TDC has an affinity for [45,46]. Therefore, it is crucial to find a suitable enzyme to increase L-tryptophan, tryptamine is readily produced as a byproduct, which can serotonin production. affect the accumulation of serotonin. Therefore, to reduce the produc­ Directed evolution can improve the catalytic activity of enzymes. tion of the by-product tryptamine, it is particularly important to select 547 X. Xie et al. Synthetic and Systems Biotechnology 7 (2022) 544–553 TDC activity [58,62,63]. The KOD-plus-neo PCR method can also be used for site-directed mutagenesis to change the size of the active pocket and achieve better binding of the substrate 5-hydroxytryptophan. A optimization through promoter engineering and confirms the interac­ tion model can be used which can also lift the rate-limiting bottleneck enzyme. Song Gao, using promoter regulation and directed evolution in the host Saccharomyces cerevisiae, established a high-throughput screening method to improve the catalytic efficiency of flavonoid 3′ -hydroxylase, so that (2S)-eriodictyol reached the current highest titer of 3.3 g/L. 3.2.3. Cofactors To increased product titers, it is necessary to not only overexpress pathway enzymes, but also boost the supply of cofactors such as BH4 and NADPH. The conversion of L-tryptophan into 5-hydroxytryptophan by TPH requires both oxygen and the cofactor BH4, which is produced from GTP. Germann et al. constructed a strain of S. cerevisiae containing heterologous genes and increased the supply of cofactors to achieve a product titer of 14.5 mg/L [18,33]. However, E. coli cannot synthesize the cofactor BH4 by itself. Directed metabolic pathway evolution was applied to achieve pterin-dependent hydroxylation of L-tryptophan in E. coli. Luo et at. used a minimum set of heterologous enzymes and a host folE (T198I) mutation for achieving pterin-dependent L-tryptophan hydroxylation by directed metabolic pathway evolution. More impor­ tant, a heterologously introduced BH4 biosynthetic pathway was deleted during adaptive laboratory evolution, the host strain repurposed one of its native cofactors, but it remained unclear exactly which E. coli pterin was repurposed as an alternative cofactor. Heterologous expression of enzymes and the enzyme mutation folE (T198I) in E. coli can be used to increase the production of GTP and BH4. A mutagenized TPH was expressed in E. coli, and 0.8 mM 5-HTP was synthesized after adding BH4 as a substrate. In addition, the BH4 regeneration pathway and glucose dehydrogenase of Bacillus subtilis were introduced to in­ crease the utilization rate of BH4, and the production of 5-HTP was increased to 2.5 mM [38,47,66]. 3.3. Melatonin synthesis Melatonin produced by a two-step enzymatic conversion of seroto­ Fig. 5. Synthesis of serotonin from L-tryptophan. nin. Serotonin is first converted by N-acetyltransferase, which replaces its hydrogen at the C3 position with N-acetyl-2-aminoethyl. Subse­ appropriate enzymes. quently, it is methylated at hydroxy position. In the pathway of TDC has different affinity for L-tryptophan and 5-hydroxytryptamine. melatonin synthesis from serotonin, ASMT is a rate-limiting enzyme. The Km value of TDC is generally higher in plants. Similar to the en­ The biosynthetic pathway of melatonin from serotonin is summarized in zymes from rice and Ophiorrhiza pumila, Withania somnifera TDC is Fig. 6. highly specific for tryptophan, and does not accept 5-hydroxytrypto­ phan to produce serotonin. The Km values of TDC enzymes from 3.3.1. Arylalkylamine N-acetyltransferase different sources are summarized in Tables 1 and 2 TDC is a homodimer Arylalkylamine N-acetyltransferase (AANAT/SNAT) is an enzyme of with strict specificity to the substrate tryptophan. The affinity between the GNAT (GCN-5 related N-acetyltransferase) superfamily, which is the enzyme and substrate is not only related to the size of the active site generally considered to be a crucial enzyme for melatonin synthesis in pocket, but is also affected by the hydrophobicity of the surrounding vertebrates. It also converts serotonin to N-acetyl-serotonin during residues. In the synthesis of serotonin, TDC can convert both microbial synthesis of melatonin. tryptophan and 5-hydroxytryptophan, but exhibits different catalytic Increasing the yield of N-acetyl serotonin requires increasing the efficiency [58–61]. Zhou et al. used protein modeling to deduce that the catalytic activity of the enzyme. Therefore, we need to understand its bottom binding pocket and active cavity of TDC were mainly composed catalytic mechanism. AANAT has a catalytic funnel combined with of hydrophobic residues of one subunit (W95, F103, F104 and L336) and reactive groups (amine and AcCoA). The catalytic histidines (His120, adjacent subunits (V125, F127, L360 and V380). In contrast to His122) at the bottom of the active site form a proton channel, which 5-hydroxytryptophan, L-tryptophan lacks a hydroxyl group on the indole deprotonates the amino group to initiate the acetyl transfer reaction. ring. Therefore, the potential reason why the TDC binding pocket can The residue corresponding to Ile160 in AANAT of sea bream (Sparus hold tryptophan is reduced steric hindrance and higher hydrophobicity. aurata) is naturally mutated to methionine, which enhances acetylation Thus, possible strategies to construct a TDC mutant that can use and reduces the Km value. However, someone studies reported that 5-hydroxytryptophan to initiate the synthesis of melatonin include the effect of the mutation at position 160 is difficult to characterize increasing the size of the active site and promoting the formation of because the enzyme has the same residue at that position. At the same hydrogen bonds between the enzyme and the substrate. It was reported time, mutation at position 114 will also cause differences in catalytic that H214 andY359 are crucial for substrate recognition and influence performance. Therefore, it was speculated that the catalytic reaction requires by the synergistic effect of the two mutations. 548 X. Xie et al. Synthetic and Systems Biotechnology 7 (2022) 544–553 Table 1 Melatonin synthesis enzymes from plants and their Km values. Enzymes Organizm Km[mM] Enzymes Organizm Km[mM] TDC Catharanthus roseus 0.075 SNAT Homo sapiens 0.06 Vinca minor 1.3 0.096 Withania coagulans 1.7 0.106 Gelatoporia subvermispora 0.16 1.23 0.16 1.35 0.29 Ovis aries 0.125 1.94 0.64 1.94 Rattus norvegicus 1.7 Bacillus sp. (in: Bacteria) 0.3 2 T5H Homo sapiens 0.0147 Sparus aurata 0.05 0.015 2.05 0.0217 Drosophila melanogaster 0.16 0.0249 Aedes aegypti 0.23 0.0265 0.42 0.0289 Arabidopsis thaliana 0.232 0.0351 Oryza sativa 0.371 0.0423 Pyropia yezoensis 0.467 0.0518 Saccharopolyspora erythraea 13 0.0684 ASMT Oncorhynchus tshawytscha 0.005 0.077 Ovibos moschatus 0.015. 0.315 Bos taurus 0.0291 Oryctolagus cuniculus 0.0142 0.054 0.031 Rattus norvegicus 0.04 0.135 0.1 Mus musculus 0.294 Oryza sativa 0.243 Gallus gallus 0.324 0.864 Schistosoma mansoni 0.0067 Arabidopsis thaliana 0.456 In addition to complex enzyme modification, heterologous expres­ (Gln310Leu), but also introduced an N single bond (Phe296),hydrogen-π sion can also increase substrate conversion. During microbial melatonin interaction and hydrophobic interaction. These changes increased the synthesis, allogenic expression of enzymes from plants and animals catalytic efficiency in the conversion of N-acetyl serotonin to melatonin. (AANAT/SNAT) can increase the production of N-acetyl serotonin and The construction of COMT variant containing C303F and V321T muta­ melatonin. Ectopic expression of SNAT from Arabidopsis, alfalfa, tomato tions increased the production of melatonin 5-fold. Molecular dy­ or apple leads to increased melatonin production in transgenic tomatoes namics simulations and binding free energy analysis resulted in the and Arabidopsis [72,73]. When the heterologous AANAT from Bos construction of a triple mutant (C296F-Q310L-V314T) with a 9.5-fold taurus was introduced into Saccharomyces cerevisiae, the highest yield of increase of activity. N-acetyl-serotonin reached 9.1 mg/L in shake-flask cultures. In addition to modifying enzymes, we can also express enzymes from Overexpression of ovine AANAT in switchgrass increased the content of different sources. ASMT/COMT orthologs from different plants and an­ melatonin three times compared with the empty vector control. imals exhibit different activities. Compared with the enzymes from an­ The Km values of animal and plant enzymes are summarized in Tables 1 imals, ASMT orthologs from plants exhibit lower Km values (Tables 1 and 2 and 2) [77,78]. 3.3.2. Acetylserotonin O-methyltransferase 3.3.3. Coenzymes N-acetyl-serotonin methyltransferase (ASMT/COMT) belongs to the In the synthesis process of serotonin to melatonin, the cofactors SAM O-methyltransferase family. The ASMT reaction seems to be the main and acetyl-CoA play a significant role. The conversion of serotonin to N- bottleneck in the biosynthesis of melatonin. The catalytic center of acetyl-serotonin requires the participation of acetyl-CoA. In the enzyme exhibits different binding affinity for compounds with Saccharomyces cerevisiae, the supply of acetyl-CoA can be increased by similar structures, resulting in variation of catalytic efficiency towards overexpressing acetaldehyde dehydrogenase or by restricting oxygen different substrates. It can recognize both N-acetyl-serotonin and. However, anaerobic conditions will affect the upstream hydrox­ serotonin, and catalyzes the methylation reaction in the core pathway of ylation reactions. Kocharin et al. increased the acetyl-CoA supply by O-melatonin biosynthesis. However, due to the presence of amide overexpressing acetaldehyde dehydrogenase, which enhanced the pro­ groups in the N-acetyl serotonin terminal chain, when the substrate ductivity of polyhydroxybutyrate approximately 16.5 times in biore­ enters the substrate binding pocket of Arabidopsis thaliana COMT, strong actor cultivations. In addition, SAM is also an important cofactor electrostatic repulsion will occur, preventing N-acetyl serotonin from for the last step of the pathway, in which ASMT methylates N-ace­ binding to the catalytic pocket. tyl-serotonin to the final product melatonin. Thus, the conversion to In order to improve the catalytic efficiency of ASMT with N-acetyl melatonin is accompanied by the concomitant conversion of SAM to serotonin, we need to improve the substrate affinity of the enzyme. S-adenosyl-L-homocysteine (SAH). The SAM cycle is native and consti­ COMT has only three highly conserved amino acids (Met-230, Asp-233 tutively expressed in budding yeast. Cofactor regulation also plays and Met-386), among which the conserved hydrophobic residues Met- a vital role in constructing a highly efficient cell factory. A com­ 230 and Met-386 are isolated in the benzene ring, and the benzene parison of the specific productivities of polyhydroxybutyrate producing ring faces the 5th position of the SAM aromatic ring provide the active strains revealed that employing the acetyl-CoA boost plasmid helps hydroxyl. Based on notable differences in the terminal structure of caf­ redirect carbon fluxes towards the polyhydroxybutyrate pathway [81, feic acid and N-acetyl serotonin, mutants were designed to strengthen 83]. the interactions between the substrate binding pocket of the enzyme and the terminal structure of the unnatural substrate N-acetyl serotonin [77, 78]. Rational design not only eliminated electrostatic repulsion 549 X. Xie et al. Synthetic and Systems Biotechnology 7 (2022) 544–553 Table 2 Melatonin synthesis enzymes from animals and their Km values. Enzymes Organizm Km Enzymes Organizm Km[mM] [mM] TPH Homo 0.0075 TPH Mus musculus 0.0075 sapiens 0.0078 0.00873 0.0106 0.0166 0.0131 0.0192 0.0132 0.031–0.033 0.015 Rattus 0.0865 norvegicus 0.017 0.119 0.0185 0.119 0.0186 Cavia 0.002 porcellus 0.02 0.3 0.0228 Oryctolagus 0.0021 cuniculus 0.0228 0.0058 0.023 0.032 0.02426 0.0479 0.0255 Gallus gallus 0.0077 0.0282 Bos taurus 0.016 Fig. 6. Synthesis of melatonin from serotonin. 0.033 Thunnus 0.023 albacares 0.0334 Schistosoma 0.022 discussed, providing a good reference for future microbial metabolic mansoni engineering projects for the biosynthesis of melatonin. 0.0403 TDC Catharanthus 1.3 Microbial metabolic networks are complex, and intermediate me­ roseus 0.0413 Gelatoporia 0.32 tabolites participate in many different metabolic modules. If the supply subvermispora of the precursor L-tryptophan or serotonin is insufficient, there will note 0.0434 0.32 be enough impetus to produce melatonin. Therefore, to improve L-tryptophan production, we can enhance the supply of precursors, such 0.0449 0.35 as glutamine, L-serine, shikimic acid and phosphoribose pyrophosphate. 0.0511 1.72 Feedback inhibition of key enzymes such as DAHP synthase (aroGFH) and anthranilate synthase (trpED) can be removed [35,134]. For the 0.0545 biosynthesis of melatonin in E. coli, TDC is the rate-limiting enzyme. However, TDC generates the by-product tryptamine, and TPH has a low 0.111 affinity for tryptamine, which leads to the accumulation of a large amount of tryptamine, which affects cell growth. Hence, to increase the precursor supply of serotonin, we can improve the activity of TPH and TDC through site-directed mutation guided by rational design or 4. Conclusions through directed evolution based on high-throughput screening. To improve TDC, it is necessary to enhance the specific binding between the In mammals, melatonin is released from the pineal gland into the enzyme and substrate. To improve the ASMT/COMT and SNA­ third ventricle and from there into circulation. It is involved in regu­ T/AANAT enzymes that synthesize melatonin, it is necessary to under­ lating the body’s sleep-wake cycle through its interactions with the su­ stand their structure, analyze their catalytic mechanism, identify the key prachiasmatic nucleus of the hypothalamus and the retina, promoting sites and carry out site-directed mutagenesis. sleep. In today’s stressful society, melatonin is a hot commodity. With the rapid development of computer technology, there are However, with the increase of annual demand, a green and efficient endless ways to modify enzymes based on in silico analysis. Some synthesis method is needed to meet the market demand, and the most scholars have combined the relevant theories of proteomics and bioin­ promising approach is microbial fermentation. The synthesis pathways formatics to use protein design software to improve enzyme activity. For of melatonin differ between species. In animals and plants, the first two example, DeepMind has been developing the Alpha-Fold system, which steps of melatonin synthesis are reversed and the Km of the involved system improves the accuracy of protein structure prediction by inte­ enzymes are significantly different (Tables 1 and 2). Among microor­ grating novel neural network architectures and training programs based ganisms, the synthetic pathways of yeast and Pseudomonas are similar on evolutionary, physical, and geometric constraints of protein struc­ to those in animals. E. coli requires the expression of heterologous en­ ture, which could be used as a computer-aided tool for enzyme modi­ zymes synthesize melatonin, such as TDC, SNAT, AANAT, ASMT and fication in the future. The development of metabolic engineering COMT. We summarized recent advances in the modification of micro­ has brought greater appreciation of its feasibility for the industrial organisms to produce melatonin. Therefore, recent and representative production of melatonin, and significant progress is expected in the publications on the subject using different strategies were selected and 550 X. Xie et al. Synthetic and Systems Biotechnology 7 (2022) 544–553 future. Morcillo-Parra MA, Gonzalez B, Beltran G, Mas A, Torija MJ. Melatonin and glycolytic protein interactions are related to yeast fermentative capacity. Food Microbiol 2020;87. Funding Fernandez-Cruz E, Alvarez-Fernandez MA, Valero E, Troncoso AM, Garcia- Parrilla MC. Melatonin and derived l-tryptophan metabolites produced during This work was supported by the National Key R&D Program of China alcoholic fermentation by different wine yeast strains. Food Chem 2017;217: 431–7. (2021YFC2100900), National Nature Science Foundation of China Sun T, Chen L, Zhang W. Microbial production of mammalian melatonin - a (32100062), Youth Innovation Promotion Association, CAS (2020182), promising solution to melatonin industry. Biotechnol J 2016;11:601–2. Tianjin Synthetic Biotechnology Inno-vation Capacity Improvement Byeon Y, Back K. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase. Appl Project (TSBICIP-CXRC-029). Microbiol Biotechnol 2016;100:6683–91. Ben-Abdallah M, Bondet V, Fauchereau F, Beguin P, Goubran-Botros H, Pagan C, Declaration of competing interest Bourgeron T, Bellalou J. Production of soluble, active acetyl serotonin methyl transferase in Leishmania tarentolae. Protein Expr Purif 2011;75:114–8. Park S, Byeon Y, Kim YS, Back K. 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