Serotonin-producing recombinant bacteria and use thereof

Abstract

The present application relates to a kind of recombinant bacteria of producing serotonin and its application, belong to fermentation technical field.The present application finds out that after the glutamic acid of the 166th of tryptamine-5-hydroxylase is mutated into lysine and the valine of the 220th is mutated into glutamic acid, the enzyme activity of the mutant of tryptamine-5-hydroxylase is significantly improved, simultaneously, the mutant of tryptamine-5-hydroxylase also significantly improves the coupling efficiency of NADPH (the ratio between the serotonin generated and the NADPH consumed), the improvement of coupling efficiency means that under the condition of same concentration of NADPH, more serotonin can be synthesized, thus it is advantageous to the fermentation of serotonin.In addition, the present application also improves the synthesis of NADPH, the synthesis of L-tryptophan and the like, and the recombinant bacteria obtained can produce serotonin, and can reduce the fermentation cost of serotonin.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a recombinant bacterium that produces serotonin and its application. Background Technology

[0002] Serotonin, also known as 5-hydroxytryptamine, is a high-value L-tryptophan derivative with effects such as regulating mood, maintaining sleep, and relieving anxiety and stress, earning it the nickname "happiness hormone." Currently, serotonin is mainly produced through chemical synthesis and extraction from plants and animals, which suffers from drawbacks such as high energy consumption, severe environmental pollution, and low extraction rates. Utilizing microbial cell factories to produce serotonin offers advantages such as high efficiency, safety, health, and sustainability, and holds promise as becoming the primary method for serotonin production.

[0003] Serotonin biosynthesis uses L-tryptophan as a substrate and can be synthesized through two steps: decarboxylation and hydroxylation. These two steps can be performed sequentially. Currently, serotonin biosynthesis mainly follows the route of hydroxylation followed by decarboxylation. However, due to the selectivity tolerance of aromatic amino acid decarboxylases (TDCs) for substrates 5-hydroxytryptophan and L-tryptophan, and the metabolic burden of tryptophan hydroxylase (TPH) on the heterologous regeneration of tetrahydropterin BH4 cofactor, the byproduct tryptophan accumulates severely, resulting in insufficient serotonin synthesis conversion [Shen, P.,Gu, S.,Jin, D.,Su, Y.,Wu, H.,Li, Q.,Yang, J.,He, W.,Huang, J., & Qi, F. (2022). Engineering Metabolic Pathways for Cofactor Self-Sufficiency and Serotonin Production in Escherichia coli. ACS syntheticbiology, 11 (8), 2889-2900]. In the biosynthesis of serotonin via decarboxylation followed by hydroxylation, insufficient enzyme activity of tryptamine-5-hydroxylase (T5H), which catalyzes the synthesis of serotonin from tryptamine, limits the efficiency of serotonin synthesis. Although studies have reported that constructing the N-terminal deletion mutant Δ40T5H-F114C / M133Q / C247N of T5H and using FdR-FdX as the reductase can significantly improve the activity of T5H enzyme, the serotonin yield remains low (998 mg / L) [Hebei Weidakang Biotechnology Co., Ltd. Tryptamine hydroxylase mutants and their applications: 202411242408.X [P]. 2024-12-06]. Studies have reported that co-expression of T5H and P450 reductase (CPR) can significantly increase T5H enzyme activity [Park, S., Kim, YS, Rupasinghe, SG, Schuler, MA, & Back, K. (2013). Rice P450 reductases differentially affect P450-mediated metabolism in bacterial expression systems. Bioprocess and biosystems engineering, 36 (3), 325-31]. Simultaneously, co-expression of T5H, CPR, and L-tryptophan decarboxylase (TDC) can construct a cell factory for the conversion of L-tryptophan to serotonin. This reaction pathway has no intermediate byproducts, and the chassis cells of *E. coli* can synthesize the cofactor NADPH themselves.However, problems such as low T5H enzyme activity, insufficient NADPH regeneration cycle, imbalance in serotonin synthesis metabolism, and insufficient supply of the substrate L-tryptophan result in low efficiency and high cost of large-scale serotonin production. Therefore, there is an urgent need to develop a method for serotonin production that improves synthesis efficiency while reducing production costs. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems of low serotonin production efficiency and high production cost in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a recombinant bacterium producing serotonin and its applications. This invention discovers that mutating glutamic acid at position 166 of tryptophan-5-hydroxylase to lysine and valine at position 220 to glutamic acid significantly increases the enzyme activity of the resulting tryptophan-5-hydroxylase mutant. More importantly, this combined mutant improves the coupling efficiency of tryptophan-5-hydroxylase to NADPH (the ratio between serotonin produced and NADPH consumed). This means that under the same NADPH concentration, more serotonin can be synthesized, which is beneficial for serotonin fermentation. Furthermore, this invention also improves NADPH synthesis and L-tryptophan synthesis, resulting in a recombinant bacterium capable of high serotonin production. Additionally, the production cost of biofermentation using the recombinant bacterium of this invention is significantly reduced.

[0006] The first objective of this invention is to provide a serotonin-producing recombinant bacterium obtained by modifying Escherichia coli by overexpressing a tryptophan-5-hydroxylase mutant, P450 reductase, membrane-bound transhydrogenase, glucose-6-phosphate-1-dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, tryptophan decarboxylase, 3-deoxy-D-arabino-heptanoate heptaphosphate synthase, and anthranilate synthase.

[0007] The tryptamine-5-hydroxylase mutant is a tryptamine-5-hydroxylase with the amino acid sequence shown in SEQ ID NO.1 modified as follows: glutamic acid at position 166 is mutated to lysine and valine at position 220 is mutated to glutamic acid.

[0008] Furthermore, tryptamine-5-hydroxylase works synergistically with cytochrome P450 reductase (CPR). Cytochrome P450 reductase transfers electrons from NADPH to tryptamine-5-hydroxylase, thereby supporting its catalytic activity. This synergistic effect is crucial for improving the enzyme's catalytic efficiency. The tryptamine-5-hydroxylase mutant of the present invention exhibits 17.16 times higher enzyme activity compared to the wild-type tryptamine-5-hydroxylase, and the tryptamine-5-hydroxylase mutant of the present invention significantly improves coupling efficiency (coupling efficiency is the ratio between serotonin generated and NADPH consumed). This increased coupling efficiency means that more serotonin can be synthesized under the same NADPH concentration, which is beneficial for serotonin fermentation. Therefore, the recombinant bacteria constructed using the tryptamine-5-hydroxylase mutant of the present invention can produce more serotonin.

[0009] Furthermore, the recombinant bacteria are expressed using a dual plasmid expression system, wherein the tryptophan-5-hydroxylase mutant, P450 reductase, tryptophan decarboxylase, 3-deoxy-D-arabino-heptanoate heptaphosphate synthase, and anthranilate synthase are expressed using the same plasmid, while the membrane-bound transhydrogenase, glucose-6-phosphate 1-dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase are expressed using another plasmid.

[0010] Furthermore, the gene encoding the tryptophan-5-hydroxylase mutant is located upstream of the gene encoding P450 reductase, the gene encoding the tryptophan decarboxylase is located downstream of the gene encoding P450 reductase, the gene encoding the 3-deoxy-D-arabino-heptanoate heptaphosphate synthase is located downstream of the gene encoding the tryptophan decarboxylase, and the gene encoding the anthranilate synthase is located downstream of the gene encoding the 3-deoxy-D-arabino-heptanoate heptaphosphate synthase. The tryptophan-5-hydroxylase, P450 reductase, and tryptophan decarboxylase are each generated by a promoter P... T7 Regulate expression.

[0011] Furthermore, the membrane-bound transhydrogenase is activated by the promoter P J23100 The expression of glucose-6-phosphate 1-dehydrogenase is regulated by the promoter P. J23106 The expression of glyceraldehyde-3-phosphate dehydrogenase is regulated via the promoter P. J23106 Regulate expression.

[0012] Furthermore, the promoter P J23100 The gene sequence is shown in SEQ ID NO.2, and the promoter P... J23106 The gene sequence is shown in SEQ ID NO.3, and the promoter P... T7 The gene sequence is shown in SEQ ID NO.4.

[0013] Furthermore, the GenBank accession number for the P450 reductase is XP_015650780.1, the GenBank accession number for the membrane-bound transaminase is X04195.1, the GenBank accession number for the glucose-6-phosphate 1-dehydrogenase is M55005.1, the GenBank accession number for the glyceraldehyde-3-phosphate dehydrogenase is AAC00355.1, the GenBank accession number for the tryptophan decarboxylase is XP_015648701.1, the GenBank accession number for 3-deoxy-D-arabino-heptanoate heptaphosphate synthase is J01591.1, and the GenBank accession number for the anthranilate synthase is V00368.1.

[0014] A second objective of this invention is to provide a method for producing serotonin, wherein the method involves inoculating the aforementioned recombinant bacteria into a fermentation medium for fermentation culture.

[0015] Furthermore, the fermentation culture includes the following steps:

[0016] S1. The above-mentioned recombinant bacteria are inoculated into the fermentation medium for a single fermentation. During the single fermentation, the concentration of glycerol is controlled to be greater than 0 g / L and less than 5 g / L.

[0017] S2. When the absorbance of the bacterial culture is 9-11, add isopropyl-β-D-thiogalactoside (IPTG) and L-tryptophan for secondary fermentation.

[0018] Furthermore, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and metal ions.

[0019] Furthermore, the fermentation medium comprises (NH4)2HPO4, KH2PO4, MgSO4·7H2O, FeSO4·7H2O, citric acid monohydrate, peptone, yeast extract, glycerol, vitamin H, vitamin B1, CaCl2, CuSO4·5H2O and ZnSO4·7H2O.

[0020] Furthermore, the concentration of the isopropyl-β-D-thiogalactoside is 0.1-0.5 mM.

[0021] Furthermore, the concentration of L-tryptophan is 20-30 g / L.

[0022] Furthermore, the fermentation culture temperature is 20-40℃.

[0023] The beneficial effects of this invention are:

[0024] This invention discovers that mutating glutamic acid at position 166 of tryptophan-5-hydroxylase to lysine and valine at position 220 to glutamic acid significantly increases the enzyme activity of the resulting mutant. More importantly, this combined mutation confers a higher coupling efficiency (the ratio between serotonin produced and NADPH consumed) to tryptophan-5-hydroxylase. This means that under the same NADPH concentration, more serotonin can be synthesized, which is beneficial for serotonin fermentation. Furthermore, this invention improves NADPH synthesis and L-tryptophan synthesis, resulting in a recombinant strain capable of producing serotonin. Additionally, the production cost of biofermentation using the recombinant strain of this invention is significantly reduced. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0026] Figure 1 It represents the total serotonin production catalyzed by the T5H mutant in the whole cell.

[0027] Figure 2 It fine-tunes serotonin production in NADPH synthesis metabolism;

[0028] Figure 3 It involves building cell factories that produce serotonin from L-tryptophan and increasing serotonin production.

[0029] Figure 4 It is to increase the supply of L-tryptophan and the production of serotonin;

[0030] Figure 5 It refers to the serotonin production under fed-batch fermentation. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0032] The culture media involved in the following examples are as follows:

[0033] LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, sterilized at 121℃ for 15 min.

[0034] TB medium: 12 g / L peptone, 5 g / L glycerol, 24 g / L yeast extract, 17 mM potassium dihydrogen phosphate, 72 mM dipotassium hydrogen phosphate, sterilized at 115℃ for 20 min.

[0035] Fermentation medium: 4 g / L (NH4)2HPO4, 13.5 g / L KH2PO4, 1.38 g / L MgSO4·7H2O, 0.2 g / L FeSO4·7H2O, 1 g / L citric acid monohydrate, 1 g / L peptone, 2 g / L yeast extract, 8 g / L glycerol, 0.2 mg / L vitamin H, 5 mg / L vitamin B1, 40 mg / L CaCl2, 60 mg / L CuSO4·5H2O, 0.11 g / L ZnSO4·7H2O.

[0036] Feeding medium: 600 g / L glycerol.

[0037] The whole-cell catalytic conditions involved in the following examples are as follows:

[0038] Recombinant *E. coli* was activated overnight in 5 mL LB liquid medium at 37°C and 220 rpm. 1% of the overnight activated strain was inoculated into 50 mL TB liquid medium and cultured at 37°C and 220 rpm until 0D. 600 =0.6;

[0039] After adding 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) to the bacterial culture, the culture was continued at 24℃ and 220 rpm for 24 h.

[0040] After centrifuging the bacterial culture at 4°C and 6000 rpm for 5 min, discard the supernatant and add BCM-1 liquid culture medium (OD200). 600 =30) Resuspend the strain;

[0041] Add 2.5-5 g / L of tryptophan or L-tryptophan to the resuspended bacterial culture, and incubate at 37℃ and 220 rpm for 72 h. Then, detect the serotonin production in the fermentation broth and bacterial cells.

[0042] The fed-batch fermentation conditions involved in the following examples are as follows: Fed-batch fermentation was carried out in a 7.5 L bioreactor. Recombinant *E. coli* was activated overnight in 200 mL LB broth at 37°C and 220 rpm. The overnight activated strain was then transferred to the fermentation medium at a 10% inoculum. Fed-batch fermentation was conducted at 37°C, 20-30% dissolved oxygen (automatically adjusted by stirring speed), and pH 7.0. During fermentation, the glycerol concentration in the fed-batch medium was controlled at approximately 2 g / L. The OD of the bacterial culture was measured... 600 When the concentration of L-tryptophan reaches 10, 0.2 mM IPTG is added and the fermentation temperature is lowered to 32℃. L-tryptophan with a final concentration of 25 g / L is then added to the reactor at a rate of 0.5–1 g / L / h. Serotonin production is measured after 48 h of fermentation.

[0043] The detection conditions for serotonin in the following examples are as follows:

[0044] Serotonin levels were detected using a reversed-phase C18 column at a temperature of 26°C and a flow rate of 0.8 mL / min. The mobile phase was 10 mM potassium phosphate buffer (pH 7.0) containing 14% ethanol.

[0045] Example 1: Improving T5H enzyme activity and serotonin production by designing T5H mutants.

[0046] (1) First, the gene encoding tryptophan-5-hydroxylase (T5H) fused with the GST tag at the N-terminus was recombined between BamH I and Hind III of the pETduet-1 plasmid to obtain the recombinant plasmid pET-T5H. Then, based on this, the gene encoding P450 reductase (OsCPR) was recombined between Nde I and Kpn I of the plasmid to obtain the recombinant plasmid pET-CPR-T5H. The recombinant plasmid pET-CPR-T5H was then introduced into Escherichia coli BL21(DE3) through chemical transformation to obtain strain WT.

[0047] (2) Based on strain WT, T5H enzyme activity and serotonin production were improved through rational design. The F / G helix of T5H is the substrate channel entrance and has a significant impact on T5H enzyme activity. Non-conserved residues A217, V220, E166, and N229 on the inner side of the F / G region were selected and mutated to larger residues or residues with the highest hydrophilicity to increase steric hindrance. Detection showed that the mutants A217V, V220E, E166K, and N229K significantly increased serotonin production under whole-cell catalytic conditions. Figure 1 As shown, the serotonin production in mutants A217V, V220E, E166K and N229K was 302 mg / L, 415 mg / L, 310 mg / L and 258 mg / L, respectively, which were 37%, 89%, 38% and 18% higher than that in WT (220 mg / L).

[0048] (3) The four dominant single-point mutations, E166K, A217V, V220E, and N229K, were combined to form two-point, three-point, and four-point mutants, and serotonin production was detected by whole-cell catalysis. Figure 1 As shown, the yield increase of the two-point mutant E166K / V220E reached up to 843 mg / L, which is 3.9 times that of WT.

[0049] (4) Crude enzyme solutions of T5H and its mutants were extracted and enzyme activity and NADPH coupling efficiency were detected. The extraction steps of crude enzyme solution were as follows: 1) The culture and induction conditions of T5H were the same as those in whole-cell catalysis of serotonin. After induction, the bacterial solution was centrifuged at 4℃ and 8000×g for 6 min. The supernatant was discarded and the cell pellet was resuspended in 10 mL of 50 mmol / L Tris-HCl buffer (pH=8.0); 2) The cells were disrupted by ultrasonic disruption for 10 min to release the protein into the buffer. The mixture was centrifuged at 4℃ and 10000×g for 20 min. The supernatant solution was taken, which is the crude enzyme solution. The method for detecting crude enzyme activity was as follows: 445 μL of crude enzyme solution was preheated in a 37℃ water bath for 3 min, 5 μL of 1 mol / L tryptophan and 50 μL of 50 mmol / L tetrasodium reduced coenzyme II were added, and the reaction was carried out at 37℃ for 30 min. Finally, the reaction was terminated with 500 μL of concentrated hydrochloric acid, with a final volume of 1 mL. The solution was then filtered through a 0.22 μm aqueous filter membrane, and the serotonin production was determined by HPLC. Enzyme activity unit (1U): The amount of enzyme required to catalyze the production of 1 μmol of serotonin per minute from tryptophan at 37℃. The method for detecting NADPH coupling efficiency was as follows: Before enzyme activity measurement, the absorbance at 340 nm was measured using a microplate reader. After the measurement under normal conditions, the absorbance at 340 nm was measured again. The consumption of tetrasodium reduced coenzyme II was determined by the change in absorbance. The ratio between serotonin production and NADPH consumption was the NADPH coupling efficiency. The test results are shown in Table 1. Compared with WT, the enzyme activities of T5H-E166K, T5H-V220E, and the optimal mutant T5H-E166K / V220E increased by approximately 1.74 times, 4.24 times, and 17.16 times, respectively, and the coupling efficiencies increased by 42%, 61%, and 75%, respectively. This indicates that the T5H mutant not only has higher catalytic activity but also higher NADPH utilization, meaning it can synthesize more serotonin under the same NADPH content.

[0050] Table 1 Enzymatic parameters of T5H and its mutants

[0051] WT 1.70±0.1 52.12±2.75 T5H-E166K 4.66±0.4 73.75±2.40 T5H-V220E 8.90±0.6 83.66±1.98 T5H-E166K / V220E 30.87±1.5 91.08±1.97

[0052] Example 2: Regulating NADPH synthesis metabolism to increase serotonin production

[0053] In the process of serotonin synthesis using tryptophan as a substrate, one molecule of NADPH is consumed for every molecule of serotonin synthesized by T5H. Therefore, after obtaining the double-point mutant strain CPR-T5H-E166K / V220E (strain S1), serotonin production was increased by fine-tuning NADPH metabolism. First, the supply of electron donor NADPH was increased by overexpressing glucose dehydrogenase (GDH), glucose-6-phosphate 1-dehydrogenase (ZWF), glyceraldehyde-3-phosphate dehydrogenase (GapB), and membrane-bound transhydrogenase (Pnt AB). After synthesizing the above-mentioned GDH, ZWF, GapB, and Pnt AB gene sequences, the GDH, Pnt AB, ZWF, and GapB gene sequences were recombined into the pACYCduet-1 plasmid through homologous recombination, respectively, to obtain the recombinant plasmids pACYC-GDH, pACYC-Pnt AB, pACYC-ZWF, and pACYC-GapB. These four recombinant plasmids were co-transformed with plasmid pET-CPR-T5H-E166K / V220E into *E. coli* BL21(DE3), yielding strains S2, S3, S4, and S5. Whole-cell catalytic fermentation revealed that the serotonin yields of strains S3, S4, and S5 were 1.05 g / L, 1.29 g / L, and 1.71 g / L, respectively, representing increases of 24.56%, 53.02%, and 103.85% compared to strain S1 (843 mg / L). Figure 2 However, the serotonin production of strain S2 was 0.68 g / L, which was approximately 19.34% lower than that of strain S1.

[0054] The PntAB, ZWF, and GapB gene sequences were homologously recombinated into the pACYCduet-1 plasmid to obtain the recombinant plasmid pACYC-PntAB-ZWF-GapB. Then, the plasmid pACYC-PntAB-ZWF-GapB was co-transformed with pET-CPR-T5H-E166K / V220E into *E. coli* BL21(DE3) to obtain strain S6. Whole-cell catalytic fermentation revealed that strain S6 produced 2.11 g / L of serotonin. Figure 2 ).

[0055] Three constitutive promoters P with different expression intensities were selected. J23100 P J23106 and P J23117 Fine-tuning the expression of Pnt AB, ZWF, and GapB. Constitutive promoter Pnt AB was expressed via fusion PCR. J23100 P J23106 and P J23117The plasmid was coupled with the Pnt AB, ZWF, and GapB genes, respectively, and the genes were introduced into the plasmid pACYCduet-1 via homologous recombination. Subsequently, the recombinant plasmid was co-transformed with pET-CPR-T5H-E166K / V220E into *E. coli* BL21(DE3). Whole-cell catalytic fermentation revealed that plasmid P... J23100 -PntAB-P J23106 -ZWF-P J23106 The serotonin production was highest in strain S7, co-expressed with GapB and pET-CPR-T5H-E166K / V220E, reaching 2.78 g / L. Figure 2 ).

[0056] Example 3: Constructing a cell factory for L-tryptophan to serotonin and regulating serotonin synthase activity to alleviate serotonin metabolic imbalance.

[0057] Codon optimization was performed on tryptophan decarboxylase (OsTDC) from *Oryza sativa*, and its related gene is shown in SEQ ID NO. 5. A strain synthesizing L-tryptophan to serotonin was constructed by expressing OsTDC, OsCPR, and mT5H (T5H-E166K / V220E). To reduce the accumulation of intermediate metabolites, the activities of key enzymes in serotonin synthesis were regulated by constructing OsCPR, mT5H, and OsTDC genes with different expression sequences. First, the expression activities of CPR and mT5H were regulated by introducing CPR and mT5H genes with different expression sequences into the pETduet-1 plasmid to construct plasmids pETduet-CPR-mT5H and pETduet-mT5H-CPR. Subsequently, these two recombinant plasmids were coupled with plasmid P... J23100 -PntAB-P J23106 -ZWF-P J23106 -GapB co-transformation of *E. coli* BL21(DE3) yielded strains S8 and S9. Strain S9 showed a higher serotonin production (3.23 g / L). Next, through homologous recombination, the OsTDC gene with the T7 promoter was inserted into the plasmid pETduet-mT5H-CPR before, during, and after CPR, constructing recombinant plasmids pET-OsTDC-mT5H-CPR, pET-mT5H-OsTDC-CPR, and pET-mT5H-CPR-OsTDC. Subsequently, these three recombinant plasmids were co-transformed with plasmid P... J23100 -PntAB-P J23106 -ZWF-P J23106-GapB co-transformation of Escherichia coli BL21(DE3) yielded strains S10, S11, and S12. Whole-cell catalysis revealed that strain S12, expressing mT5H, CPR, and OsTDC in that order, produced the highest serotonin yield, reaching 3.54 g / L. Figure 3 ).

[0058] Example 3: Regulation of L-tryptophan synthesis and metabolism

[0059] The amino acid sequence (P0AB91) of 3-deoxy-D-arabino-heptanoate heptaphosphate synthase (DAHPS), a key enzyme in tryptophan synthesis, was retrieved from the Uniprot (https: / / www.uniprot.org / ) website. Optimized DAHPS nucleotide sequence (SEQ ID NO.6) was obtained through E. coli codon optimization. The amino acid sequence (P00895) of anthranilate synthase (AS), another key enzyme in tryptophan synthesis, was retrieved from the Uniprot (https: / / www.uniprot.org / ) website. Optimized AS nucleotide sequence (SEQ ID NO.7) was obtained through E. coli codon optimization. The DAHPS and AS nucleotide sequences were then recombined between BamH I and Hind III of the pETduet-1 plasmid to obtain the recombinant plasmids pET-DAHPS and pET-AS, respectively. Then, the nucleotide sequences of DAHPS and AS, containing the T7 promoter and lactose inducer, respectively, were recombined into plasmid pET-mT5H-CPR-OsTDC to obtain recombinant plasmids pET-mT5H-CPR-OsTDC-DAHPS and pET-mT5H-CPR-OsTDC-AS. These two recombinant plasmids were then combined with plasmid P... J23100 -PntAB-P J23106 -ZWF-P J23106 -GapB co-transformation of Escherichia coli BL21(DE3) yielded strains S13 and S14. Whole-cell catalysis revealed serotonin production in strains S13 and S14 to be 4.03 g / L and 4.12 g / L, respectively. Figure 4 ).

[0060] The DAHPS and AS nucleotide sequences containing the T7 promoter and lactose inducer were recombined into a plasmid expressing mT5H, CPR, and OsTDC in that order, resulting in the recombinant plasmid pET-mT5H-CPR-OsTDC-DAHPS-AS. Then, this recombinant plasmid was combined with plasmid P... J23100 -PntAB-P J23106 -ZWF-P J23106-GapB co-transformation of Escherichia coli BL21(DE3) yielded strain S15. Whole-cell catalysis revealed that strain S15 produced 4.42 g / L of serotonin, an increase of approximately 24.86% compared to strain S12.

[0061] Example 4: Fed-fed fermentation for efficient serotonin synthesis

[0062] The optimal strain S15 was obtained and subjected to fed-batch fermentation in a 7.5 L bioreactor. After 48 hours of fed-batch fermentation, the final serotonin titer was 20.76 g / L, with a production efficiency of 0.43 g / L / h and a conversion rate of 0.83 g / g L-tryptophan. Figure 5 ).

[0063] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant bacterium producing serotonin, characterized in that, The recombinant bacteria were obtained by targeting Escherichia coli (E. coli) Escherichia coli The following modifications were performed to obtain: an overexpression of tryptophan-5-hydroxylase mutant, P450 reductase, membrane-bound transhydrogenase, glucose-6-phosphate 1-dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, tryptophan decarboxylase, 3-deoxy-D-arabino-heptanoate heptaphosphate synthase, and anthranilate synthase. The tryptamine-5-hydroxylase mutant is a tryptamine-5-hydroxylase with the amino acid sequence shown in SEQ ID NO.1 modified as follows: glutamic acid at position 166 is mutated to lysine and valine at position 220 is mutated to glutamic acid.

2. The recombinant bacteria according to claim 1, characterized in that, The recombinant bacteria are expressed using a dual plasmid expression system. The tryptophan-5-hydroxylase mutant, P450 reductase, tryptophan decarboxylase, 3-deoxy-D-arabino-heptanoate heptaphosphate synthase, and anthranilate synthase are expressed using the same plasmid, while the membrane-bound transhydrogenase, glucose-6-phosphate 1-dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase are expressed using another plasmid.

3. The recombinant bacteria according to claim 2, characterized in that, The gene encoding the tryptophan-5-hydroxylase mutant is located upstream of the gene encoding P450 reductase; the gene encoding the tryptophan decarboxylase is located downstream of the gene encoding P450 reductase; the gene encoding the 3-deoxy-D-arabino-heptanoate heptaphosphate synthase is located downstream of the gene encoding the tryptophan decarboxylase; and the gene encoding the anthranilate synthase is located downstream of the gene encoding the 3-deoxy-D-arabino-heptanoate heptaphosphate synthase. The tryptophan-5-hydroxylase, P450 reductase, and tryptophan decarboxylase are each generated by a promoter P. T7 Regulate expression.

4. The recombinant bacteria according to claim 1, characterized in that, The membrane-bound transhydrogenase is activated by promoter P J23100 The expression of glucose-6-phosphate 1-dehydrogenase is regulated by the promoter P. J23106 The expression of glyceraldehyde-3-phosphate dehydrogenase is regulated via the promoter P. J23106 Regulate expression.

5. The recombinant bacteria according to claim 4, characterized in that, The promoter P J23100 The gene sequence is shown in SEQ ID NO.2, and the promoter P... J23106 The gene sequence is shown in SEQ ID NO.

3.

6. The recombinant bacteria according to claim 1, characterized in that, The GenBank accession number for the P450 reductase is XP_015650780.1, the GenBank accession number for the membrane-bound transaminase is X04195.1, the GenBank accession number for the glucose-6-phosphate 1-dehydrogenase is M55005.1, the GenBank accession number for the glyceraldehyde-3-phosphate dehydrogenase is AAC00355.1, the GenBank accession number for the tryptophan decarboxylase is XP_015648701.1, the GenBank accession number for the 3-deoxy-D-arabino-heptanoate heptaphosphate synthase is J01591.1, and the GenBank accession number for the anthranilate synthase is V00368.

1.

7. A method for producing serotonin, characterized in that, The method involves inoculating the recombinant bacteria according to any one of claims 1-6 into a fermentation medium for fermentation culture.

8. The method according to claim 7, characterized in that, The fermentation culture includes the following steps: S1. The recombinant bacteria according to any one of claims 1-6 are inoculated into a fermentation medium for a single fermentation, wherein the concentration of glycerol is controlled to be greater than 0 g / L and less than 5 g / L during the single fermentation. S2. When the absorbance of the bacterial culture after the first fermentation is 9-11, add isopropyl-β-D-thiogalactoside and L-tryptophan and carry out a second fermentation.

9. The method according to claim 8, characterized in that, The concentration of the isopropyl-β-D-thiogalactoside is 0.1-0.5 mM.

10. The method according to claim 8, characterized in that, The concentration of L-tryptophan is 20-30 g / L.

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