Engineered bacteria for heterogenous synthesis of melatonin and construction method and application thereof

Abstract

The application discloses a genetically engineered bacterium for heterologous synthesis of melatonin as well as a construction method and application thereof, and belongs to the field of microbial engineering and synthetic biology. The genetically engineered bacterium comprises melatonin synthesis pathway genes, BH4 synthesis and circulation genes, SAM synthesis genes, a heterologous SAHase gene and an adenosine kinase gene, and further comprises knockout of SAM competitive consumption genes and adenosine degradation genes. The application combines a BH4 supply module and a SAM-ATP cofactor circulation module, solves the bottleneck problems of insufficient cofactor supply and energy imbalance in the synthesis of melatonin, and realizes a 2.43 g / L melatonin yield.

Description

Technical Field

[0001] This invention belongs to the fields of microbial engineering and synthetic biology, specifically relating to a genetically engineered bacterium that synthesizes melatonin heterologously, its construction method, and its application. Background Technology

[0002] Melatonin is an important indoleamine hormone with physiological functions such as regulating circadian rhythms, anti-oxidation, and neuroprotection, and it has wide applications in the pharmaceutical and health product fields. Currently, the industrial production of melatonin mainly relies on chemical synthesis, which has drawbacks such as long production routes and heavy pollution. Synthesizing melatonin using microbial cell factories provides a green alternative.

[0003] The melatonin biosynthetic pathway was heterologously reconstructed in *E. coli*, using tryptophan as a precursor and generating melatonin through a four-step enzymatic reaction involving hydroxylation, decarboxylation, acetylation, and methylation. In this pathway, O Methylation catalyzed by methyltransferases is a key rate-limiting step, relying on S-adenosylmethionine (SAM) as a methyl donor. However, the efficiency of methylation reactions in the prior art is limited by several factors: (1) Insufficient SAM supply. SAM synthesis requires a large amount of ATP, and the host's endogenous ATP supply capacity is limited, making it difficult to support efficient and continuous SAM synthesis. (2) SAH product inhibition. The byproduct of methylation, S-adenosylhomocysteine ​​(SAH), inhibits the synthesis of SAM. O -Methyltransferases have a strong competitive inhibitory effect, causing the reaction rate to decrease rapidly with the extension of reaction time. (3) Adenosine cannot be effectively recycled. After SAH hydrolysis, adenosine is generated. Adenosine is mainly consumed in E. coli through degradation pathways and cannot be effectively recycled into ATP, resulting in energy waste. (4) SAM is consumed by endogenous competitive pathways. Cyclopropane fatty acid synthase (CPA) in E. coli cfa ) and SAM-dependent methyltransferases ( tehB Endogenous pathways such as tryptophan hydroxylase consume SAM, competing with melatonin synthesis for methyl donors. Furthermore, the hydroxylation reaction catalyzed by tryptophan hydroxylase depends on tetrahydrobiopterin (BH4) as a cofactor, but E. coli lacks an endogenous BH4 synthesis pathway.

[0004] To address the aforementioned technical challenges, some studies have attempted to express single cofactor synthesis genes or add exogenous cofactors. However, these fragmented strategies cannot synergistically resolve the multiple constraints among SAM supply, SAH clearance, ATP reuse, and BH4 supply. Therefore, developing an engineered strain that systematically addresses these bottlenecks is of significant technological innovation importance. Summary of the Invention

[0005] This invention provides a genetically engineered bacterium for heterologous melatonin synthesis, its construction method, and its applications. The technical problem to be solved is to systematically overcome multiple bottlenecks in heterologous melatonin-synthesizing *E. coli*, such as insufficient cofactor supply, product inhibition, and energy metabolism imbalance, to achieve efficient melatonin biosynthesis. Specifically, in existing technologies, the BH4 cofactor required for tryptophan hydroxylase is absent in the synthetic pathway of *E. coli*; the supply of the SAM methyl donor required for methylation is limited due to the large ATP consumption during synthesis; and the methylation byproduct SAH... O -Methyltransferases produce a strong product inhibition effect; adenosine generated by SAH hydrolysis cannot be effectively recycled into ATP, resulting in energy waste; at the same time, endogenous E. coli... cfa and tehB These pathways lead to the non-productive consumption of SAM, further exacerbating the shortage of methyl donors. This invention aims to address these multiple constraints simultaneously through systematic genetic engineering.

[0006] Technical solution: The complete technical means and methods of this invention.

[0007] This invention provides a genetically engineered bacterium that synthesizes melatonin heterologously.

[0008] The genetically engineered bacteria include: (1) A heterologous melatonin synthesis pathway module, wherein the module comprises a tryptophan hydroxylase gene, an aromatic amino acid decarboxylase gene, and an aromatic alkylamine. N - Acetyltransferase gene and O -Methyltransferase gene; (2) BH4 synthesis and regeneration genes folE , ptps , spr , pcd , dhpr ; (3) S-adenosylmethionine synthase gene metK ; (4) Heterologous S-adenosine homocysteine ​​hydrolase gene sah1 ; (5) Adenosine kinase gene ado1 ; Preferably, the recombinant Escherichia coli knockout strain is selected from... cfa , tehB At least one SAM competitive depletion pathway gene.

[0009] Preferably, the recombinant Escherichia coli knockout strain is selected from... add , ushA , amn At least one adenosine / AMP degradation pathway gene.

[0010] Preferably, the tryptophan hydroxylase gene is a human tryptophan hydroxylase mutant, HsTPH. MS4 .

[0011] Preferably, the aromatic amino acid decarboxylase gene is derived from *Streptomyces glaucus*. Streptomyces griseofuscus of Sg TDC, by psmH Gene encoding.

[0012] Preferably, the aromatic alkylamine N-acetyltransferase gene is derived from Drosophila melanogaster. Drosophila melanogaster of Dm AANAT.

[0013] Preferably, the O-methyltransferase gene is derived from perennial ryegrass. Lolium perenne of Lp COMT or mutants with enhanced activity, more preferably mutant M2 containing a combination of mutations of T32V, S52F, I316S, and N321T.

[0014] Preferably, the S-adenosylmethionine synthase gene is endogenous in Escherichia coli. metK Gene.

[0015] Preferably, the heterologous S-adenosylhomocysteine ​​hydrolase gene is derived from... Saccharomyces cerevisiae of sah1 Gene.

[0016] Preferably, the adenosine kinase gene is derived from Saccharomyces cerevisiae of ado1 Gene.

[0017] The present invention also provides a method for constructing the above-mentioned genetically engineered bacteria.

[0018] The construction method includes the following steps: (1) The tryptophan hydroxylase gene Hs tph MS4 Cloned into the first expression vector; (2) Aromatic amino acid decarboxylase gene psmH Aromatic alkylamine N-acetyltransferase gene Dmaanat and O -Methyltransferase gene Lpcomt Or a mutant thereof, cloned into a second expression vector; (3) The S-adenosylmethionine synthase gene metK It is expressed in tandem with the O-methyltransferase gene; (4) BH4 synthesis and regeneration genes folE ,ptps , spr , pcd , dhpr Three copies were integrated into the genome in a cistron configuration; (5) The heterologous S-adenosine homocysteine ​​hydrolase gene sah1 Integrate into the genome; (6) Adenosine kinase gene ado1 Integrate into the genome; (7) Knockout using gene editing systems cfa and / or tehB Gene; (8) Knock out a using a gene editing system dd , ushA and / or amn Gene; (9) Transform each expression vector into Escherichia coli host cells and screen for positive transformants.

[0019] Preferably, the gene editing system is a CRISPR-Cas9 system.

[0020] This invention provides a recombinant Escherichia coli expressing an O-methyltransferase mutant. The O-methyltransferase mutant is formed by mutating threonine at position 32 to valine, serine at position 52 to phenylalanine, isoleucine at position 316 to serine, and asparagine at position 321 to threonine.

[0021] In one embodiment of the present invention, the recombinant Escherichia coli has the genome knocked out tnaA The gene also overexpresses the tryptophan hydroxylase mutant HsTPH MS4 Aromatic amino acid decarboxylase psmH N - Acetyltransferase, endogenous E. coli metK Genes, cyclized hydrolases folE 6 Pyruvyltetrahydrobiopterin synthase ptps guanopterin reductase spr Pterin 4a Methanolamine dehydratase pcd dihydropteridine reductase dhpr .

[0022] In one embodiment of the present invention, the tryptophan hydroxylase mutant HsTPH is encoded. MS4The nucleotide sequence is shown in SEQ ID NO.3, and the nucleotide sequence encoding the aromatic amino acid decarboxylase psmH is shown in SEQ ID NO.4, which encodes the... N - Acetyltransferase Dmaanat The nucleotide sequence is shown in SEQ ID NO.6, encoding the endogenous E. coli. metK The nucleotide sequence of the gene is shown in SEQ ID NO.7, encoding the cyclization hydrolase. fole The nucleotide sequence is shown in SEQ ID NO.8, encoding the 6 Pyruvyltetrahydrobiopterin synthase ptps The nucleotide sequence is shown in SEQ ID NO. 9, encoding the guanopterin reductase. spr The nucleotide sequence is shown in SEQ ID NO.10, encoding the pterin. 4a Methanolamine dehydratase pcd The nucleotide sequence is shown in SEQ ID NO.11, encoding the dihydropteridine reductase. dhpr The nucleotide sequence is shown in SEQ ID NO.12.

[0023] In one embodiment of the present invention, the recombinant Escherichia coli is: [a strain with knocked-out genome]. tnaA Gene( tnaA The NCBI number is: ACT45387.1 E. coli BL21(DE3) serves as the host cell and expresses the tryptophan hydroxylase mutant HsTPH in free form. MS4 Aromatic amino acid decarboxylase psmH, O-methyltransferase mutant, and endogenous E. coli metK Gene, N - Acetyltransferase Dmaanat , respectively in ykgH , yghE , yeeL The sites (NCBI IDs: ACT42165.1, ACT44635.1, and ACT43746.1) integrated and expressed cyclases. folE 6 Pyruvyltetrahydrobiopterin synthase ptps guanopterin reductase spr Pterin 4a Methanolamine dehydratase pcd dihydropteridine reductase dhpr The recombinant Escherichia coli is: E. coli BL21(DE3), Δ tnaA Δ ykgH ::PT7 - fole - spr - ptps -P T7 - pcd -P T7 - dhpr Δ yghE ::P T7 - fole - spr - ptps -P T7 - pcd -P T7 - dhpr Δ yeeL ::P T7 - fole - spr - ptps -P T7 - pcd -P T7 - dhpr pRSF-P T7 - Hstph MS4 -P T7 - psmH pCDF-P T7 - Lpcomt M2 - Ecmetk -P T7 - Dmaanat It was named M01.

[0024] In one embodiment of the present invention, the recombinant Escherichia coli also overexpresses a strain derived from... Saccharomyces cerevisiae S-adenosylhomocysteine ​​hydrolase sah1 Origin Saccharomyces cerevisiae adenosine kinase ado1 .

[0025] In one embodiment of the present invention, the recombinant Escherichia coli is, using M01 as the host, derived from... Saccharomyces cerevisiae S-adenosylhomocysteine ​​hydrolase sah1 Gene integration into the genome yoeA The locus (NCBI ID: ACT42368) was named M02 (M01, Δ yoeA :: Scsah1 ).

[0026] In one embodiment of the present invention, the recombinant Escherichia coli is, using MO2 as the host, derived from... Saccharomyces cerevisiae adenosine kinase ado1 Integration into the genome mbhAThe locus (NCBI ID: ACT42125) was named M03 (M02, Δ mbhA :: P tac - Scado1 ).

[0027] In one embodiment of the present invention, the S-adenosylhomocysteine ​​hydrolase is encoded Scsah1 The nucleotide sequence is shown in SEQ ID NO.13.

[0028] Encoding the adenosine kinase Scado1 The nucleotide sequence is shown in SEQ ID NO.14.

[0029] In one embodiment of the present invention, the recombinant Escherichia coli also knocked out the genome encoding adenosine deaminase. add Gene, encoding 5'-nucleotidase ushA Genes and encoding AMP nucleosylase amn Gene; In one embodiment of the present invention, an adenosine deaminase is encoded. add The nucleotide sequence is shown in SEQ ID NO.15; Encoding 5'-nucleotidase ushA The nucleotide sequence is shown in SEQ ID NO.16; Encoding AMP nucleoside enzyme amn The nucleotide sequence is shown in SEQ ID NO.17.

[0030] In one embodiment of the present invention, the recombinant Escherichia coli is a strain of M03 whose genome has been sequentially knocked out of adenosine deaminase. add 5'-nucleotidase ushA and AMP nuclease amn The gene was named M04 (M03, Δ add Δ ushA Δ amn ).

[0031] In one embodiment of the present invention, the recombinant E. coli also knocked out the following gene segments: cfa Genes and tehB Gene; In one embodiment of the present invention, the cfa The nucleotide sequence of the gene is shown in SEQ ID NO.18; The tehB The nucleotide sequence of the gene is shown in SEQ ID NO.19.

[0032] In one embodiment of the present invention, the recombinant Escherichia coli is a strain with the M04 gene knocked out.cfa and tehB The gene was named M05 (M04, Δ cfa Δ tehB ).

[0033] The present invention also provides a method for preparing melatonin, wherein the above-mentioned recombinant Escherichia coli is added to a system containing tryptophan and fermented to prepare melatonin.

[0034] In one embodiment of the present invention, the amount of tryptophan added is 1~5g / L tryptophan.

[0035] In one embodiment of the present invention, the fermentation conditions are as follows: the prepared recombinant Escherichia coli seed liquid is inoculated into the fermentation medium and cultured at 35-37°C and 200-220 rpm in a shaker until the OD600 reaches 6-8; then IPTG is added to a final concentration of 0.1-0.2 mM to induce heterologous expression, and the temperature is lowered to 25-30°C for fermentation to prepare melatonin.

[0036] The present invention also provides the application of the above-mentioned recombinant Escherichia coli in the preparation of melatonin or products containing melatonin.

[0037] The present invention also provides a method for the biosynthesis of melatonin.

[0038] The method includes culturing the above-mentioned genetically engineered bacteria, fermenting them with tryptophan as a substrate, and collecting melatonin from the product.

[0039] Preferably, the method uses glycerol as a carbon source.

[0040] Preferably, the fermentation temperature of the method is 30-37℃, and the induction temperature is 25-30℃.

[0041] Preferably, the pH of the method is maintained at 6.5-7.5.

[0042] Preferably, the method involves feeding and fermenting in batches in a 5-L fermenter.

[0043] Beneficial effects This invention achieves endogenous supply and cyclic regeneration of two key cofactors by simultaneously introducing a BH4 synthesis module and a SAM supply and regeneration module into *E. coli* that heterologously synthesizes melatonin, avoiding the cost and complexity of exogenous addition. For the first time, a complete SAM-ATP coupled cycle is constructed in the melatonin synthesis system. By introducing heterologous SAHase and adenosine kinase and combining them with blocking the adenosine degradation pathway, efficient reuse of SAH to ATP is achieved, providing sufficient energy for SAM synthesis while relieving SAH product inhibition. By knocking out… cfa andtehB By blocking the endogenous competitive consumption of SAM through gene therapy, the effective utilization rate of SAM was further improved. The systematic combination of the above strategies produced a significant synergistic effect, with a melatonin yield of 660.02 mg / L in shake-flask fermentation and 2.43 g / L in a 5-L fermenter, providing an efficient solution for the industrial production of heterologous melatonin synthesis. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the plasmid construction of the genetically engineered bacteria of the present invention.

[0045] Figure 2 The yield of melatonin synthesized by shake-flask fermentation of engineered bacteria M01-M05. Detailed Implementation

[0046] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0047] The detection methods involved in the following embodiments are as follows: Melatonin production testing: HPLC detection method: Dilute the reaction solution 5 times with methanol, centrifuge at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm membrane. Chromatographic column: Dimosoil C18 (5 μL, 250 mm × 4.6 mm), mobile phase: A: 0.1% formic acid aqueous solution, B: 100% methanol, detector: UV detector, detection wavelength: 276 nm, column temperature: 30℃, injection volume: 10 μL, flow rate: 1 mL / min.

[0048] The genotypes of the strains involved in the following examples are shown in Table 1 below: Table 1: Genotypes of different strains

[0049] The method for integrating genes into the genome using the CRISPR-Cas9 system in the following examples is as follows: Gene knockout and genome integration were achieved in *E. coli* BL21(DE3) using pREDCas9 and pGRB plasmids based on the CRISPR-Cas9 system. First, a pGRB-sgRNA plasmid containing the target sequence was constructed, and a donor DNA fragment with a homologous arm (approximately 500 bp) was prepared by PCR. The pREDCas9 plasmid was transformed into the strain, and after L-arabinose-induced expression of Red recombinase, electrocompetent cells were prepared. These cells were then co-transformed with the donor DNA and pGRB-sgRNA plasmid. Positive clones were screened by plate coating, and after PCR sequencing confirmed correct integration, the plasmid was eliminated at 42°C.

[0050] The primer sequences involved in gene editing are shown in Table 2 below: Table 2: Primers

[0051] Example 1: O-methyltransferase mutant Lp Design and preparation of COMT mutant M2 1. Obtaining the target gene The maternal gene used in this invention is derived from perennial ryegrass ( Lolium perenne Caffeic acid O-methyltransferase gene Lp COMT (GenBank accession number: NC_067247.2). This gene was synthesized by a commercial company after codon optimization in E. coli. The nucleotide sequence is shown in SEQ ID NO:2, and the encoded amino acid sequence is shown in SEQ ID NO:1.

[0052] Caffeic acid O-methyltransferase amino acid sequence (SEQ ID NO.1): MGSTAADMAASADEEACMFALQLASSSILPMTLKNAIELGLLEILVAAGGKSLTPTEVAAKLPSAANPEAPDMVDRMLRLLASYNVVSCLVEEGKDGRLSRSYGAAPVCKFLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMSAFEYHGTDPRFNRVFNEGMKNH SIIITKKLLELYHGFQGLGTLVDVGGGVGATVAAITAHYPAIKGVNFDLPHVISEAPPFPGVTHVGGDMFKEVPSGDAILMKWILHDWSDQHCATLLKNCYDALPAHGKVVLVECILPVNPEAKPSSQGVFHVDMIMLAHNPGGRERYEREFEALARGAGFTGVKSTYIYANAWAIEFTK The nucleotide sequence encoding caffeic acid O-methyltransferase (SEQ ID NO.2): 2. O-methyltransferase mutant Lp Preparation of COMT mutant M2 (T32V / S52F / I316S / N321T) 1) Construction of recombinant plasmids containing wild-type enzymes: The LpCOMT gene, with a chemically synthesized nucleotide sequence as shown in SEQ ID NO:2, was then ligated into the pET-28a plasmid. Bam HI、 Han After dIII restriction site removal, the recombinant plasmid pET-28a-LpCOMT was prepared.

[0053] 2) Construction of recombinant vectors containing mutant enzymes: Using the pET-28a plasmid carrying the LpCOMT gene as a template, site-directed mutagenesis was introduced via inverse plasmid PCR. The primers shown in Table 3 were used for mutagenesis at the corresponding sites. The PCR reaction mixture was as follows: 1 μL upstream primer, 1 μL downstream primer, 1 μL template plasmid, 25 μL 2 × Phanta Max Master Mix, and 22 μL ddH2O. The reaction conditions were: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 2 min, and final extension at 72℃ for 5 min, for a total of 30 cycles.

[0054] Table 3: Mutation sites and primers

[0055] The PCR product was digested with Dpn I, self-ligated, and transformed into Escherichia coli JM109. Sequencing confirmed the correctness of the mutant sequence, and recombinant vectors expressing different mutants were prepared.

[0056] 3) Construction of recombinant strains The recombinant vectors containing wild-type enzymes and the recombinant vectors containing mutants obtained in steps 1) and 2) were respectively transformed into... E. coli Recombinant strains were prepared from BL21 (DE3) competent cells: E. coli BL21 (DE3) / pET-28a-LpCOMT, E. coli BL21 (DE3) / pET-28a-M2 (T32V / S52F / I316S / F321T).

[0057] 3. The enzyme activities of the wild-type enzyme and the mutant enzyme prepared in Example 1 were detected respectively. The recombinant strains prepared in the above steps were inoculated into 10 mL of liquid LB medium containing kanamycin (concentration: 50 μg / mL) and cultured at 37℃ for 12 h to prepare seed culture. The seed culture was then transferred to 50 mL of LB liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ until OD600 = 0.6-0.8. IPTG was added to a final concentration of 0.2 mM, and induction was performed at 25℃ for 12 h to prepare fermentation broth. The fermentation broth was centrifuged, the cell pellet was collected, sonicated, and the target protein was purified by Ni-NTA affinity chromatography to prepare wild-type pure enzyme solution and M2 mutant pure enzyme solution, respectively.

[0058] Reaction system (1 mL): 50 mM Tris-HCl (pH 7.4), 1 mM SAM (S adenosine L Methionine), 2 mMNAS (N Acetylserotonin), 100 μL of purified enzyme solution. React at 37℃ for 10 minutes, then terminate the reaction with methanol. HPLC was used for quantitative detection of melatonin production.

[0059] Enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of melatonin per minute under the above conditions. Specific enzyme activity is defined as the activity per milligram of protein.

[0060] The results show: The specific activity of wild-type LpCOMT was 2.58 ± 0.12 U / mg, and the specific activity of mutant M2 (T32V / S52F / I316S / F321T) was 20.84 ± 0.85 U / mg. The above results show that the specific activity of the optimal mutant M2 of this invention is 8.10 times that of wild-type, which is significantly better than other O-methyltransferase mutants in the prior art.

[0061] Example 2: Construction of melatonin-producing genetically engineered bacteria The specific steps are as follows: 1. Preparation of genetically engineered bacteria (1) The tryptophan hydroxylase mutant gene Hs TPH MS4 and decarboxylase gene psmH Cloned into the pRSFDuet-1 vector Bam HI / Hin dIII site and Eco rV / kpn I site, to obtain pRSF-TP plasmid (pRSF-P) T7 - Hstph MS4 -P T7- psmH ).

[0062] Tryptophan hydroxylase mutant gene Hs TPH MS4 The sequence is as follows (SEQ ID NO.3): atggaggatggaatggagacagtgccttggttcccaaaaaaaatcagtgatttagatcattgcgcgaatcgcgttttattgtatgggatcgaactggatgcggaccaccctggctttaaggataatgtttaccgtaaacgccgtaaatattttgccgatttggcaatgaactataaacatggcgacccgattcctaaggtcgagtttacagaagaagagatcaaaacctgggggactgtattccaggagttgaataagctgtatccaacccacgcctgtcgtgagtacttgaagaacctgccattactgagtaaatactgcggttaccgcgaggataacattccacaattggaggatgtttcaaactttctgaaagagcgcacaggcttttcaatccgcccagttgccggctcattatcccctcgtgactttttgtcgggcttagccttccgcgtgttccattgtacgcagtacgttcgtcatagtagcgatccgttctacacaccggaacccgatacctgccacgagttattaggacacgtccccttgttggccgaaccttcgtttgctcagtttagccaagagatcggattggcgtctttaggcgccagcgaggaggctgtgcagaagctggcaacgtgctatttcttcacggtagagtttggactttgcaagcaagatggtcaacttcgtgtctttggggcaggtttattaagctcaatttcggaactgaagcacgctcttagtgggcacgccaaggtgaaaccctttgatccgaagatcacttgcaaacaagagtgcttaatcacaaccttccaggatgtctatttcgtctcagaatcctttgaggacgctaaggagaaaatgcgtgagtttaccaaaactatcaagcgtccgtttggagttaaatacaatccttacacgcgttcgattcaaatcctgaaggatactaaatcaattactagcgctatgtaa psmHThe gene sequence is as follows (SEQ ID NO.4): (2) Dm AANAT and Lp The COMT mutant M2 gene was assembled into the BamHI / HindIII and EcoRV / kpnI sites of the pCDF-Fuet-1 vector, respectively, to obtain the pCDF-CA plasmid (pCDF-P). T7 - Lpcomt M2 -P T7 - Dmaanat ).

[0063] Lp The sequence of the COMT mutant M2 gene is as follows (SEQ ID NO.5): Dm The sequence of the AANAT gene is as follows (SEQ ID NO.6): atggaagatgccctgaccgtatctggcaaaccggctgcctgcccagttgatcaggattgcccgtaccatcgaactgattcagccggaggatggtgaagcggttatcgcgatgctgaagaccttcttcttcaaagatgagccgctgaacacctttctggatctgggtgaatgcaaagaa ctggagaaatattctctgaaaccgctgccggataactgctcctacaaagctgtgaacaaaaagggcgagatcatcggcgtgttcctgaac ggcctgatgcgtcgcccgtctcctgacgacgtgccggaaaaagctgctgactcttgtgaacacccgaagtttaaaaaaatcctgtctctga tggatcacgtcgaggagcagtttaacatcttcgatgtttacccagatgaagaactgatcctggacggtaaaattctgtccgttgacaccaattaccgtggcctgggcatcgcgggccgtctgactgaacgcgcctacgaatacatgcgtgaaaacggcatcaacgtttaccacgttctgtg ttctagccattactccgctcgcgttatggagaaactgggtttccacgaagtcttccgcatgcagttcgcggattacaagccgcagggtgaagttgtattcaaaccggccgctccgcacgttggcatccaggttatggctaaggaagtcggtccggctaaagcggcccagacgaaactgtaa (3) Introducing endogenous Escherichia coli metK The gene was cloned into the pCDF-CA obtained in step (2) and placed in... Lp Downstream of COMT, tandem expression yields the pCDF-CAM plasmid (pCDF-P). T7 - Lpcomt M2 - Ecmetk -P T7 - Dmaanat A schematic diagram of plasmid construction is shown below. Figure 1 As shown.

[0064] endogenous E. coli metK The gene sequence is as follows (SEQ ID NO.7): (4) Using the CRISPR-Cas9 system to synthesize and regenerate the BH4 gene, including the cyclization hydrolase. folE 6 Pyruvyltetrahydrobiopterin synthase ptps guanopterin reductase spr Pterin 4a Methanolamine dehydratase pcd dihydropteridine reductase dhpr Integrate into three copies respectively E. coli BL21(DE3)(Δ tnaA ) strain genome ykgH , yghE , yeeL The loci (NCBI numbers are: ACT42165.1, ACT44635.1, and ACT43746.1).

[0065] Based on the CRISPR-Cas9 system, using the primers in Table 2, the following preparations were obtained: E. coli BL21(DE3) Δ tnaA ( tnaA The NCBI number is ACT45387.1) of the chassis strain. Simultaneously, based on the CRISPR-Cas9 system, using the primers in Table 2, P was sequentially... T7 , fole , spr , ptps P T7 , pcd P T7 , dhpr Integrate separately into E. coli BL21(DE3) Δ tnaA Chassis strains ykgH , yghE , yeeL The sites were prepared separately: E. coli BL21(DE3), Δ tnaA Δ ykgH ::P T7 - fole - spr - ptps -P T7 - pcd -P T7 - dhpr Δ yghE ::P T7 - fole - spr - ptps -P T7 - pcd -P T7 -dhpr , Δ yeeL ::P T7 - fole - spr - ptps -P T7 - pcd -P T7 - dhpr .

[0066] folE The gene sequence is as follows (SEQ ID NO.8): atgaaagaagttaataaagagcaaatcgaacaagctgttcgtcaaattttagaagcgatcggagaagacccgaatagagaagggcttcttgatactccgaaaagagtcgcaaagatgtatgccgaagtattctccggcttgaatgaagatccaaaagaacatttccagactatcttcggtgaaaaccatgaggagcttgttcttgtaaaagatatagcgtttcattctatgtgtgagcatcaccttgttcccttttatggaaaagcacatgttgcatatatcccgcgaggcggaaaggtcacaggactcagcaaactggcacgtgccgttgaagccgttgcaaagcgcccgcagcttcaggaacgcatcacttctacaattgcagaaagcatcgtagaaacgcttgatccgcatggcgtaatggtagtggttgaagcggaacacatgtgcatgacgatgcgcggtgtaagaaaaccgggtgcgaaaactgtgacttcagcagtcagaggcgtttttaaagatgatgccgctgcccgtgcagaagtattggaacatattaaacgccaggactaa ptps The gene sequence is as follows (SEQ ID NO.9): atgagcactgaaggtggtggtcgtcgttgccaggcccaagtatcccgccgcatctctttttctgcatcccaccgcctgtactctaagttcctgtctgacgaagaaaacctgaagctgttcggtaagtgcaacaacccgaacggtcacggtcacaattataaagttgtagtaaccgtgcacggtgaaatcgatccggcaaccggtatggttatgaacctggctgatctgaaaaaatacatggaggaagcgatcatgcaaccgctggaccataaaaacctggatatggacgtaccgtattttgcggacgttgtctccactactgaaaacgttgcggtctacatttgggataacctgcagaaagtactgccggttggcgttctgtacaaagtgaaagtgtacgagacggacaataacattgtggtttacaaaggcgaataa spr The gene sequence is as follows (SEQ ID NO. 10): atggaaggcggcctgggccgtgctgtctgtctgctgaccggtgctagccgcggctttggccgtaccctggccccactgctggctagcctgctgagcccaggtagcgtgctggtgctgtccgcccgtaacgatgaagctctgcgccagctggaagccgaactgggcgcggagcgttctggtctgcgtgttgttcgcgttccggctgatctgggtgcggaagcgggtctgcagcaactgctgggtgcactgcgtgaactgccacgtccgaaaggcctgcaacgtctgctgctgatcaataacgccggttccctgggcgacgtttctaaaggtttcgttgatctgtccgactccacccaggtaaacaactactgggctctgaacctgacctctatgctgtgtctgacgtcttctgtgctgaaagcatttccggactctccgggcctgaatcgcactgttgttaacatcagctccctgtgtgctctgcagccatttaagggttgggccctgtactgcgctggtaaagcagcgcgtgatatgctgttccaggttctggccctggaagaaccgaacgtgcgtgtgctgaactacgcgccgggtccgctggacactgacatgcagcagctggctcgtgagacctccgttgacccggatatgcgtaaaggtctgcaggagctgaaagccaaaggcaaactggtggactgtaaggtaagcgcacagaaactgctgtccctgctggagaaagatgagttcaaaagcggcgctcacgtagacttctatgacaaataa pcd The gene sequence is as follows (SEQ ID NO. 11): atgtctactctgaatcaagctcactgcgaagcatgccgtgcagacgcccctcaggtgtccgaagctgaactgccggagctgctgaaacaaatcccggattggaacatcgaagttcgcgacggtgtgatgcagctggagaaagtctttctgttcaagaacttcaaattcgccctggcgttcactaatgcagttggtgagattgctgaagcagaaggtcaccacccaggtctgctgactgaatggggcaaagttaccgttacttggtggtcccacagcatcaaaggtctgcaccgtaacgatttcatcatggctgcgcgtaccgatggtgttgcttctggtgctgaaggccgtaaataa dhpr The gene sequence is as follows (SEQ ID NO. 12): atggatattatcagcgttgcgctgaaacgtcatagcaccaaagcctttgatgcaagcaaaaagctgaccccggaacaggcagaacagattaaaacgctgctgcagtatagcccgagcagcaccaacagccagccgtggcattttattgtcgcaagcaccgaag aaggtaaagcacgtgttgcaaaaagcgcagcaggtaattatgtttttaatgaacgtaaaatgctggatgcaagccatgtggttgtattttgtgtgcaaaaaccgcaatggatgatgtgtggctgaaactggttgttgatcaggaagatgcagatggccgttttgcc accccggaagccaaagcagcaaatgataaaggtcgtaaattttttgcagatatgcatcgtaaagatttacatgatgatgcagaatggatggcaaaacaggtatatctgaatgttggtaactttctgctgggtgttgcagcactgggtctggatgccgttccga ttgaaggttttgatgcagcaattctggatgcagaatttggtctgaaagaaaaaggttatacctccctggttgttgttcctgttggtcatcattcagttgaagattttaatgcaaccctgccgaaatctcgtctgccgcagaatattacactgacggaagtttaa P7 promoter (the enzyme expression in this invention uses the P7 promoter inherent to the vector plasmid): TAATACGACTCACTATAGG (5) The pRSF-TP plasmid obtained in step (1) and the pCDF-CAM plasmid obtained in step (3) were co-transformed into the engineered bacteria that integrated the BH4 synthesis and regeneration genes in step (4), and positive clones were screened to obtain engineered bacteria M01:BL21(DE3), Δ tnaA Δ ykgH ::P T7 - folE - spr - ptps -P T7 - pcd -P T7 - dhpr Δ yghE::P T7 - folE - spr - ptps -P T7 - pcd -P T7 - dhpr Δ yeeL ::P T7 - folE - spr - ptps -P T7 - pcd -P T7 - dhpr pRSF-P T7 - Hstph MS4 -P T7 - psmH pCDF-P T7 - Lpcomt M2 - Ecmetk -P T7 - Dmaanat .

[0067] 2. Preparation of melatonin by shake-flask fermentation Shake-flask fermentation was carried out using 2 g / L tryptophan as a substrate, as detailed below: The prepared engineered bacteria M01 was inoculated into LB medium and cultured at 37℃ for 12 h to prepare seed culture. The prepared seed culture was inoculated into the fermentation medium at an inoculum of 5% (v / v) and cultured at 37°C and 220 rpm on a shaker until the OD600 reached 6-8. Then, IPTG was added to a final concentration of 0.2 mM to induce heterologous expression, and the temperature was lowered to 30°C for production.

[0068] The fermentation medium consisted of: 10 g / L glycerol, 5 g / L yeast extract, 10 g / L peptone, 2 g / L tryptophan, 2 g / L citrate monohydrate, 4 g / L ammonium sulfate, 1 g / L magnesium sulfate heptahydrate, 4 g / L dipotassium hydrogen phosphate, 100 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, and 1 ml of a vitamin mixture (2.5 mg / L thiamine, 2.5 mg / L niacin, 2.5 mg / L pantothenic acid, 2.5 mg / L biotin, and 2.5 mg / L vitamin B12). The results are shown in the attached figure. Figure 2 As shown, the yield of melatonin synthesized by M01 bacteria after 48 h of fermentation was 354.62 mg / L.

[0069] Example 3: Construction of the SAM-ATP cofactor circulation system The specific steps are as follows: 1. Preparation of strains (1) Based on M01, using the CRISPR-Cas9 system, the source of Saccharomyces cerevisiae S-adenosylhomocysteine ​​hydrolase sah1 Genes consist of a constitutive promoter P tac Driven expression, integration into the genome yoeA Locus (NCBI ID: ACT42368), obtained M02 (M01, Δ yoeA :: Scsah1 ).

[0070] sah1 The gene sequence is as follows (SEQ ID NO.13): (2) The source Saccharomyces cerevisiae adenosine kinase ado1 Genes consist of a constitutive promoter P tac Driven expression, integration into the genome mbhA Locus (NCBI ID: ACT42125), obtained M03 (M02, Δ mbhA ::P tac - Scado1 ).

[0071] ado1 The gene sequence is as follows (SEQ ID NO.14): (3) Adenosine deaminase on the genome of strain M03 was knocked out sequentially using the CRISPR-Cas9 system and the primers in Table 2. add 5'-nucleotidase ushA and AMP nuclease amn Gene, obtain M04 (M03, Δ add Δ ushA Δ amn ).

[0072] (4) Using the CRISPR-Cas9 system and the primers in Table 2, the M04 strain genome was knocked out sequentially. cfa and tehB Gene, obtain M05 (M04, Δ cfa Δ tehB ).

[0073] tehB sequence (SEQ ID NO.19): atgatcattcgtgacgaaaactattttactgataaatatgaattaacccgcacacactctgaagtaatggaagcggtgaaagtggttaaaccgggtaaaacgctggatctgggctgtggcaatggtcgtaacagtctttacctggcag ccaatggttatgatgttgacgcatgggataaaaatgccatgagtatcgccaacgtcgagcgcattaaatctattgaaaatctggataatttacacactcgggtcgttgatctgaataacctcacatttgctggacagtacgattttatt ctttcgactgtggtgctgatgttccttgaggctaaaaccatcccagggctgattgccaatatgcaacgttgcacgaaacctggcggttacaacctgattgtggcggcgatggatagcgctgattatccatgtaccgtcggcttcccgt ttgccttcaaagagagaaattacgtcgatattacgaaggctgggagatggtgaaatacaatgaagacgtcggcgagctgcaccgcaccgacgccaacggtaatcgtattaaactgcgtttcgccacgatgctggcacgtaaaaaatga 2. Preparation of melatonin The yield of melatonin prepared from different strains was measured separately: Specifically, the process is the same as step 2 in Example 2, except that the strains are adjusted to M02, M03, M04, and M05 respectively; the yield of the prepared melatonin is detected according to the method in step 2. The prepared engineered bacteria M02, M03, M04 or M05 were inoculated into LB medium and cultured at 37℃ for 12 h to prepare seed culture. The prepared seed culture was inoculated into the fermentation medium at an inoculum of 5% (v / v) and cultured at 37°C and 220 rpm on a shaker until the OD600 reached 6-8. Then, IPTG was added to a final concentration of 0.2 mM to induce heterologous expression, and the temperature was lowered to 30°C to prepare melatonin.

[0074] The fermentation medium consisted of: 10 g / L glycerol, 5 g / L yeast extract, 10 g / L peptone, 2 g / L tryptophan, 2 g / L citrate monohydrate, 4 g / L ammonium sulfate, 1 g / L magnesium sulfate heptahydrate, 4 g / L dipotassium hydrogen phosphate, 100 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, and 1 ml of a vitamin mixture (2.5 mg / L thiamine, 2.5 mg / L niacin, 2.5 mg / L pantothenic acid, 2.5 mg / L biotin and 2.5 mg / L vitamin B12).

[0075] The results are shown in the attached figure. Figure 2 As shown.

[0076] The results show: The melatonin yields of M02, M03, and M04 after 48 h of fermentation were 469.31, 547.65, and 594.41 mg / L, respectively.

[0077] like Figure 2 As shown, the melatonin production of M05 after 48 h of fermentation was further increased to 660.02 mg / L.

[0078] Comparative example: The strain was constructed in the same manner as in Examples 2 and 3, with the difference being adjustments. Lp COMT mutant M2 is wild-type. Lp The yield of melatonin prepared by COMT enzyme was detected according to step 3 of Example 2. The results showed that: expression of wild-type Lp The COMT enzyme strain yielded 197.55 mg / L after 48 h of fermentation.

[0079] 3. Horizontal preparation of melatonin in fermentation tanks The M05 strain was subjected to fed-batch fermentation in a 5-L scale bioreactor, and the specific steps are as follows: The M05 strain was inoculated into LB medium in a bioreactor and cultured at 37°C until the OD600 was 20-25.

[0080] The excess seed culture medium was then drained, leaving approximately 400 mL. 1.6 L of fermentation medium was added to bring the initial working volume to 2 L, and the culture was carried out at 37°C.

[0081] The fermentation medium consisted of: 10 g / L glycerol, 5 g / L yeast extract, 10 g / L peptone, 3 g / L tryptophan, 2 g / L citrate monohydrate, 4 g / L ammonium sulfate, 1 g / L magnesium sulfate heptahydrate, 4 g / L dipotassium hydrogen phosphate, 100 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, and 1 mL of a vitamin mixture (2.5 mg / L thiamine, 2.5 mg / L niacin, pantothenic acid, biotin, and vitamin B12 2.5 mg / L).

[0082] During fermentation, 50% (v / v) glycerol containing 8 g / L tryptophan was fed. The pH was maintained at 7.0 and dissolved oxygen at 20%-30% by automatic addition of ammonia. When the OD600 reached 30, IPTG was added to a final concentration of 0.2 mM, and the temperature was adjusted from 37℃ to 30℃ to continue fermentation for a total of 60 h.

[0083] The results showed that the melatonin yield reached its highest level of 2.43 g / L after 48 hours of fermentation.

[0084] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An O-methyltransferase mutant, characterized in that, The O-methyltransferase mutant is formed by mutating threonine at position 32 to valine, serine at position 52 to phenylalanine, isoleucine at position 316 to serine, and asparagine at position 321 to threonine.

2. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli expressed an O-methyltransferase mutant, which was formed by mutating threonine at position 32 to valine, serine at position 52 to phenylalanine, isoleucine at position 316 to serine, and asparagine at position 321 to threonine in the amino acid sequence shown in SEQ ID NO.

1. Preferably, the recombinant E. coli has the following genome knocked out: tnaA The gene also overexpresses the tryptophan hydroxylase mutant HsTPH MS4 Aromatic amino acid decarboxylase psmH N - Acetyltransferase, endogenous E. coli metK Genes, cyclized hydrolases folE 6 Pyruvyltetrahydrobiopterin synthase ptps guanopterin reductase spr Pterin 4a Methanolamine dehydratase pcd dihydropteridine reductase dhpr ; Preferably, the cyclized hydrolase folE 6 Pyruvyltetrahydrobiopterin synthase ptps guanopterin reductase spr Pterin 4a Methanolamine dehydratase pcd dihydropteridine reductase dhpr It has 3 copies.

3. The recombinant Escherichia coli according to claim 2, characterized in that, Encoding the tryptophan hydroxylase mutant HsTPH MS4 The nucleotide sequence is shown in SEQ ID NO.3, and the nucleotide sequence encoding the aromatic amino acid decarboxylase psmH is shown in SEQ ID NO.4, which encodes the... N The nucleotide sequence of the α-acetyltransferase is shown in SEQ ID NO.6, encoding the endogenous α-acetyltransferase in the *E. coli*. metK The nucleotide sequence of the gene is shown in SEQ ID NO.7, encoding the cyclization hydrolase. folE The nucleotide sequence is shown in SEQ ID NO.8, encoding the 6 Pyruvyltetrahydrobiopterin synthase ptps The nucleotide sequence is shown in SEQ ID NO.9, encoding the guanopterin reductase. spr The nucleotide sequence is shown in SEQ ID NO.10, encoding the pterin. 4a Methanolamine dehydratase pcd The nucleotide sequence is shown in SEQ ID NO.11, encoding the dihydropteridine reductase. dhpr The nucleotide sequence is shown in SEQ ID NO.

12.

4. The recombinant Escherichia coli according to claim 3, characterized in that, The recombinant E. coli also overexpressed a gene derived from... Saccharomyces cerevisiae S-adenosylhomocysteine ​​hydrolase, derived from Saccharomyces cerevisiae adenosine kinase ado1 ; Preferably, the nucleotide sequence encoding the S-adenosylhomocysteine ​​hydrolase is shown in SEQ ID NO.13; The nucleotide sequence encoding the adenosine kinase is shown in SEQ ID NO.

14.

5. The recombinant Escherichia coli according to claim 4, characterized in that, The recombinant E. coli also had the genome encoding adenosine deaminase knocked out. add Gene, encoding 5'-nucleotidase ushA Genes and encoding AMP nucleosylase amn Gene; Preferably, the nucleotide sequence encoding adenosine deaminase is shown in SEQ ID NO.15; The nucleotide sequence encoding the 5'-nucleotidase is shown in SEQ ID NO.16; The nucleotide sequence encoding the AMP nucleoside enzyme is shown in SEQ ID NO.

17.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The recombinant E. coli also knocked out the genome. CFA Genes and tehB Gene; Preferably, the CFA The nucleotide sequence of the gene is shown in SEQ ID NO.18; The tehB The nucleotide sequence of the gene is shown in SEQ ID NO.

19.

7. A method for preparing melatonin, characterized in that, The method involves adding the recombinant Escherichia coli according to any one of claims 1 to 6 to a system containing tryptophan, and then fermenting it to prepare melatonin.

8. The preparation method according to claim 7, characterized in that, The amount of tryptophan added is 1~5g / L.

9. The preparation method according to claim 7 or 8, characterized in that, The fermentation conditions are as follows: the prepared recombinant Escherichia coli seed liquid is inoculated into the fermentation medium and cultured at 35-37℃ and 200-220 rpm in a shaker until the OD600 reaches 6-8; then IPTG is added to a final concentration of 0.1-0.2 mM to induce heterologous expression, and the temperature is lowered to 25-30℃ for fermentation to prepare melatonin.

10. The use of the recombinant Escherichia coli according to any one of claims 1 to 6 in the preparation of melatonin or products containing melatonin.

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