Application of a carnosine N-methyltransferase gene in increasing the fermentation yield of L-anserine

Abstract

The present invention belongs to the field of biotechnology, and in particular to an application of a carnosine N-methyltransferase gene in increasing the fermentation yield of L-anserine. The carnosine N-methyltransferase gene provided by the present invention includes a gene expressing TaNMT, GgNMT2, AcNMT or AfNMT, which is combined with a pTrc99a plasmid transport system to form a recombinant plasmid, and then transformed into a genetically engineered strain obtained by knocking out the L-carnosine degradation gene in Escherichia coli, obtaining a recombinant strain and fermenting the substrate L-carnosine. In the final fermentation product, the yield of L-anserine is significantly higher than that of the original strain, has the advantage of industrial amplification, and reduces costs.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a carnosine N-methyltransferase gene in improving the fermentation yield of L-anserine. Background Art

[0002] Anserine (β-alanyl-1-methyl L-histidine) is a highly stable water-soluble dipeptide that naturally exists in the skeletal muscle tissue and brain tissue of vertebrates. It has the effects of promoting uric acid excretion and relieving hyperuricemia. It has strong antioxidant and anti-aging functions and can be used as a natural antioxidant and uric acid lowering agent.

[0003] Existing Literature et al. [1] In a study by Cao et al., the UPF0586 protein C9orf41 homolog was identified as a carnosine N-methyltransferase responsible for the production of anserine in rat muscle. The study expressed and purified rat, yeast, chicken, and human UPF0586 protein homologs and confirmed their activity in the production of anserine. Cao et al. (2018) [2] The study explored the molecular basis of specific methylation of histidine N1 site by carnosine N-methyltransferase 1 (CARNMT1). [3] , presented a dipeptide hydrolase BmPepD from Bacillus subtilis, which achieved efficient synthesis of L-carnosine through reverse hydrolysis. Through site-directed mutagenesis, they obtained a more active variant T171N, which provides an efficient pathway for the biosynthesis of L-carnosine. The study by Cheng Schwank-Xu et al. (2021) [4] , explored the effect of L-carnosine on the biosynthesis of coenzyme Q10. In a type 2 diabetic mouse model, L-carnosine enhanced CoQ gene expression and increased liver CoQ biosynthesis, showing the potential role of L-carnosine in regulating mitochondrial function and oxidative stress. However, the enzyme activity of previous studies was limited, and the yield of anserine was also limited.

[0004] [1] J.,Piecuch,M.,Poleszak,O.,Kozlowski,P.,Chrobok,L.,Baelde,H.,&de Heer,E.(2015).UPF0586 Protein C9orf41 Homolog Is Anserine-producingMethyltransferase.Journal of Biological Chemistry,290(28),17190-17203.

[0005] [2]Cao, R., Zhang, X., Liu,

[0006] [3]Guan,B.-H.,Yin,W.-T.,Cao,B.,Zhao,C.,Pan,J.,&Xu,J.-H.(2023).Characterization and mutagenesis of a high-activity and highly substrate-tolerant dipeptidase for L-carnosine biosynthesis via reversedhydrolysis.Molecular Catalysis,113500.

[0007] [4]Cheng Schwank-Xu, Forsberg, E., Bentinger, M., Zhao, A., Ansurudeen, I., Dallner, G., Catrina, S., Brismar, K., & Tekle, M. (2021). L-Carnosine Stimulation ofCoenzyme Q10 Biosynthesis Promotes Improved Mitochondrial Function and Decreases Hepatic Steatosis in Diabetic Conditions.Antioxidants,10(5),793. Summary of the invention

[0008] The carnosine N-methyltransferase gene provided by the present invention, i.e., a gene expressing TaNMT, GgNMT2, AcNMT or AfNMT, is combined with a pTrc99a plasmid transport system to form a recombinant plasmid, which is then transformed into a genetically engineered strain SHK19K obtained by knocking out the L-carnosine degradation gene of Escherichia coli to obtain a recombinant bacterium and ferment the substrate L-carnosine. In the final fermentation product, the yield of L-anserine is significantly higher than that of the original strain, which has the advantage of industrial amplification and reduces the cost.

[0009] the term:

[0010] In the present invention, Carnosine NMT (N-methyltransferase) is an enzyme responsible for catalyzing the conversion of L-carnosine into L-anserine. This conversion involves a methyl transfer process, in which the NMT enzyme plays a key role.

[0011] In the present invention, TaNMT (Tyto alba Carnosine NMT) is a specific Carnosine NMT enzyme extracted from a certain organism (Tyto alba, i.e. the common barn owl).

[0012] In the present invention, GgNMT2 (Gallus gallus Carnosine NMT2) is another Carnosine NMT enzyme extracted from another organism (Gallus gallus, i.e. domestic chicken).

[0013] In the present invention, AcNMT (Athene cunicularia Carnosine NMT) is Carnosine NMT enzyme extracted from Athene cunicularia (cave owl).

[0014] In the present invention, AfNMT (Aythya fuligula Carnosine NMT) is Carnosine NMT enzyme extracted from Aythya fuligula (crested duck).

[0015] In the present invention, recombinant strains are microbial strains modified by genetic engineering, which are designed to overexpress (ie, produce at high levels) specific enzymes, such as Carnosine NMT, to improve production efficiency.

[0016] In the present invention, shake flask fermentation: This is a laboratory-scale fermentation method, usually used for small-scale production or testing. In this method, bottles containing microorganisms and nutrients are shaken under controlled conditions to promote growth and metabolism. In the present invention, L-anserine is produced by shake flask fermentation, in which genetically modified microorganisms (such as bacteria or yeast) are used to efficiently produce the target compound, i.e., L-anserine.

[0017] In one aspect, the present invention provides an application of a carnosine N-methyltransferase gene in increasing the fermentation yield of L-anserine.

[0018] The carnosine N-methyltransferase gene is selected from any one or more of the genes expressing GgNMT2, TaNMT, AcNMT and AfNMT. Preferably, it is selected from any one of them.

[0019] Specifically, the amino acid sequence of GgNMT2 includes SEQ ID NO.1, or a sequence with a sequence homology of more than 60% to SEQ ID NO.1;

[0020] The amino acid sequence of TaNMT includes SEQ ID NO.2, or a sequence with a sequence homology of more than 60% to SEQ ID NO.2;

[0021] The amino acid sequence of AcNMT includes SEQ ID NO.3, or a sequence with a sequence homology of more than 60% to SEQ ID NO.3;

[0022] The amino acid sequence of AfNMT includes SEQ ID NO.4, or a sequence with a sequence homology of more than 60% with SEQ ID NO.4.

[0023] SEQ ID NO.1:

[0024] MEPTPEMKRNRLPSMNFEAEILADPHDNSELYVIPSMRSLTAEEYVEAFQSFLDHSTEHQCMDEFNKEVMPHIMAGLGNGKSTINILGVGSGTGEQDLKMIQILQAAHPGVLINNEIIEPNPQHVAAYKELVNRAPDLQGVSFTWHQLTSSEYEQQVKEKN THKKFDFIHMIQMLYRVEDIPNTIKFFHSCLNHQGKLLIIILSDSSGWASLWKKYRHCLPSTDSGHYITSDSITAVLRKLGIKYHVYEFPSGWDITECFIEGDPAGGHMMDFLTGTKNFLGTAPAALRSRLQEALCQPECSSRKDGRVIFCNNLSMIVAES;

[0025] SEQ ID NO.2:

[0026] MEGKRFNLANGELQLSQEPRQDQLPTSIPLPVSHRDPGMDSTSPKNRFPSMNFEAEILTDPHDNSELYVIPSMRSLTAEEYVEAFKSFLDHSTEHQCMDEFNKEEMPNIMAGLGNGRSTINVLGVGSGTGEQDLKMIRILQAAHPGVLIDNEIIEPNPQHVAAYKELVNQAPDLQNVSFIWHQLTSSEYEQQVKEKGAHKKFDFIHMIQMLYRVEDIPNTIKFFHSCLNHHGKLLIIILSDSSGWASLWKKYRHCLPSTDSGH YITSNGITDVLKRLGVEYHVYEFPSGWDITECFIEGDPVGSRMMDFLTGTKNFLGTAPAGLRQRLQEALCQPECSSKKDGRIIFSNNLSMIVVES;

[0027] SEQ ID NO.3:

[0028] MDPTPEMKRNRFPSMNFEAEILTASHDNSELYVIPSMRSLTAEEYVEAFKSFLDHSTEHQCMDQFNKEAMPNIMAGLGNGKSTINVLGVGSGTGEQDLKMIRILQAAHPGVLIDNEIIEPNPRHVAAYKELVNQAPDLQNVSFIWHQFTSSEYEQQMKEKGAHKKFDFIHMIQMLYRVEDIPNTIKYFHSCLDHHGKLLIIILSDSSGWASLWKKYRHCLPSTDSGHYITSNSITDVLKRLGVEYHVYEFPSGWDITECFIEGDPVGGHMMDFLTGTKNFLGTAPPGLRQRLQEALCQPECSSKKDGRIIFSNNLSMIVVES;

[0029] SEQ ID NO.4:

[0030] MEPAPEMKRNRFPSMNFEAEILAAPHDNSELYVVPSMRSLTAEEYVEAFKSFLDHSTEHQCMDEFNKEEMPRIMAGLGNGKSTINVLGVGSGTGEQDLKMLRILQAAHPGVLINNEIVEPNPQHVAAYKELVNQAPDLQGVSFIWHQLTSSEYEQQVKEKC INKKFDFIHMIQMLYRVEDIPNTIKFFHSCLDHQGKLLIIILSDSSGWASLWKKYRHCLPSTDSGHYITSDSITAVLRRLGIQYRVHEFPSGWDITECFVEGDPAGGQMMDFLTGTKNFLGTAPPALRRRLQEALCQPECSSKKDGRVIFCNNLSMIVVES.

[0031] According to the codon degeneracy, those skilled in the art can arbitrarily design the specific sequence of the expressed gene, provided that the amino acid sequence is known. As an example, the present invention provides genes including but not limited to the following:

[0032] The gene expressing GgNMT2 comprises the nucleotide sequence shown in SEQ ID NO.5, or a sequence encoding the same carnosine N-methyltransferase with a homology of more than 60% to the nucleotide sequence shown in SEQ ID NO.5;

[0033] The gene expressing TaNMT comprises the nucleotide sequence shown in SEQ ID NO.6, or a sequence encoding the same carnosine N-methyltransferase with a homology of more than 60% to the nucleotide sequence shown in SEQ ID NO.6;

[0034] The gene expressing AcNMT comprises the nucleotide sequence shown in SEQ ID NO.7, or a sequence encoding the same carnosine N-methyltransferase with a homology of more than 60% to the nucleotide sequence shown in SEQ ID NO.7;

[0035] The gene expressing AfNMT comprises the nucleotide sequence shown in SEQ ID NO.8, or a sequence encoding the same carnosine N-methyltransferase with a homology of more than 60% to the nucleotide sequence shown in SEQ ID NO.8.

[0036] SEQ ID NO.5:

[0037] ATGGAACCGACCCCGGAAATGAAACGCAACCGTTTACCGAGCATGAACTTTGAAGCGGAAATTCTGGCCGATCCGCATGATAACAGCGAACTGTATGTGATTCCGAGCATGCGCAGCCTGACCGCGGAAGAATATGTGGAAGCGTTTCAGAGCTTTCTGGATCATAGCACCGAACATCAGTGCATGGATGAATTTAACAAAGAAGTGATGCCGCATATTATGGCGGGCCTGGGCAACGGCAAAAGCACCATTAACATTCTGGGCGTGGGCAGCGGCACCGGCGAACAAGACCTGAAAATGATTCAGATTCTGCAAGCGGCGCATCCGGGCGTGCTGATTAACAACGAAATTATTGAACCGAACCCGCAGCATGTGGCGGCGTATAAAGAACTGGTGAACCGCGCGCCGGATCTGCAAGGCGTGAGCTTTACCTGGCATCAACTGACGAGCAGCGAATATGAACAGCAAGTGAAAGAAAAAAACACCCATAAAAAATTTGATTTTATTCATATGATTCAGATGCTGTATCGCGTGGAAGATATTCCGAACACCATTAAATTTTTTCATAGCTGCCTGAACCATCAAGGCAAACTGCTGATTATCATTCTGAGCGATAGCAGCGGCTGGGCGAGCCTGTGGAAAAAATATCGCCATTGCCTGCCGAGCACCGACAGCGGCCATTATATTACGAGCGATAGCATTACCGCGGTGCTGCGCAAACTGGGCATTAAATATCATGTGTATGAATTTCCGAGCGGCTGGGATATTACCGAATGCTTTATTGAAGGCGATCCGGCGGGCGGCCATATGATGGATTTTCTGACCGGCACCAAAAACTTTCTGGGCACCGCGCCGGCGGCGCTGCGCAGCCGCCTGCAAGAAGCGCTGTGTCAGCCGGAATGCAGCAGCCGCAAAGATGGCCGCGTGATTTTTTGCAACAACCTGAGCATGATTGTGGCGGAAAGCTAA;

[0038] SEQ ID NO.6:

[0039] ATGGAAGGCAAACGCTTTAACCTGGCGAACGGCGAATTACAACTGAGCCAAGAACCGCGCCAAGATCAACTGCCGACGAGCATTCCGCTGCCGGTGAGCCATCGCGATCCGGGCATGGATAGCACGAGCCCGAAAAACCGCTTTCCGAGCATGAACTTTGAAGCGGAAATTCTGACCGATCCGCATGATAACAGCGAACTGTATGTGATTCCGAGCATGCGCAGCCTGACCGCGGAAGAATATGTGGAAGCGTTTAAGAGCTTTCTGGATCATAGCACCGAACATCAGTGCATGGATGAATTTAACAAAGAAGAAATGCCGAACATTATGGCGGGCCTGGGCAACGGCCGCAGCACCATTAACGTGCTGGGCGTGGGCAGCGGCACCGGCGAACAAGACCTGAAAATGATTCGCATTCTGCAAGCGGCGCATCCGGGCGTGCTGATTGATAACGAAATTATTGAACCGAACCCGCAGCATGTGGCGGCGTATAAAGAACTGGTGAACCAAGCGCCGGATTTACAGAACGTGAGCTTTATTTGGCATCAACTGACGAGCAGCGAATATGAACAGCAAGTGAAAGAAAAAGGCGCGCATAAAAAATTTGATTTTATTCATATGATTCAGATGCTGTATCGCGTGGAAGATATTCCGAACACCATTAAATTTTTTCATAGCTGCCTGAACCATCATGGCAAACTGCTGATTATCATTCTGAGCGATAGCAGCGGCTGGGCGAGCCTGTGGAAAAAATATCGCCATTGCCTGCCGAGCACCGATAGCGGCCATTATATTACGAGCAACGGCATTACCGATGTGCTGAAACGCCTGGGCGTGGAATATCATGTGTATGAATTTCCGAGCGGCTGGGATATTACCGAATGCTTTATTGAAGGCGATCCGGTGGGCAGCCGCATGATGGATTTTCTGACCGGCACCAAAAACTTCCTGGGCACGGCGCCGGCGGGCTTACGTCAGCGTCTGCAAGAAGCGCTGTGTCAGCCGGAATGCAGCAGCAAAAAAGATGGCCGCATTATTTTTAGCAACAACCTGAGCATGATTGTGGTGGAAAGCTAA;

[0040] SEQ ID NO.7:

[0041] ATGGACCCGACCCCGGAAATGAAACGCAACCGCTTTCCGAGCATGAACTTTGAAGCGGAAATTCTGACCGCGAGCCATGATAACAGCGAACTGTATGTGATTCCGAGCATGCGCAGCCTGACCGCGGAAGAATATGTGGAAGCGTTTAAGAGCTTTCTGGATCATAGCACCGAACATCAGTGCATGGATCAGTTTAACAAAGAAGCGATGCCGAACATTATGGCGGGCCTGGGCAACGGCAAAAGCACCATTAACGTGCTGGGCGTGGGCAGCGGCACCGGCGAACAAGACCTGAAAATGATTCGCATTCTGCAAGCGGCGCATCCGGGCGTGCTGATTGATAACGAAATTATTGAACCGAACCCGCGCCATGTGGCGGCGTATAAAGAACTGGTGAACCAAGCGCCGGATTTACAGAACGTGAGCTTTATTTGGCATCAGTTTACGAGCAGC GAATATGAACAGCAGATGAAAGAAAAAGGCGCGCATAAAAAATTTGATTTTATTCATATGATTCAGATGCTGTATCGCGTGGAAGATATTCCGAACACCATTAAATATTTTCATAGCTGCCTGGATCATCATGGCAAACTGCTGATTATCATTCTGAGCGATAGCAGCGGCTGGGCGAGCCTGTGGAAAAAATATCGCCATTGCCTGCCGAGCACCGATAGCGGCCATTATATTACGAGCAACAGCATTACCGATGTGCTGAAACGCCTGGGCGTGGAATATCATGTGTATGAATTTCCGAGCGGCTGGGATATTACCGAATGCTTTATTGAAGGCGATCCGGTGGGCGGCCATATGATGGATTTTCTGACGGGCACCAAAAACTTTCTGGGCACCGCGCCGCCGGGCCTGCGCCAACGCCTGCAAGAAGCGCTGTGTCAGCCGGAATGCAGCAGCAAAAAAGATGGCCGCATTATTTTTAGCAACAACCTGAGCATGATTGTGGTGGAAAGCTAA;

[0042] SEQ ID NO.8:

[0043] ATGGAACCGGCGCCGGAAATGAAACGCAACCGCTTTCCGAGCATGAACTTTGAAGCGGAAATTCTGGCGGCGCCGCATGATAACAGCGAACTGTATGTGGTGCCGAGCATGCGCAGCCTGACCGCGGAAGAATATGTGGAAGCGTTTAAGAGCTTTCTGGATCATAGCACCGAACATCAGTGCATGGATGAATTTAACAAAGAAGAAATGCCGCGCATTATGGCGGGCCTGGGCAACGGCAAAAGCACCATTAACGTGCTGGGCGTGGGCAGCGGCACCGGCGAACAAGACCTGAAAATGCTGCGCATTCTGCAAGCGGCGCATCCGGGCGTGCTGATTAACAACGAAATTGTGGAACCGAACCCGCAGCATGTGGCGGCGTATAAAGAACTGGTGAACCAAGCGCCGGATCTGCAAGGCGTGAGCTTTATTTGGCATCAACTGACGAGCAGCGAGTACGAACAGCAAGTGAAAGAGAAATGCATTAATAAAAAATTTGATTTTATTCATATGATTCAGATGCTGTATCGCGTGGAAGATATTCCGAACACCATTAAATTTTTTCATAGCTGCCTGGATCATCAAGGCAAACTGCTGATTATCATTCTGAGCGATAGCAGCGGCTGGGCGAGCCTGTGGAAAAAATATCGCCATTGCCTGCCGAGCACCGATAGCGGCCATTATATTACGAGCGATAGCATTACCGCGGTGCTGCGCCGCCTGGGCATTCAGTATCGCGTGCATGAATTTCCGAGCGGCTGGGATATTACCGAATGCTTTGTGGAAGGCGATCCGGCGGGCGGTCAGATGATGGATTTTCTGACGGGCACCAAAAACTTTTTAGGCACCGCGCCGCCGGCGCTGCGCCGTCGCCTGCAAGAAGCGCTGTGTCAGCCGGAATGCAGCAGCAAAA AAGATGGCCGCGTGATTTTTTGCAACAACCTGAGCATGATTGTGGTGGAAAGCTAA。

[0044] In the present invention, the homology may be more than 60%, and the possible range covered includes but is not limited to: any one or more of more than 70%, more than 72%, more than 75%, more than 78%, more than 80%, more than 85%, more than 90%, more than 92%, more than 93%, more than 95%, more than 96%, more than 98% or more than 99%.

[0045] In yet another aspect, the present invention also provides a primer for amplifying the aforementioned carnosine N-methyltransferase gene fragment;

[0046] The primers for expressing the gene of GgNMT2, the nucleotide sequences of which include SEQ ID NO.9 and SEQ ID NO.10;

[0047] The primers for expressing the gene of TaNMT, the nucleotide sequences of which include SEQ ID NO.11 and SEQ ID NO.12;

[0048] The primers for expressing the gene of AcNMT, the nucleotide sequences of which include SEQ ID NO.13 and SEQ ID NO.14;

[0049] The primers for expressing the gene of AfNM have nucleotide sequences including SEQ ID NO.15 and SEQ ID NO.16.

[0050] SEQ ID NO.9: cacacaggaaacagaccATGGAACCGACCCGGAAATG;

[0051] SEQ ID NO.10: caggtcgactctagaggatccTTAGCTTCCGCCACAATCATGCT C;

[0052] SEQ ID NO.11: cacacaggaaacagaccATGGAAGGCAAACGCTTTAACCTG;

[0053] SEQ ID NO.12: caggtcgactctagaggatccTTAGCTTTCCACCACAATCATGCT CAGG;

[0054] SEQ ID NO.13: cacacaggaaacagaccATGGACCCGACCCCGGAAATG;

[0055] SEQ ID NO.14:caggtcgactctagaggatccTTAGCTTTCCACCACAATCATGCT CAG;

[0056] SEQ ID NO.15: cacacaggaaacagaccATGGAACCGGCGCCGGAAATG;

[0057] SEQ ID NO. 16: caggtcgactctagaggatccTTAGCTTTCCACCACAATCATGCT CAGG.

[0058] In yet another aspect, the present invention also provides a method for increasing the fermentation yield of L-angesone.

[0059] The method comprises: overexpressing the aforementioned carnosine N-methyltransferase gene in a genetically engineered bacterium in which the L-carnosine degradation gene is knocked out, wherein the N-methyltransferase gene comprises a gene expressing GgNMT2, TaNMT, AcNMT or AfNMT.

[0060] Specifically, the overexpression is achieved by constructing a pTrc99a plasmid vector.

[0061] Specifically, the basic starting bacteria of the genetically engineered bacteria for knocking out the L-carnosine degradation gene is Escherichia coli W3110.

[0062] Preferably, the deposit number of the genetically engineered bacteria with knocked-out L-carnosine degradation gene is CGMCC No.28838.

[0063] In another aspect, the present invention also provides a carnosine N-methyltransferase gene for increasing the fermentation yield of L-angesone.

[0064] Specifically, it is selected from SEQ ID NO. 5-8, or a gene having a homology of more than 60% with SEQ ID NO. 5-8.

[0065] In yet another aspect, the present invention also provides a recombinant plasmid comprising and expressing any one of the aforementioned carnosine N-methyltransferase genes.

[0066] Specifically, the host cell of the recombinant plasmid is an Escherichia coli cell.

[0067] Specifically, the backbone plasmid is an Escherichia coli expression plasmid vector.

[0068] Specifically, the backbone plasmid is selected from pEZ07, pACYC, pETrc, pUC19, pBR322 or pTrc99a.

[0069] Preferably, the backbone plasmid is pTrc99a.

[0070] Specifically, the insertion site of the carnosine N-methyltransferase gene in the pTrc99a plasmid is NcoI / BamHI.

[0071] Specifically, the overexpression of the carnosine N-methyltransferase gene is achieved by constructing a recombinant plasmid.

[0072] In some specific embodiments, the method for constructing a recombinant plasmid comprises the following steps:

[0073] (1) The carnosine N-methyltransferase gene fragment was mixed with the pTrc99a vector fragment recovered by NcoI / BamHI digestion at a ratio of 1:2 for EZ cloning construction;

[0074] (2) Warm the recombinant cloning reaction solution in a 45°C water bath for 30 min, then transfer to ice and place for 5 min;

[0075] (3) Add TG1 transformation competent cells, mix well and place for 5 min, heat shock at 42°C for 2 min, ice bath for 2 min, and then add 800 μL of recovery liquid culture medium LB;

[0076] (4) After 1 h of recovery culture, the cells were centrifuged and coated on LB plates containing 150 mg / L ampicillin antibiotics. The next day, clones were picked and cultured overnight. The plasmids were extracted and verified by enzyme digestion. Finally, plasmids pTrc99a-GgNMT2, pTrc99a-TaNMT, pTrc99a-AcNMT, and pTrc99a-AfNMT were constructed and numbered pHE592, pHE593, pHE595, and pHE596.

[0077] Based on the above, the present invention also provides a genetically engineered strain comprising the aforementioned recombinant plasmid.

[0078] Beneficial effects of the present invention:

[0079] The present invention screens out a carnosine N-methyltransferase gene that helps to increase the fermentation yield of L-angesine. After the screened gene expressing TaNMT, GgNMT2, AcNMT or AfNMT is overexpressed in a recombinant strain, L-carnosine is converted into L-angesine by shake flask fermentation. The yield of L-angesine is improved compared with the prior art, and the invention has the advantage of industrial scale-up.

[0080] Deposit certificate

[0081] Deposited strain: SHK19K;

[0082] Classification and nomenclature: Escherichia coli;

[0083] Deposit number: CGMCC No.28838;

[0084] Deposit date: November 2, 2023;

[0085] Depository: General Microbiology Center, China Culture Collection Administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 It is an NMT screened from the NCBI Carnosine NMT sequence library.

[0087] Figure 2 This is a comparison chart of NMT HPLC peaks.

[0088] Figure 3 This is the result of NMT fermentation. DETAILED DESCRIPTION

[0089] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0090] In the following embodiments:

[0091] LB medium: Each liter of culture medium contains 5g yeast powder, 10g sodium chloride, 10g peptone, and deionized water to make up to 1L (author: [US] J. Shambrook, translated by Huang Peitang, Molecular Cloning Guide 2002, 1595).

[0092] The above solution is sterilized by high pressure steam at a sterilization temperature of 121° C. for 20-30 min.

[0093] Fermentation medium: 30g glucose per liter, 200mL 5N-5 times salt solution, 1mL TM3 solution, 10mg ferric citrate, 246mg magnesium sulfate heptahydrate, 111mg calcium chloride, 1μg thiamine, and dilute to 1L with sterile deionized water. The 5N-5 times salt solution is 75.6g disodium hydrogen phosphate dodecahydrate, 15g potassium dihydrogen phosphate per liter, 2.5g sodium chloride, 25g ammonium chloride, and dilute to 1L with deionized water; TM3 solution is 2.0g zinc chloride tetrahydrate, 2.0g calcium chloride hexahydrate, 2.0g sodium molybdate dihydrate, 1.9g copper sulfate pentahydrate, 0.5g boric acid, 100mL hydrochloric acid, and dilute to 1L with deionized water.

[0094] The above solution is sterilized by high pressure steam at a temperature of 121° C. for 20-30 min. At the same time, empty shake bottles are prepared, and 0.4 g of calcium carbonate is weighed into each bottle to make the final concentration of calcium carbonate 20 g / L.

[0095] pTrc99a: purchased from Miaoling Biotechnology Co., Ltd.

[0096] Gel recovery and purification kit: purchased from Shanghai Jierui Bioengineering Technology Co., Ltd.

[0097] GBclonart seamless cloning kit: purchased from Suzhou Shenzhou Gene Co., Ltd.

[0098] Basic Example 1 Shake flask fermentation to verify the method for producing L-anserine by recombinant strains

[0099] 1. Instrument: Constant temperature shaking incubator.

[0100] 2. Methods:

[0101] Shake flask fermentation process: (1) inoculate the recombinant strain into 3 mL of LB medium containing antibiotics, and culture it in a shaker at 37°C at 250 rpm; (2) take 200 μL of the seeds after 16 hours of culture and transfer them to 2 mL of LB liquid medium containing antibiotics, and culture them in a shaker at 37°C at 250 rpm for 4 hours; (3) transfer all 2 mL of secondary seeds into a shake flask containing 18 mL of fermentation medium, and culture it in a shaker at 37°C at 250 rpm for 4 hours; (4) add IPTG to a final concentration of 1 mM and add 5 g / L L-Car + 1 g / LMet to adjust the shaker temperature to 34°C, continue to culture for about 20 hours, take 0.25 mL of fermentation broth and 0.75 mL of water, mix and centrifuge (12000 rpm, 1 minute), and take the supernatant for detection. The detection method is detailed in Basic Example 2.

[0102] Basic Example 2 HPLC determination of L-anserine in fermentation broth

[0103] The fermentation broth was diluted 4 times with sterile water, centrifuged through a 0.22 μm filter membrane, and detected by high performance liquid chromatography (HPLC).

[0104] The parameters of HPLC are as follows: Ultimate AQ-C18, 4.6*250*5μm; mobile phase A: acetonitrile, B: 10mM sodium octane sulfonate + 50mM potassium dihydrogen phosphate solution, pH3.0, A:B=15:85; column flow rate is 1.0mL / min, column temperature is 30℃; wavelength is 210nm, injection volume is 5μL (after dilution 4 times); detection time is 16min. The detection wavelength is 210nm using an ultraviolet detector; the initial mobile phase flow rate is 1.0mL / min, the fermentation broth loading volume is 5μL, and the column temperature is 30℃. The elution time of L-angesone is 9 minutes. The HPLC spectrum is shown in Figure 2 shown.

[0105] Example 1 Selection of Carnosine NMT from different sources

[0106] Some NMTs were screened from the NCBI Carnosine NMT sequence library (see Table 1 ), and NMTs with low amino acid sequence identity to ScNMT, RnNMT, hNMT, and GgNMT1 reported in the literature were selected through sequence alignment (see Figure 1 ), except for ScNMT, the remaining genes were synthesized by Suzhou GeneWeizhi after the sequences were optimized for Escherichia coli codons.

[0107] Table 1

[0108]

[0109] Example 2 Construction of NMT expression plasmid and genetically engineered strain

[0110] Nine NMTs from different sources (for details, see Table 1) were constructed into the medium copy vector pTrc99a by seamless cloning using primers, and 12 NMT expression plasmids pHE589-pHE600 (plasmid construction information is shown in Table 2) were obtained, including pHE592, pHE593, pHE595, and pHE596 (i.e., pTrc99a-GgNMT 2. The construction of pTrc99a-TaNMT, pTrc99a-AcNMT, and pTrc99a-AfNMT) was taken as an example: the synthetic gene was used as a template, and the primer pairs pHE592-F / pHE592-R, pHE593-F / pHE593-R, pHE595-F / pHE595-R, and pHE596-F / pHE596-R were used to amplify the gene fragments of GgNMT2, TaNMT, AcNMT, and AfNMT (primer pairs are shown in Table 3), and fragments of 1007 bp, 1115 bp, 1007 bp, and 1007 bp were obtained. The fragments were recovered and purified using a Jeremycin recovery and purification kit, and the obtained fragments were mixed with Nc The pTrc99a vector fragment recovered by oI / BamHI digestion was constructed by EZ cloning using the GBclonart seamless cloning kit. The recombinant cloning reaction solution was incubated in a 45°C water bath for 30 minutes, then transferred to ice for 5 minutes, added to TG1 transformation competent cells, mixed and placed for 5 minutes, heat shocked at 42°C for 2 minutes, and added with 800 μL of recovery medium LB after 2 minutes of ice bath. After 1 hour of recovery culture, the plate was centrifuged and coated on an LB plate containing 150 mg / L ampicillin antibiotic. The next day, clones were picked and cultured overnight, and the plasmids were extracted for enzyme digestion verification. Finally, the plasmids pTrc99a-GgNMT2, pTrc99a-TaNMT, pTrc99a-AcNMT and pTrc99a-AfNMT were constructed and numbered pHE592, pHE593, pHE595 and pHE596. Among them, the amino acid sequences of GgNMT2, TaNMT, AcN MT, and AfNMT are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, and the gene sequences of GgNMT2, TaNMT, AcNMT, and AfNMT are SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8. Then, the transformed strain SHK19K was subjected to shake flask fermentation.

[0111] This laboratory uses Escherichia coli W3110 (ATCC 27325) as the starting strain, with the genotype: F-mcr AmcrBIN (rrnD-rrnE) 1lambda-. After special modification, the degradation gene of L-carnosine was knocked out to obtain the genetically engineered strain SHK19K, which is classified and named Escherichia coli. The strain has been deposited in the General Microbiology Center of China National Microbiological Culture Collection on November 2, 2023, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, and the deposit number is CGMCC No.28838.

[0112] The NMT-related plasmids constructed as above were transformed into the host SHK19K (it was difficult to transform pHE591 into a competent state made with calcium chloride, and it was necessary to transform it into an electroporation competent state made with 10% glycerol), and recombinant strains containing NMT were obtained. These strains were fermented in shake flasks together with the control strain SHK19K / pTrc99a to screen NMT that was helpful in increasing the production of L-angesone.

[0113] Table 2

[0114]

[0115]

[0116] Table 3

[0117]

[0118] Example 3 Screening of NMT

[0119] NMT was screened in accordance with the shake flask fermentation comparison method of Basic Example 1: the recombinant strains containing NMT from different sources and the control bacteria SHK19K / pTrc99a were inoculated and cloned into LB test tubes containing 150 mg / L ampicillin antibiotics, 200 μL of seeds cultured overnight were transferred to 2 mL of LB liquid culture medium containing ampicillin antibiotics, and all were transferred to shake flasks containing 18 mL of fermentation medium after culturing at 37°C and 250 rpm for 4 hours in a shaker at 37°C, cultured at 250 rpm for 4 hours, induced by IPTG, cultured at 34°C with 5 g / L L-Car+1 g / L Met overnight, and continued to culture for about 20 hours, 0.25 mL of fermentation broth was diluted 4 times with 0.75 mL of ionized water, centrifuged at 12000 rpm for 1 minute, and the supernatant was tested. The detection method is detailed in Basic Example 2. Three clones were selected for each strain for parallel fermentation, and the results were averaged.

[0120] Results Figure 3, showing that the production of L-angesone was 0.18 g / L-0.64 g / L after overexpression of genes expressing GgNMT2, TaNMT, AcNMT, and AfNMT.

[0121] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Application of carnosine N-methyltransferase gene in improving the fermentation yield of L-anserine in Escherichia coli SHK19K, characterized in that: The deposit number of the Escherichia coli SHK19K is CGMCC No.28838; The carnosine N-methyltransferase gene is selected from any one or more of the genes expressing GgNMT2, TaNMT, AcNMT and AfNMT; The amino acid sequence of the carnosine N-methyltransferase GgNMT2 is shown in SEQ ID NO.1; The amino acid sequence of the carnosine N-methyltransferase TaNMT is shown in SEQ ID NO.2; The amino acid sequence of the carnosine N-methyltransferase AcNMT is shown in SEQ ID NO.3; The amino acid sequence of the carnosine N-methyltransferase AfNMT is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene expressing GgNMT2 is shown in SEQ ID NO.5; The nucleotide sequence of the gene expressing TaNMT is shown in SEQ ID NO.6; The nucleotide sequence of the gene expressing AcNMT is shown in SEQ ID NO.7; The nucleotide sequence of the gene expressing AfNMT is shown in SEQ ID NO.

8.

3. A method for increasing the fermentation yield of L-angesone, characterized in that: include: The carnosine N-methyltransferase gene is overexpressed in a genetically engineered strain in which the L-carnosine degradation gene is knocked out. The genetically engineered strain is Escherichia coli SHK19K, and the deposit number is CGMCC No. 28838. The carnosine N-methyltransferase gene is a gene expressing the GgNMT2, TaNMT, AcNMT or AfNMT described in claim 1.

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