Method for producing L-arginine using recombinant Escherichia coli and recombinant bacteria used

Abstract

This invention relates to a method for producing L-arginine using recombinant Escherichia coli and the recombinant bacteria used. Specifically, this invention relates to a method for producing L-arginine using recombinant Escherichia coli and the recombinant bacteria used. Recombinant enterobacteria are recombinant bacteria obtained by overexpressing a gene encoding a cyanate transporter in receptor enterobacteria, or recombinant bacteria obtained by overexpressing a gene encoding carbonic anhydrase in receptor enterobacteria. The cyanate transporter is a protein having the amino acid sequence shown in SEQ ID No. 4, and the carbonic anhydrase is a protein having the amino acid sequence shown in SEQ ID No. 5. By controlling the expression of the cyanate transporter or carbonic anhydrase, the production of arginine can be significantly increased, bringing a new approach to the synthesis of bio-based L-arginine and offering broad applicability and market potential.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to two Chinese patent applications filed with the China Patent Office on July 18, 2023, application number 202310879981.0, with the title of the invention "Method for producing L-arginine using Escherichia coli with overexpression of the cynX gene and recombinant bacteria used therein," and another Chinese patent application filed with the China Patent Office on July 18, 2023, application number 202310879978.9, with the title of the invention "Method for producing L-arginine using Escherichia coli with overexpression of the cynT gene and recombinant bacteria used therein." The entire contents of these applications are incorporated into this application by reference.

[0002] This invention belongs to the field of genetic engineering, and more specifically, relates to a method for producing L-arginine using recombinant Escherichia coli and the recombinant bacteria used. [Background technology]

[0003] L-arginine is widely used in fields such as pharmaceuticals, industry, food, cosmetics, and livestock farming, and has significant economic and social value.

[0004] Currently, L-arginine production methods mainly include microbial fermentation and protein hydrolysis. Compared to protein hydrolysis, microbial fermentation has advantages such as a wide range of raw material sources, a simple production process, less environmental impact, and higher product purity, making it suitable for large-scale industrial production.

[0005] Breeding efficient strains capable of producing L-arginine is key to the industrial application of microbial fermentation. Currently, breeding methods for L-amino acid-producing strains are mainly divided into two types: 1) Irrational mutagenesis screening: This method mainly involves mutagenesis treatment of wild-type chassis microorganisms using physical or chemical methods, and in combination with L-arginine analog resistance screening methods, selecting mutagenesis strains that have antagonistic effects against L-arginine. After multiple mutagenesis cycles, superior production strains with L-arginine synthesis ability are screened. 2) Rational metabolic engineering modification: This method primarily utilizes efficient genome editing technology to systematically modify the L-arginine synthesis network in chassis microorganisms to maximize the redirection of carbon metabolic fluxes to the L-arginine synthesis pathway. This mainly includes blocking the L-arginine degradation pathway, releasing the feedback inhibition control mechanism of key enzymes in the synthesis pathway, increasing the supply of the precursor carbamoyl phosphate, optimizing the supply balance of coenzymes in chassis cells, and modifying the L-arginine transmembrane transport system. With the rapid development of genetic engineering technology, methods for constructing efficient L-arginine-producing strains through rational metabolic engineering modification are gradually replacing mutagenesis screening methods and becoming the mainstream method for breeding highly efficient and stable L-arginine-producing strains.

[0006] Improving the L-arginine production performance of chassis microorganisms is a sustained goal in rational metabolic engineering breeding. In the process of systematically remodeling the metabolic network of chassis microorganisms, increasing or decreasing the expression intensity of target genes is a common strategy for improving L-arginine production performance. For example, reducing the expression of one or more genes involved in L-arginine degradation, one or more genes in the competitive pathway of L-arginine synthesis, or genes involved in carbon and nitrogen flux redistribution significantly promotes L-arginine synthesis. Furthermore, increasing the expression of key enzymes in the L-arginine synthesis pathway, increasing the expression of the efflux membrane protein of the target product, and increasing the expression of key enzymes in the precursor synthesis pathway of the target product have significant and beneficial effects on improving L-arginine synthesis in chassis microorganisms.

[0007] The cynX gene encodes a cyanate transporter. The effect of increasing cynX gene expression on L-arginine production has not yet been investigated, and in particular, there are currently no reports on L-arginine production using Enterobacteriaceae that overexpress the cynX gene.

[0008] The cynT gene encodes carbonic anhydrase that catalyzes the reaction between CO2 and H2O to synthesize H2CO3. The effect of increasing cynT gene expression on L-arginine production has not yet been investigated, and in particular, there are currently no reports on L-arginine production using E. coli that overexpress the cynT gene. [Overview of the project]

[0009] The problem that this invention aims to solve is to improve the L-arginine production capacity of microorganisms.

[0010] To solve the above problems, the present invention provides recombinant intestinal bacteria.

[0011] The recombinant enterobacteria provided in the present invention are recombinant enterobacteria A or recombinant enterobacteria B, wherein recombinant enterobacteria A is a recombinant obtained by overexpressing a gene encoding a cyanate transporter in a receptor enterobacteria, and recombinant enterobacteria B is a recombinant obtained by overexpressing a gene encoding carbonic anhydrase in a receptor enterobacteria.

[0012] In the recombinant Enterobacteriaceae described above, the cyanate transporter gene is derived from Escherichia coli, and the carbonic anhydrase gene is also derived from Escherichia coli.

[0013] In the recombinant Enterobacteria described above, the cyanate transporter is one of the following proteins: A1) A protein having the amino acid sequence shown in SEQ ID No. 4, encoded by a gene encoding a cyanate transporter. A2) A protein having 80% or more identity with the protein described in A1) and possessing cyanate transporter activity, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence of A1), A3) A fusion protein having cyanate transporter activity, obtained by ligating a tag to the N-terminus and / or C-terminus of A1) or A2).

[0014] In the recombinant Enterobacteria described above, carbonic anhydrase is one of the following proteins: B1) A protein having the amino acid sequence shown in SEQ ID No. 5, encoded by the gene encoding carbonic anhydrase. A protein having 75% or more identity with the protein described in B1) and possessing carbonic anhydrase activity, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence of B2)B1). A fusion protein having carbonic anhydrase activity, obtained by ligating a tag to the N-terminus and / or C-terminus of B3)B1) or B2).

[0015] The protein having the amino acid sequence shown in SEQ ID No. 4 above is a cyanate transporter (abbreviated as cynX).

[0016] In one specific embodiment, the amino acid sequence of the cyanate transporter is as follows: MLLVLVLIGLNMRPLLTSVGPLLPQLRQASGMSFSVAALLTALPVVTMGGLALAGSWLHQHVSERRSVAISLLLIAVGALMRELYPQSALLLSSALLGGVGIGIIQAVMPSVIKRRFQQRTPLVMGLWSAALMGGGGLGAAITPWLVQHSETWYQTLAWWALPAVVALFAWWWQSAREVASSHKTTTTPVRVVFTPRAWTLGVYFGLINGGYASLIAWLPAFYIEIGASAQYSGSLLALMTLGQAAGALLMPAMARHQDRRKLLMLALVLQLVGFCGFIWLPMQLPVLWAMVCGLGLGGAFPLCLLLALDHSVQPAIAGKLVAFMQGIGFIIAGLAPWFSGVLRSISGNYLMDWAFHALCVVGLMIITLRFAPVRFPQLWVKEA (SEQ ID No. 4).

[0017] The protein having the amino acid sequence shown in SEQ ID No. 5 above is a carbonic anhydrase (abbreviated as cynT).

[0018] In one specific embodiment, the amino acid sequence of the carbonic anhydrase is as follows: MKEIIDGFLKFQREAFPKREALFKQLATQQSPRTLFISCSDSRLVPELVTQREPGDLFVIRNAGNIVPSYGPEPGGVSASVEYAVAALRVSDIVICGHSNCGAMTAIASCQ CMDHMPAVSHWLRYADSARVVNEARPHSDLPSKAAAMVRENVIAQLANLQTHPSVRLALEEGRIALHGWVYDIESGSIAAFDGATRQFVPLAANPRVCAIPLRQPTAA(SEQ ID No.5).

[0019] In the recombinant Enterobacteria described above, the gene encoding the cyanate transporter may be any one of the following: C1) A DNA molecule whose nucleotide sequence is SEQ ID No. 2, A DNA molecule that has more than 80% identity with the nucleotide sequence defined in C2)C1) and encodes a cyanate transporter. C3) A DNA molecule that hybridizes with a nucleotide sequence defined by either C1) or C2) under stringent conditions and encodes a cyanate transporter.

[0020] In the recombinant Enterobacteria described above, the gene encoding carbonic anhydrase may be any one of the following: D1) A DNA molecule whose nucleotide sequence is SEQ ID No. 3, A DNA molecule that has 75% or more identity with the nucleotide sequence defined in D2)D1) and encodes a carbonic anhydrase, D3) A DNA molecule that hybridizes with a nucleotide sequence defined by either D1) or D2) under stringent conditions and encodes a carbonic anhydrase.

[0021] Those skilled in the art can easily mutate the nucleotide sequence encoding the cyanate transporter or carbonic anhydrase of the present invention using known methods such as directed evolution or point mutation. An artificially modified nucleotide sequence having 75% or more identity with the nucleotide sequence encoding the cyanate transporter or carbonic anhydrase of the present invention is derived from and equivalent to the nucleotide sequence of the present invention, insofar as it encodes a cyanate transporter or carbonic anhydrase and has the function of a cyanate transporter or carbonic anhydrase.

[0022] As used herein, the term "identity" means sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or more, 85% or more, 90% or more, or 95% or more identity with the nucleotide sequence encoding the cyanate transporter or carbonic anhydrase of the present invention. Identity can be assessed visually or by computer software. When using computer software, identity between two or more sequences can be expressed as a percentage (%) and can be used to assess identity between related sequences.

[0023] The above-mentioned 75% or higher identity may be 80%, 85%, 90%, or 95% or higher identity.

[0024] In the recombinant Enterobacteriaceae described above, the expression levels of genes encoding cyanate transporters or carbonic anhydrases in the recombinant Enterobacteriaceae are higher than those in the receptor Enterobacteriaceae.

[0025] In the recombinant enterobacteria described above, overexpression of a gene encoding a cyanate transporter or carbonic anhydrase in the recipient enterobacteria may be equivalent to replacing the ygaY gene in the recipient enterobacteria with a gene encoding a cyanate transporter or carbonic anhydrase.

[0026] The method for replacing the ygaY gene in the above-mentioned receptor enterobacteria with a gene encoding a cyanate transporter or carbonic anhydrase includes inserting the gene encoding a cyanate transporter or carbonic anhydrase into the ygaY gene locus of the receptor enterobacteria and knocking out the ygaY gene of the receptor enterobacteria.

[0027] In the recombinant Enterobacteriaceae described above, the putative transporter ygaY gene encodes one of the following proteins: H1) A protein having the amino acid sequence shown in SEQ ID No. 6, encoded by the gene ygaY which encodes a putative transporter. A protein having 80% or more identity with the protein described in H1) and possessing putative transporter activity, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence of H2)H1). A fusion protein having putative transporter activity, obtained by ligating a tag to the N-terminus and / or C-terminus of H3)H1) or H2).

[0028] In one specific embodiment, the amino acid sequence encoded by ygaY is as follows: MILGTALTGLFSVVAQILVPLAATLASPDKRGKVVGTIMSGLLLGILLARTVAGLLANLGGWRTVFWVASVLMALMALALWRGLPQMKSETHLNYPQLLGSVFSMFISDKILRTRALLGCLTFANFSILWTSMAFLLAAPPFNYSDGV IGLFGLAGAAGALGARPAGGFADKGKSHHTTTFGLLLLLLSWLAIWFGHTSVLALIIGILVLDLTVQGVHITNQTVIYRIHPDARNRLTAGYMTSYFIGGAAGSLISASAWQHGGWAGVCLAGATIALVNLLVWWRGFHRQEAAN(SEQ ID No.6).

[0029] In this specification, the ygaY gene may be one of the following: P1) A DNA molecule whose nucleotide sequence is SEQ ID No. 7, DNA molecules having more than 75% identity with the nucleotide sequence defined in P2)P1), P3) A DNA molecule that hybridizes with a nucleotide sequence defined by either P1) or P2) under stringent conditions.

[0030] In one specific embodiment, the nucleotide sequence of ygaY is as follows: 5'--3' (SEQ ID No. 7).

[0031] Furthermore, the recombinant E. coli described above contains promoters that drive the transcription of genes encoding cyanate transporters or carbonic anhydrases.

[0032] The above promoter may be the Ptrc promoter. The Ptrc promoter is one of the following DNA molecules: 1) A DNA molecule whose single-stranded nucleotide sequence is shown as SEQ ID No. 1 in the sequence listing. 2) A DNA molecule that has 80% or more identity with the DNA molecule in 1) and also possesses promoter function.

[0033] In one specific embodiment, the nucleotide sequence of the Ptrc promoter is as follows: 5'-TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACC-3' (SEQ ID No. 1).

[0034] The above recombinant enterobacteria may be recombinant Escherichia coli, and the above enterobacteria may be Escherichia coli.

[0035] The above-mentioned recipient Escherichia coli may be wild-type Escherichia coli or modified Escherichia coli.

[0036] The wild-type E. coli mentioned above may be E. coli W3110.

[0037] The modified Escherichia coli described above may be the genetically engineered bacterium E. coli W3110 (E. coli E. coli W3110 ARG10).

[0038] The above recombinant Escherichia coli may be ARG-cynX, W3110-cynX, ARG-pZ8-cynX, and W3110-pZ8-cynX.

[0039] The expression level of the cyanate transporter-encoding gene cynX in the recombinant E. coli ARG-cynX described above is higher than that of the recipient E. coli ARG10.

[0040] The expression level of the cyanate transporter-encoding gene cynX in the recombinant E. coli W3110-cynX is higher than that in the recipient E. coli W3110.

[0041] The expression level of the gene encoding the cyanate transporter in the recombinant E. coli ARG-pZ8-cynX is higher than that of the recipient E. coli ARG10.

[0042] The expression level of the gene encoding the cyanate transporter in the recombinant E. coli W3110-pZ8-cynX is higher than that in the recipient E. coli W3110.

[0043] The recombinant E. coli strains ARG-cynX, W3110-cynX, ARG-pZ8-cynX, and W3110-pZ8-cynX described above do not contain the complete coding region of the gene ygaY, which encodes the putative transporter. The complete coding region of ygaY is 882 bp, and positions 215-405 of the ygaY coding region (positions 215-405 of SEQ ID No. 7) are replaced with Ptrc-driven cynX.

[0044] The above recombinant Escherichia coli may be ARG-cynT, W3110-cynT, ARG-pZ8-cynT, and W3110-pZ8-cynT.

[0045] The expression level of the gene cynT, which encodes carbonic anhydrase, in the recombinant E. coli ARG-cynT is higher than that in the recipient E. coli ARG10.

[0046] The expression level of the gene cynT, which encodes carbonic anhydrase, in recombinant E. coli W3110-cynT is higher than that in recipient E. coli W3110.

[0047] The expression level of the gene encoding carbonic anhydrase in the recombinant E. coli ARG-pZ8-cynT is higher than that in the recipient E. coli ARG10.

[0048] The expression level of the gene encoding carbonic anhydrase in the recombinant E. coli W3110-pZ8-cynT is higher than that in the recipient E. coli W3110.

[0049] The recombinant E. coli strains ARG-cynT, W3110-cynT, ARG-pZ8-cynT, and W3110-pZ8-cynT described above do not contain the complete coding region of the gene ygaY, which encodes the putative transporter. The complete coding region of ygaY is 882 bp, and positions 215-405 of the ygaY coding region (positions 215-405 of SEQ ID No. 7) are replaced with Ptrc-driven cynT.

[0050] The vectors described herein mean vectors that can introduce, amplify, and express foreign DNA or a target gene into host cells. Such vectors may be cloning vectors or expression vectors, and include, but are not limited to, plasmids, phages (such as λ phages or M13 filamentous phages), cosmids, or viral vectors.

[0051] The microorganisms described herein may be yeasts, bacteria, algae, or fungi. Here, the bacteria belong to the genera Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., Brevibacterium sp., Corynebacterium sp., Aerobacter sp., Enterobacteria sp., Micrococcus sp., Serratia sp., Salmonella sp., Streptomyces sp., and Providencia. It may originate from, but is not limited to, sp., etc.

[0052] Furthermore, the above-mentioned bacteria may be Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, or Corynebacterium pekinense.

[0053] The cells described herein may be plant cells or animal cells. The cells may be any biological cells capable of synthesizing the target amino acid.

[0054] The present invention also provides a method for constructing the recombinant Escherichia coli described above.

[0055] The present invention provides a method for constructing the above-mentioned recombinant E. coli. The method is either Method A or Method B. Method A described above is a method for controlling the arginine production of microorganisms by controlling the expression of a gene encoding a cyanate transporter, or by controlling the activity or content of a cyanate transporter, thereby obtaining microorganisms with altered arginine production. Method B described above is a method for controlling the arginine production of microorganisms by controlling the expression of a gene encoding carbonic anhydrase, or by controlling the activity or content of carbonic anhydrase, thereby obtaining microorganisms with altered arginine production.

[0056] By controlling the expression of a protein-coding gene in the L-arginine metabolic pathway, or by controlling the activity or content of the protein in the L-arginine metabolic pathway, the arginine production of microorganisms is controlled, and microorganisms with altered arginine production are obtained.

[0057] In the above method, the method for controlling the expression of the gene encoding the cyanate transporter, or for controlling the activity or content of the cyanate transporter, may be any one of the following: M1) A method for introducing a gene encoding a cyanate transporter into a target microorganism. M2) A method for introducing a gene encoding the amino acid sequence shown in SEQ ID No. 4 into a target microorganism. In the above method, the method for controlling the expression of the gene encoding carbonic anhydrase, or for controlling the activity or content of carbonic anhydrase, may be any one of the following: N1) A method for introducing a gene encoding carbonic anhydrase into a target microorganism. N2) A method for introducing a gene encoding the amino acid sequence shown in SEQ ID No. 5 into a target microorganism.

[0058] In one embodiment, recombinant E. coli is constructed according to a method comprising the following steps: positions 215-405 of SEQ ID No. 7, which are nucleotides of the ygaY gene of the recipient E. coli, are replaced with a gene encoding a cyanate transporter or carbonic anhydrase.

[0059] The above ygaY gene encodes the putative transporter YgaY.

[0060] In the above method, the gene encoding the cyanate transporter may be any one of the following: C1) A DNA molecule whose nucleotide sequence is SEQ ID No. 2, A DNA molecule that has 75% or more identity with the nucleotide sequence defined in C2)C1) and encodes a cyanate transporter. C3) A DNA molecule that hybridizes with a nucleotide sequence defined by any one of C1) to C2) under stringent conditions and encodes a cyanate transporter.

[0061] In the above method, the gene encoding carbonic anhydrase may be any one of the following: D1) A DNA molecule whose nucleotide sequence is SEQ ID No. 3, A DNA molecule that has 75% or more identity with the nucleotide sequence defined in D2)D1) and encodes a carbonic anhydrase, D3) A DNA molecule that hybridizes with a nucleotide sequence defined by any one of D1) to D2) under stringent conditions and encodes a carbonic anhydrase.

[0062] In the above method, the putative transporter ygaY gene encodes one of the following proteins: H1) A protein having the amino acid sequence shown in SEQ ID No. 6, encoded by the gene ygaY which encodes a putative transporter. A protein having 80% or more identity with the protein described in H1), wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence of H2)H1), A fusion protein having putative transporter activity, obtained by ligating a tag to the N-terminus and / or C-terminus of H3)H1) or H2).

[0063] The present invention also provides a use of the above strain in any one of the following: E1) Use in controlling arginine production by microorganisms, E2) Use in the production of arginine, E3) Use of arginine in the construction of genetically engineered microorganisms.

[0064] The present invention also provides the use of the above-mentioned strain in the production of arginine or arginine-containing foods, pharmaceuticals and / or feeds.

[0065] The present invention also provides a use of protein in any one of the following: F1) Use in controlling arginine production by microorganisms. F2) Use in the manufacture of arginine, F3) Use of arginine in the construction of genetically engineered microorganisms.

[0066] The above protein may be either protein A or protein B. Protein A is a cyanate transporter having the amino acid sequence shown in SEQ ID No. 4, and protein B is a carbonic anhydrase having the amino acid sequence shown in SEQ ID No. 5.

[0067] The present invention also provides use of the above construction method in any one of the following: E1) Use in controlling arginine production by microorganisms, E2) Use in the production of arginine, E3) Use of arginine in the construction of genetically engineered microorganisms.

[0068] The present invention also provides the use of the above-described construction method in the production of arginine or arginine-containing foods, pharmaceuticals and / or feeds.

[0069] The present invention also provides nucleic acid molecules, which are nucleic acid molecule A or nucleic acid molecule B, wherein nucleic acid molecule A encodes a cyanate transporter in the above-mentioned intestinal bacteria, and nucleic acid molecule B encodes a carbonic anhydrase in the above-mentioned intestinal bacteria.

[0070] The present invention also provides an expression cassette, which is either expression cassette A or expression cassette B, where expression cassette A contains nucleic acid molecule A and expression cassette B contains nucleic acid molecule B.

[0071] The present invention also provides recombinant vectors, which are recombinant vector A or recombinant vector B, wherein recombinant vector A comprises nucleic acid molecule A or expression cassette, and recombinant vector B comprises nucleic acid molecule B or expression cassette.

[0072] The present invention also provides a whole-cell catalyst, which is either whole-cell catalyst A or whole-cell catalyst B, where whole-cell catalyst A comprises nucleic acid molecule A and whole-cell catalyst B comprises nucleic acid molecule B.

[0073] The present invention also provides a method for producing L-arginine. The method includes the steps of culturing the recombinant Escherichia coli to obtain a fermentation product and obtaining L-arginine from the fermentation product.

[0074] Recombinant microorganisms or recombinant cells constructed by overexpressing cynX or cynT in the present invention can also be used to produce a variety of products, including, but not limited to, lysine, glutamic acid, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, α-ketoglutaric acid, citric acid, ornithine and / or citrulline. [Modes for carrying out the invention]

[0075] The present invention will be described in detail below with reference to specific embodiments. The examples shown are for illustrative purposes only and do not limit the scope of the present invention. The examples provided below can be used by those skilled in the art as a guide for further improvements, but do not limit the present invention in any way.

[0076] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were carried out in accordance with the techniques or conditions described in the literature in this art, or in the product manuals. Unless otherwise specified, the materials, reagents, etc., used in the following examples are all commercially available.

[0077] Unless otherwise specified, the quantitative tests in the following examples all involve setting up three repeated experiments, and the average value is used as the result.

[0078] The pREDCas9 plasmid used in the following examples is described in "Jiang W, Bikard D, Cox D, Zhang F, Marrafni LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233-239. https: / / doi.org / 10.1038 / nbt.2508". The public can obtain this biomaterial from the applicant. This biomaterial is to be used solely for the purpose of replicating the experiments of the present invention and not for any other purpose.

[0079] The shuttle expression vector pZ8 used in the following examples is described in "Huang Qinqin, Wang Huimei, Liang Ling, Huang Qingeng, Wu Songgang, E., Effects of site-directed mutation of lysC and tandem expression of lysC and asdA on L-threonine accumulation in Corynebacterium glutamicum, Biotechnology Bulletin [J].2019(035)002." The public can obtain the biomaterial from the applicant. This biomaterial is to be used solely to reproduce the experiments of the present invention and not for any other purpose.

[0080] The modified Escherichia coli W3110 ARG10 in the following examples is also known as genetically engineered E. coli W3110 ARG10 and was constructed according to the method described in Example 1 of Patent CN110964683B.

[0081] The wild-type E. coli W3110 used in the following examples was purchased from Bai'ao Laibo and designated as catalog number BTN12-170201y.

[0082] In the following examples, data were processed using SPSS 11.5 statistical software, and experimental results were expressed as mean ± standard deviation. One-way ANOVA tests were used, with P<0.05(*) considered statistically significant, P<0.01(**) considered very statistically significant, and P<0.001(***) considered extremely statistically significant.

[0083] In the following examples, each primer was designed based on the principle of homologous recombination, and the synthesis and sequencing of the primers were performed by Suzhou Genewiz Biotechnology Co., Ltd. Refer to Table 1 below for the specific sequences. TIFF2026525966000001.tif252170TIFF2026525966000002.tif254170TIFF2026525966000003.tif84170

[0084] Specific information on the strains used in the present invention is shown in Table 2. TIFF2026525966000004.tif104170

[0085] In the following examples, the composition of the slant medium is 1 - 5 g / L glucose, 5 - 10 g / L tryptone, 5 - 10 g / L beef extract, 1 - 5 g / L yeast powder, 1 - 2.5 g / L NaCl, 15 - 20 g / L agar, the balance being water, and the pH is 7.0 - 7.2.

[0086] In the following examples, the composition of the seed medium is 20 - 40 g / L glucose, 2 - 5 g / L yeast extract, 2 - 4 g / L tryptone, 1 - 3 g / L K2HPO4, 1 - 2 g / L MgSO4·7H2O, 15 - 20 mg / L FeSO4·7H2O, 15 - 20 mg / L MnSO4·7H2O, B1 V B3 V B5 V B12 V H 1 - 3 mg / L each, the balance being water, and the pH is 7.0 - 7.2.

[0087] In the following examples, the composition of the fermentation medium is 20 - 40 g / L glucose, 1 - 3 g / L yeast extract, 2 - 3 g / L tryptone, 3 - 6 g / L K2HPO4, 1 - 2 g / L MgSO4·7H2O, 15 - 20 mg / L FeSO4·7H2O, 15 - 20 mg / L MnSO4·7H2O, B1 V B3 V B5 V B12 V HEach component contains 1-3 mg / L, with the remainder being water, and the pH is 7.0-7.2.

[0088] Unless otherwise noted, the experimental methods in the following examples are all conventional methods and were carried out in accordance with the techniques or conditions described in the literature in this art, or in the product's instruction manual.

[0089] Detailed procedures for some of the experimental methods in the following examples are as follows: I. Genome editing method: This was performed in reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic engineering, 2015, 31:13-21.). Here, pREDCas9 possesses a gRNA expression plasmid pGRB removal system, a λ phage Red recombination system, and a Cas9 protein expression system, is resistant to spectinomycin (concentration: 100 mg / L), and is cultured at 32°C. pGRB contains promoter J23100, gRNA-Cas9 binding site sequence, and terminator sequence, is resistant to ampicillin (concentration: 100 mg / L), and is cultured at 37°C.

[0090] The specific procedure for constructing the pGRB plasmid according to the present invention is as follows: 1. Construction of the pGRB plasmid The purpose of constructing the pGRB plasmid is to transcribe the corresponding gRNA, form a complex with the Cas9 protein, recognize the target site of the target gene via base pairing and PAM, and achieve a double-strand break of the target DNA. The pGRB plasmid was constructed by recombining a DNA fragment containing the target sequence with a linearization vector fragment.

[0091] 1.1 Design of the target sequence The target sequence (PAM:5'-NGG-3') was designed using CRISPR RGEN Tools.

[0092] 1.2 Preparation of DNA fragments containing the target sequence Primers were designed as follows: 5'-linearization vector terminal sequence (15 bp)-restriction enzyme site-target sequence (excluding PAM sequence)-linearization vector terminal sequence (15 bp)-3' and its reverse primer. DNA fragments containing the target sequences were prepared by annealing single-stranded DNA. Specifically, the target sequences of the present invention are as follows: 5'-CTCAACTACCCACAGTTGTT-3' (for ygaY gene knockout, SEQ ID No. 46), 5'-ATTTGTGGTCATTCCAACTG-3' (for cynT gene knockout, SEQ ID No. 47), and 5'-ACTGCTACCGCAATTGCGCC-3' (for cynX gene knockout, SEQ ID No. 48).

[0093] Reaction conditions: Pre-denaturation (95°C, 5 minutes), annealing (30-50°C, 1 minute). The 20 μL annealing system is as follows: TIFF2026525966000005.tif26170

[0094] 1.3 Preparation of linearization vectors Vector linearization was performed using inverse PCR amplification. Plasmid pGRB was amplified using primers YPpGRBF and YPpGRBR.

[0095] 1.4 Recombination Reactions The recombinant systems are shown in Table 4. All recombinant enzymes used are from the ClonExpress® II One Step Cloning Kit series. Recombination conditions: 37°C, 30 minutes.

[0096] TIFF2026525966000006.tif46170

[0097] 1.5 Plasmid Transformation 10 μL of the recombinant reaction solution from step 1.4 was added to 100 mL of competent cells for DH5α transformation, gently mixed uniformly, then bathed in ice for 20 minutes, subjected to heat shock at 42°C for 45-90 seconds, immediately bathed in ice for 2-3 minutes, added 900 μL of SOC medium, and recovered cultured at 37°C for 1 hour. The cells were centrifuged at 8000 rpm for 2 minutes, a portion of the supernatant was discarded, and the cells were resuspended, leaving approximately 200 μL. This was spread onto a plate containing 100 mg / L ampicillin, and the plate was inverted and cultured overnight at 37°C. After single colonies formed on the plate, colony PCR was performed to identify and select positive recombinants.

[0098] 1.6 Identification of Clones PCR-positive colonies were inoculated into LB medium containing 100 mg / L ampicillin, cultured overnight, and then stored. Plasmids were extracted and identified by restriction enzyme digestion to obtain recombinant plasmid pGRB-sgRNA.

[0099] The structure of the recombinant plasmid pGRB-sgRNA is described as follows: It is a recombinant expression vector obtained by replacing a small fragment between the homology arms of the pGRB vector, 5'-GCTCAGTCCTAGGTATAATACTAGT-3' (SEQ ID No. 49) and 5'-GTTTTAGAGCTAGAAATAGCAAGT-3' (SEQ ID No. 31), with a DNA fragment having a nucleotide sequence containing the target sequence 5'-CTCAACTACCCACAGTTGTT-3' (for ygaY gene knockout, SEQ ID No. 46), 5'-ATTTGTGGTCATTCCAACTG-3' (for cynT gene knockout, SEQ ID No. 47), or 5'-ACTGCTACCGCAATTGCGCC-3' (for cynX gene knockout, SEQ ID No. 48), while retaining the other sequences of the pGRB vector without modification.

[0100] II. Transformation of pREDCas9 vector and preparation of competent cells of target strains containing pREDCas9 1. Electrotransformation of pREDCas9 The pREDCas9 plasmid was introduced into electrotransformation-competent cells of the starting strain by electrotransmission. After recovery culture of the cells, they were spread on LB plates containing spectinomycin (concentration: 100 mg / L) and incubated overnight at 32°C. Colony PCR was performed on single colonies grown on the resistance plates using the identification primer set pCasF / pCasR (see Table 1 for specific sequences) to screen for positive recombinants.

[0101] 2. Preparation of competent cells of target strains containing pREDCas9 The target strain containing pREDCas9 was subjected to OD at 32°C. 600 = When the culture level reaches 0.1~0.2, add 0.1M IPTG (so that the final concentration is 0.1mM) and OD 600 Competent cells were prepared after continuous culture until the pH reached 0.6-0.7. The purpose of adding IPTG was to induce the expression of recombinant enzyme on the pREDCas9 plasmid. The culture medium and preparation steps required for competent cell preparation followed standard procedures.

[0102] Example 1: Use of the cynX protein and the gene encoding it in controlling arginine production. A. Construction of the genetically engineered bacterium ARG-cynX 1. Preparation of recombinant DNA fragments The recombinant fragment for overexpressing the cynX gene consists of the upstream and downstream homology arms of the cynX gene (ygaY::P trc-cynX). Using primer design software primer5, primers (amplification length approximately 400-800 bp) for the upstream and downstream homology arms were designed using the upstream and downstream sequences of the cynX gene as templates. The upstream and downstream primers for the upstream homology arm are UP-ygaY-S / UP-ygaY-A (see Table 1 for specific sequences), the upstream and downstream primer sequences for cynX are cynX-S / cynX-A (see Table 1 for specific sequences), and the upstream and downstream primers for the downstream homology arm are DN-ygaY-S / DN-ygaY-A (see Table 1 for specific sequences). After amplifying the upstream and downstream homology arms and the target gene fragment by PCR, the recombinant fragment ygaY::P was obtained by overlap PCR. trc -cynX was prepared. Recombinant fragment ygaY::P trc The nucleotide sequence of -cynX is sequence 8 in the sequence listing.

[0103] The PCR system and method for 50 μL are shown in Table 5 below. TIFF2026525966000007.tif58170

[0104] The 50 μL overlap PCR system is shown in Table 6 below. TIFF2026525966000008.tif71170 Note: The template consists of equimolar amplified fragments of the upstream and downstream homology arms and the target gene, and the total amount does not exceed 10 ng.

[0105] PCR reaction conditions (PrimeSTAR HS enzyme from Takara Bio): Pre-denaturation (95°C, 5 minutes), followed by 30 cycles: denaturation (98°C, 10 seconds), annealing ((Tm-3 / 5)°C, 15 seconds), extension at 72°C (this enzyme activity extends by approximately 1 kb per minute), final extension at 72°C for 10 minutes, maintenance (4°C).

[0106] 2. Preparation of competent cells for electrotransformation containing pREDCas9 For specific instructions on preparing competent cells for electrotransformation containing pREDCas9, please refer to Experimental Method II above.

[0107] 3. Transformation of pGRBs and recombinant DNA fragments The pGRB prepared in experimental method I above and the recombinant DNA fragment obtained in step 1 of A in this example were simultaneously electrotransmitted into competent cells for electrotransmission containing pREDCas9 prepared in step 2. After electrotransmission, the recovered cultured cells were spread onto LB plates containing ampicillin (concentration: 100 mg / L) and spectinomycin (concentration: 100 mg / L) and incubated overnight at 32°C. Colony PCR was performed using specially designed identification primer sets UP-ygaY-S and DN-ygaY-A (see Table 1 for specific sequences), and strains with 2462 bp were designated as positive strains. The screened positive recombinants were stored as strains.

[0108] 4. Plasmid removal 1) Removal of pGRB The positive recombinants obtained in step 3 were cultured overnight in LB medium containing 0.2% arabinose, diluted as needed, and then spread onto spectinomycin-resistant LB plates and cultured overnight at 32°C. Spotting was performed on ampicillin-containing LB plates and spectinomycin-resistant LB plates. Single colonies that did not grow on the ampicillin-containing plates but grew on the spectinomycin-resistant plates were selected and stored as bacterial strains.

[0109] 2) Removal of the pREDCas9 plasmid Positive recombinants were transferred to antibiotic-free LB liquid medium and incubated overnight at 42°C. After appropriate dilution, they were spread onto antibiotic-free LB plates and incubated overnight at 37°C. Spectinomycin-resistant LB plates and antibiotic-free LB plates were spotted, and single colonies that did not grow on the spectinomycin-resistant plates but grew on the antibiotic-free plates were selected and stored as bacterial strains.

[0110] 5. ygaY::P trc -cynX genome editing 1) Using the E. coli W3110 genome as a template, primers for the upstream homology arm (UP-ygaY-S, UP-ygaY-A) and the downstream homology arm (DN-ygaY-S, DN-ygaY-A) were designed based on the upstream and downstream sequences of its ygaY gene (NCBI GeneID:2847696), yielding the upstream homology arm fragment UP-ygaY and the downstream homology arm fragment DN-ygaY.

[0111] 2) Using the E. coli W3110 genome as a template, primers cynX-S and cynX-A, necessary for amplifying the cynX gene, were designed based on the upstream and downstream sequences of the cynX gene (NCBI GeneID: 948767) and the Ptrc promoter sequence, and the recombinant fragment Ptrc-cynX was obtained.

[0112] 3) The upstream homology arm fragment UP-ygaY, the downstream homology arm fragment DN-ygaY, and the recombinant fragment Ptrc-cynX are fused by overlap PCR to form ygaY::P trc -cynX (upstream homology arm-Ptrc-cynX-downstream homology arm) was obtained. Here, the length of the upstream homology arm is 616 bp, the length of the Ptrc promoter is 74 bp, the length of the cynX fragment is 1155 bp, the length of the downstream homology arm is 617 bp, the total length of the overlapping fragment is 2462 bp, and the nucleotide sequence of the overlapping fragment is SEQ ID No. 8 in the sequence listing.

[0113]

[0114] 4) Construction of pGRB-ygaY: Recombinant plasmid pGRB-ygaY was constructed by annealing target sequence-containing DNA fragments obtained from primers gRNA-ygaY-S and gRNA-ygaY-A with plasmid pGRB.

[0115] The structure of recombinant plasmid pGRB-ygaY is described as follows: A recombinant plasmid obtained by substituting the fragment between 5'-GCTCAGTCCTAGGTATAATACTAGT-3' (SEQ ID No. 49) and 5'-GTTTTAGAGCTAGAAATAGCAAGT-3' (SEQ ID No. 31) in pGRB with a DNA molecule whose nucleotide sequence is 5'-CTCAACTACCCACAGTTGTT-3' (SEQ ID No. 46), while retaining the other nucleotides of pGRB without modification.

[0116] 5) Competent cells of the genetically modified strain ARG10 (derived from patent CN110964683B) and the wild-type strain W3110 were prepared and introduced into the pREDCas9 plasmid. The target strains containing pREDCas9 were then subjected to OD at 32°C. 600 = When the culture level reaches 0.1~0.2, add 0.1M IPTG (so that the final concentration is 0.1mM) and OD 600 Competent cells were prepared after continuous culture until the pH reached 0.6-0.7. The purpose of adding IPTG was to induce the expression of recombinant enzyme on the pREDCas9 plasmid. The culture medium and preparation steps required for the preparation of competent cells followed standard procedures. For details, please refer to Experimental Method II above.

[0117] Colony PCR was performed using the primer sets UP-ygaY-S and DN-ygaY-A (see Table 1 for specific sequences). Strains yielding 2462 bp were designated as positive strains, while strains yielding 1424 bp were designated as the original starting strain ARG10 and the wild-type strain W3110, respectively, which did not incorporate the primers. The nucleotide sequences obtained by sequencing the PCR products produced by colony PCR using UP-ygaY-S and DN-ygaY-A are listed as SEQ ID No. 8 in the sequence listing.

[0118] Genetically modified strains ARG-cynX and W3110-cynX, in which the expression level of the cynX gene was enhanced, were sequentially constructed. In step 1, the pGRB-cynX vector was constructed, and in step 2, ygaY::P was generated by PCR. trc -Amplified the cynX-integrated fragment, in step 3 prepared competent cells of ARG10 and W3110 and transformed them with pREDCas9, in step 4 prepared competent cells of target strains ARG10 and W3110 containing pREDCas9 and pGRB-cynX vector and ygaY::P trc -Transform the embedded fragment of -cynX, and in step 5, ygaY::P trc - Since the strains ARG-cynX and W3110-cynX incorporating -cynX contain the pGRB-cynX vector, the pGRB-cynX vector is removed. In step 6, the pREDCas9 vector is removed from the target strains ARG-cynX and W3110-cynX from which the pGRB-cynX vector has been removed, thereby obtaining genetically modified strains ARG-cynX and W3110-cynX that do not contain the pGRB-cynX and pREDCas9 vectors and have enhanced expression of the cynX gene. For specific procedures, please refer to steps 1 to 5 of this example.

[0119] The expression level of the cyanate transporter-encoding gene cynX in the genetically modified strain ARG-cynX is higher than that of the receptor E. coli ARG10. The expression level of the cyanate transporter-encoding gene cynX in the genetically modified strain W3110-cynX is higher than that of the receptor E. coli W3110.

[0120] B. Construction of genetically engineered bacteria ARG-pZ8-cynX and W3110-pZ8-cynX The tac promoter on the pZ8 vector drives cynX expression, as demonstrated by constructing the cynX low-copy recombinant expression vector pZ8-cynX using the shuttle expression vector pZ8, introducing it into the genetically modified strain ARG10 and the wild-type strain W3110, and improving the L-arginine yield.

[0121] 1. Construction of the recombinant expression vector pZ8-cynX Based on the upstream and downstream sequences of the cynX gene (NCBI GeneID: 948767), primers cynX-pS and cynX-pA (see Table 1 for specific sequences) necessary for amplifying the cynX gene were designed. Using the E. coli W3110 genome as a template, the cynX gene was amplified by PCR using the cynX-pS and cynX-pA primers, purified, and recovered. The nucleotide sequence of the obtained cynX fragment is SEQ ID No. 2 in the sequence listing. For details on the PCR system and program, please refer to Procedure I of the experimental method described above.

[0122] After purifying the pZ8 vector by digestion with EcoR I and Sal I, the above-mentioned cynX PCR purified product was recombined with the linearized pZ8 vector to obtain the recombinant vector pZ8-cynX of cynX and pZ8.

[0123] The structure of the recombinant vector pZ8-cynX is described as follows: A recombinant plasmid obtained by replacing the fragment between 5'-TGAGCGGATAACAATTTCACACAGGAAACAGAATTC-3' (SEQ ID No. 50) and 5'-TCTCATCCGCCAAAACAGAAGCTTGGCTGCAGGTCGAC-3' (SEQ ID No. 51) of pZ8 with a DNA molecule having the nucleotide sequence shown in SEQ ID No. 2, while retaining the other nucleotides of pZ8 without modification.

[0124]

[0125] Recombinant vector pZ8-cynX was used to transform DH5α-competent cells, and PCR amplification was performed on kanamycin-resistant plates using primers pZ8F / pZ8R (see Table 1 for specific sequences). Positive transformants were then screened. For specific plasmid transformation methods, please refer to step 1.5 in procedure I of the experimental method described above.

[0126] PCR product sequencing revealed that the pZ8-cynX plasmid-positive transformant was 1243 bp, and no PCR amplification band was obtained in the DH5α strain.

[0127] 2. Construction of genetically modified bacterial strains ARG-pZ8-cynX and W3110-pZ8-cynX For the preparation of competent cells of the genetically modified strain ARG10 and the wild-type strain W3110, please refer to Procedure II of the experimental method described above. Competent cells of the starting strain ARG10 and the wild-type strain W3110 were transformed with the recombinant vector pZ8-cynX, respectively. PCR amplification was performed on a kanamycin-resistant plate using primers pZ8F / pZ8R (see Table 1 for specific sequences), and positive transformants were screened to sequentially construct the genetically modified strains ARG-pZ8-cynX and W3110-pZ8-cynX, which showed enhanced expression of the cynX gene.

[0128] PCR validation of positive strains containing the pZ8-cynX plasmid revealed a bp expression of 1243 bp, and no PCR amplification band was obtained in the starting strain. The expression level of the gene encoding the cyanate transporter in recombinant E. coli ARG-pZ8-cynX containing the pZ8-cynX plasmid was higher than that of the recipient E. coli ARG10. The expression level of the gene encoding the cyanate transporter in recombinant E. coli W3110-pZ8-cynX containing the pZ8-cynX plasmid was higher than that of the recipient E. coli W3110.

[0129] C. Construction of the genetically engineered bacterium ARG:ΔcynX 1. Obtaining the recombinant plasmid pGRB-cynX 1) ΔcynX genome editing Using the E. coli W3110 genome as a template, primers for the upstream homology arm (UP-cynX-S / UP-cynX-A (see Table 1 for specific sequences)) and the downstream homology arm (DN-cynX-S / DN-cynX-A (see Table 1 for specific sequences)) were designed based on the upstream and downstream sequences outside the coding region of the cynX gene (NCBI GeneID: 948767). The above fragments were fused by overlap PCR to obtain ΔcynX (cynX upstream homology arm - downstream homology arm).

[0130] 2) Construction of pGRB-cynX A DNA fragment containing the target sequence (nucleotide sequence 5'-ACTGCTACCGCAATTGCGCC-3', SEQ ID No. 48) obtained by annealing primers gRNA-cynX-S and gRNA-cynX-A (see Table 1 for specific sequences) was ligated with plasmid pGRB to construct recombinant plasmid pGRB-cynX.

[0131] The structure of the recombinant plasmid pGRB-cynX is described as follows: A recombinant plasmid obtained by substituting the fragment between 5'-GCTCAGTCCTAGGTATAATACTAGT-3' (SEQ ID No. 49) and 5'-GTTTTAGAGCTAGAAATAGCAAGT-3' (SEQ ID No. 31) of pGRB with a DNA molecule whose nucleotide sequence is 5'-ACTGCTACCGCAATTGCGCC-3' (SEQ ID No. 48), while retaining the other nucleotides of pGRB without modification.

[0132] 2. Construction of genetically modified bacterial strains ARG:ΔcynX and W3110:ΔcynX For specific instructions on preparing competent cells for the genetically modified strain ARG10 and the wild-type strain W3110, please refer to Procedure II of the experimental method described above.

[0133] Recombinant vector pGRB-cynX and ΔcynX fragments were used to transform competent cells of the starting strain ARG10 and the wild-type strain W3110, respectively. PCR amplification was performed on ampicillin (concentration: 100 mg / L) and spectinomycin (concentration: 100 mg / L) resistant plates using primers UP-cynX-S and DN-cynX-A (see Table 1 for specific sequences). Positive transformants were screened, and genetically modified strains ARG:ΔcynX and W3110:ΔcynX with reduced cynX gene expression were sequentially constructed.

[0134] Here, the length of the upstream homology arm is 572 bp, the length of the downstream homology arm is 527 bp, and the total length of the overlapping fragment is 1099 bp. Verification by PCR revealed that the PCR amplification fragment length of the positive strain was 1099 bp, while the PCR amplification product length of the parent strain was 2254 bp. The nucleotide sequence of the ΔcynX fragment is SEQ ID No. 9 in the sequence listing.

[0135]

[0136] Fermentation production of L-arginine using genetically modified strains overexpressing the D,cynX gene and genetically modified strains knocking out the cynX gene. Test strains: Modified strains overexpressing the cynX gene ARG-cynX, ARG-pZ8-cynX, W3110-cynX, W3110-pZ8-cynX; strain ARG:ΔcynX with the cynX gene knocked out; and the starting strain ARG10 (control strain).

[0137] The fermentation procedure is as follows: 1) Slant culture: Strains stored at -80°C were streaked onto activated slants, cultured at 37°C for 12 hours, and subcultured once. 2) Spawn culture by shaking: Spawn was scraped from the slant using an inoculation loop, inoculated into a 500 mL Erlenmeyer flask containing 30 mL of spawn medium, sealed with 9 layers of gauze, and cultured at 37°C and 200 rpm for 7-10 hours. 3) Fermentation culture by shaking: Inoculation of 10-15% of the volume of the starter culture solution was placed in a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL), sealed with 9 layers of gauze, and cultured with shaking at 37°C and 200 r / min. During the fermentation process, the pH was maintained at 7.0-7.2 by adding ammonia water, and fermentation was maintained by adding 60% (m / v) glucose solution. The fermentation cycle was set to 26-30 hours.

[0138] The above bacterial strains were subjected to shaking fermentation culture, and the L-arginine concentration in the fermentation supernatant was measured. The method for measuring L-arginine concentration followed industry standard GB 36897-2018.

[0139] TIFF2026525966000009.tif70170

[0140] The results are shown in Table 7. After 26-30 hours of fermentation, increasing the expression level of the cynX gene did not significantly affect the growth of the genetically modified strain ARG10. On the other hand, in ARG-cynX, the L-arginine concentration increased from 26.3 g / L to 29.4 g / L, and the L-arginine yield improved by 11.8%. In ARG-pZ8-cynX, the L-arginine concentration increased from 26.3 g / L to 29.5 g / L, and the L-arginine yield improved by 12.2%. When the expression level of the cynX gene was reduced, the L-arginine concentration in ARG:ΔcynX decreased from 26.3 g / L to 24.7 g / L, and the L-arginine yield decreased by 6.1%. The L-arginine production by W3110 with enhanced cynX expression was 0.35-0.41 g / L.

[0141] The results showed that enhancing the expression level of the cynX gene in E. coli significantly improved the L-arginine production capacity of genetically modified strains. Conversely, knocking out the cynX gene did not affect the normal growth of the strains, but it significantly reduced the L-arginine production capacity of the genetically modified strains, suggesting that the cynX gene contributes to the improvement of L-arginine production capacity.

[0142] Example 2: Use of carbonic anhydrase (cynT) and the gene encoding it in controlling arginine production. A. Preparation of recombinant DNA fragments The recombinant fragment for overexpressing cynT consists of the upstream and downstream homology arms of the cynT gene (ygaY::P trc-cynT). Using primer design software primer5, primers (amplification length approximately 400-800 bp) for the upstream and downstream homology arms were designed using the upstream and downstream sequences of the cynT gene as templates. The primer for the upstream homology arm is UP-ygaY-S / UP-ygaY-A (see Table 1 for specific sequences), the primer sequence for the downstream homology arm is cynT-S / cynT-A (see Table 1 for specific sequences), and the primer for the downstream homology arm is DN-ygaY-S / DN-ygaY-A (see Table 1 for specific sequences). After amplifying the upstream and downstream homology arms and the target gene fragment by PCR, the recombinant fragment ygaY::P was obtained by overlap PCR. trc -cynT was prepared. Recombinant fragment ygaY::P trc The nucleotide sequence of -cynT is sequence 10 in the sequence listing.

[0143] The 50 μL PCR system and method are shown in Table 8 below. TIFF2026525966000010.tif58170

[0144] The 50 μL overlap PCR system is shown in Table 9 below. TIFF2026525966000011.tif71170 Note: The template consists of equimolar amplified fragments of the upstream and downstream homology arms and the target gene, and the total amount does not exceed 10 ng.

[0145] PCR reaction conditions (PrimeSTAR HS enzyme from Takara Bio): Pre-denaturation (95°C, 5 minutes), followed by 30 cycles: denaturation (98°C, 10 seconds), annealing ((Tm-3 / 5)°C, 15 seconds), extension at 72°C (this enzyme activity extends by approximately 1 kb per minute), final extension at 72°C for 10 minutes, maintenance (4°C).

[0146] 2. Preparation of competent cells for electrotransformation containing pREDCas9 For specific instructions on preparing competent cells for electrotransformation containing pREDCas9, please refer to Experimental Method II above.

[0147] 3. Transformation of pGRBs and recombinant DNA fragments The pGRB prepared in experimental method I above and the recombinant DNA fragment obtained in step 1 of A in this example were simultaneously electrotransmitted into competent cells for electrotransmission containing pREDCas9 prepared in step 2. After electrotransmission, the recovered cultured cells were spread onto LB plates containing ampicillin (concentration: 100 mg / L) and spectinomycin (concentration: 100 mg / L) and incubated overnight at 32°C. Verification was performed by colony PCR using specially designed identification primer sets UP-ygaY-S and DN-ygaY-A (see Table 1 for specific sequences), and positive recombinants were screened and the strains were preserved.

[0148] 4. Plasmid removal 1) Removal of pGRB The positive recombinants obtained in step 3 were cultured overnight in LB medium containing 0.2% arabinose, diluted as needed, and then spread onto spectinomycin-resistant LB plates and cultured overnight at 32°C. Spotting was performed on ampicillin-containing LB plates and spectinomycin-resistant LB plates. Single colonies that did not grow on the ampicillin-containing plates but grew on the spectinomycin-resistant plates were selected and stored as bacterial strains.

[0149] 2) Removal of the pREDCas9 plasmid Positive recombinants were transferred to antibiotic-free LB liquid medium and incubated overnight at 42°C. After appropriate dilution, they were spread onto antibiotic-free LB plates and incubated overnight at 37°C. Spectinomycin-resistant LB plates and antibiotic-free LB plates were spotted, and single colonies that did not grow on the spectinomycin-resistant plates but grew on the antibiotic-free plates were selected and stored as bacterial strains.

[0150] 5. ygaY::P trc -cynT genome editing 1) Using the E. coli W3110 genome as a template, primers for the upstream homology arm (UP-ygaY-S, UP-ygaY-A) and the downstream homology arm (DN-ygaY-S, DN-ygaY-A) were designed based on the upstream and downstream sequences of its ygaY gene (NCBI GeneID:2847696), thereby obtaining the upstream homology arm UP-ygaY and the downstream homology arm DN-ygaY.

[0151] 2) Using the E. coli W3110 genome as a template, primers cynT-S and cynT-A, necessary for amplifying the cynT gene, were designed based on the upstream and downstream sequences of the cynT gene (NCBI GeneID: 946548) and the Ptrc promoter sequence, and recombinant fragment 3 was obtained.

[0152] 3) The upstream homology arm UP-ygaY, the downstream homology arm DN-ygaY, and recombinant fragment 3 are fused by overlap PCR to form ygaY::P trc -cynT (upstream homology arm-Ptrc-cynT-downstream homology arm) was obtained. Here, the length of the upstream homology arm is 616 bp, the length of the Ptrc promoter is 74 bp, the length of the cynT fragment is 660 bp, the length of the downstream homology arm is 617 bp, the total length of the overlapping fragment is 1967 bp, and the nucleotide sequence of the overlapping fragment is SEQ ID No. 10 in the sequence listing.

[0153]

[0154] 4) Construction of pGRB-ygaY: Recombinant plasmid pGRB-ygaY was constructed by annealing target sequence-containing DNA fragments obtained from primers gRNA-ygaY-S and gRNA-ygaY-A with plasmid pGRB.

[0155] The structure of recombinant plasmid pGRB-ygaY is described as follows: A recombinant plasmid obtained by substituting the fragment between 5'-GCTCAGTCCTAGGTATAATACTAGT-3' (SEQ ID No. 49) and 5'-GTTTTAGAGCTAGAAATAGCAAGT-3' (SEQ ID No. 31) in pGRB with a DNA molecule whose nucleotide sequence is 5'-CTCAACTACCCACAGTTGTT-3' (SEQ ID No. 46), while retaining the other nucleotides of pGRB without modification.

[0156] 5) Competent cells were prepared from the genetically modified strain ARG10 (derived from patent CN110964683B) and the wild-type strain W3110. For details, please refer to Experimental Method II above.

[0157] ygaY::P trc -When a positive bacterial strain incorporating -cynT was validated by PCR, its PCR amplification was 1967 bp, while the parent strain's PCR amplification was 1424 bp. ygaY::P trc The nucleotide sequence of the -cynT fragment is SEQ ID No. 10 in the sequence listing.

[0158] Genetically modified bacterial strains ARG-cynT and W3110-cynT, in which the expression level of the cynT gene was enhanced, were sequentially constructed. In Step 1, a pGRB-cynT vector is constructed, and in Step 2, ygaY::P is generated by PCR. trc-Amplified the cynT integration fragment, in step 3 prepared competent cells of ARG10 and W3110 and transformed them with pREDCas9, in step 4 prepared competent cells of target strains ARG10 and W3110 containing pREDCas9 and pGRB-cynT vector and ygaY::P trc -Transform the embedded fragment of -cynT, and in step 5, ygaY::P trc - Since the strains ARG-cynT and W3110-cynT incorporating -cynT contain the pGRB-cynT vector, the pGRB-cynT vector was removed. In step 6, the pREDCas9 vector was removed from the target strains ARG-cynT and W3110-cynT from which the pGRB-cynT vector had been removed, thereby obtaining genetically modified strains ARG-cynT and W3110-cynT that do not contain pGRB-cynT or the pREDCas9 vector and have enhanced cynX gene expression. For specific procedures, please refer to steps 1 to 5 of this example.

[0159] The expression level of the gene cynT, which encodes carbonic anhydrase, in the genetically modified strain ARG-cynT is higher than that of the receptor E. coli ARG10. The expression level of the gene cynT, which encodes carbonic anhydrase, in the genetically modified strain W3110-cynT is higher than that of the receptor E. coli W3110.

[0160] B. Construction of genetically engineered bacteria ARG-pZ8-cynT and W3110-pZ8-cynT The tac promoter on the pZ8 vector drives cynT expression, as demonstrated by constructing the low-copy recombinant cynT expression vector pZ8-cynT using the shuttle expression vector pZ8 and introducing it into the genetically modified strain ARG10 and the wild-type strain W3110, thereby improving the L-arginine yield.

[0161] 1. Construction of the recombinant expression vector pZ8-cynT Based on the upstream and downstream sequences of the cynT gene (NCBI GeneID: 946548), primers cynT-pS and cynT-pA (see Table 1 for specific sequences) necessary for amplifying the cynT gene were designed. Using the E. coli W3110 genome as a template, the cynT gene was amplified by PCR using the cynT-pS and cynT-pA primers, purified, and recovered. The nucleotide sequence of the obtained cynT fragment is SEQ ID No. 3 in the sequence listing. For details on the PCR system and program, please refer to Procedure I of the experimental method described above.

[0162] After purifying the pZ8 vector by digestion with EcoR I and Sal I, the above-mentioned cynT PCR purified product was recombined with the linearized pZ8 vector to obtain the recombinant vector pZ8-cynT of cynT and pZ8.

[0163] The structure of the recombinant vector pZ8-cynT is described as follows: A recombinant plasmid obtained by substituting the fragment between 5'-TGAGCGGATAACAATTTCACACAGGAAACAGAATTC-3' (SEQ ID No. 50) and 5'-TCTCATCCGCCAAAACAGAAGCTTGGCTGCAGGTCGAC-3' (SEQ ID No. 51) of pZ8 with a DNA molecule having the nucleotide sequence shown in SEQ ID No. 3, while retaining the other nucleotides of pZ8 without modification.

[0164] The nucleotide sequence of the cynT fragment is 5'--3' (SEQ ID No. 3).

[0165] Recombinant vector pZ8-cynT was used to transform DH5α-competent cells, and PCR amplification was performed on kanamycin-resistant plates using primers pZ8F / pZ8R (see Table 1 for specific sequences). Positive transformants were then screened. For specific plasmid transformation methods, please refer to step 1.5 in procedure I of the experimental method described above.

[0166] Sequencing of the PCR product revealed that the length of the PCR product of a pZ8-cynT plasmid-positive transformant was 748 bp, and no PCR amplification band was obtained in the DH5α strain.

[0167] 2. Construction of genetically modified bacterial strains ARG-pZ8-cynT and W3110-pZ8-cynT For the preparation of competent cells for the genetically modified strain ARG10 and the wild-type strain W3110, please refer to Procedure II of the experimental method described above. Competent cells of the starting strain ARG10 and the wild-type strain W3110 were transformed with the recombinant vector pZ8-cynT, respectively. PCR amplification was performed on a kanamycin-resistant plate using primers pZ8F / pZ8R (see Table 1 for specific sequences), and positive transformants were screened to sequentially construct the genetically modified strains ARG-pZ8-cynT and W3110-pZ8-cynT, which showed enhanced expression of the cynT gene.

[0168] The length of the PCR product of the positive strain containing the pZ8-cynT plasmid was 748 bp, and no PCR amplification band was obtained in the starting strain. The expression level of the gene encoding carbonic anhydrase in recombinant E. coli ARG-pZ8-cynT containing the pZ8-cynT plasmid was higher than that of the recipient E. coli ARG10. The expression level of the gene encoding carbonic anhydrase in recombinant E. coli W3110-pZ8-cynT containing the pZ8-cynT plasmid was higher than that of the recipient E. coli W3110.

[0169] C. Construction of genetically engineered bacteria ARG:ΔcynT 1. Obtaining the recombinant plasmid pGRB-cynT 1) ΔcynT genome editing Using the E. coli W3110 genome as a template, primers for the upstream homology arm (UP-cynT-S / UP-cynT-A (see Table 1 for specific sequences)) and the downstream homology arm (DN-cynT-S / DN-cynT-A) were designed based on the upstream and downstream sequences outside the coding region of the cynT gene (NCBI GeneID: 946548). The above fragments were fused by overlap PCR to obtain ΔcynT (cynT upstream homology arm - downstream homology arm).

[0170] 2) Construction of pGRB-cynT A DNA fragment containing the target sequence (target sequence: nucleotide sequence 5'-ATTTGTGGTCATTCCAACTG-3', SEQ ID No. 47) obtained by annealing primers gRNA-cynT-S and gRNA-cynT-A was ligated with plasmid pGRB to construct recombinant plasmid pGRB-cynT.

[0171] The structure of the recombinant plasmid pGRB-cynT is described as follows: A recombinant plasmid obtained by substituting the fragment between 5'-GCTCAGTCCTAGGTATAATACTAGT-3' (SEQ ID No. 49) and 5'-GTTTTAGAGCTAGAAATAGCAAGT-3' (SEQ ID No. 31) of pGRB with a DNA molecule whose nucleotide sequence is 5'-ATTTGTGGTCATTCCAACTG-3' (SEQ ID No. 47), while retaining the other nucleotides of pGRB without modification.

[0172] 2. Construction of genetically modified bacterial strains ARG:ΔcynT and W3110:ΔcynT For specific instructions on preparing competent cells for the genetically modified strain ARG10 and the wild-type strain W3110, please refer to Procedure II of the experimental method described above.

[0173] Recombinant vector pGRB-cynT and ΔcynT fragments were used to transform competent cells of the starting strain ARG10 and wild-type strain W3110, respectively. PCR amplification was performed on kanamycin-resistant plates using primers UP-cynT-S / DN-cynT-A (see Table 1 for specific sequences), and positive transformants were screened to sequentially construct genetically modified strains ARG:ΔcynT and W3110:ΔcynT, which exhibit reduced cynT gene expression.

[0174] Here, the length of the upstream homology arm is 543 bp, the length of the downstream homology arm is 519 bp, and the total length of the overlapping fragment is 1062 bp. Verification by PCR revealed that the PCR amplification fragment length of the positive strain with cynT knockout was 1062 bp, while the PCR amplification product length of the parent strain was 1722 bp. The nucleotide sequence of the ΔcynT fragment is SEQ ID No. 11 in the sequence listing.

[0175]

[0176] Fermentation production of L-arginine using genetically modified bacterial strains overexpressing the D,cynT gene and genetically modified bacterial strains knocking out the cynT gene. Test strains: Modified strains overexpressing the cynT gene ARG-cynT, ARG-pZ8-cynT, W3110-cynT, W3110-pZ8-cynT; strain W3110:ΔcynT with the cynT gene knocked out; and starting strains ARG10 and W3110 (control strains).

[0177] The fermentation procedure is as follows: 1) Slant culture: Strains stored at -80°C were streaked onto activated slants, cultured at 37°C for 12 hours, and subcultured once. 2) Spawn culture by shaking: Spawn was scraped from the slant using an inoculation loop, inoculated into a 500 mL Erlenmeyer flask containing 30 mL of spawn medium, sealed with 9 layers of gauze, and cultured at 37°C and 200 rpm for 7-10 hours. 3) Fermentation culture by shaking: Inoculation of 10-15% of the volume of the starter culture solution was placed in a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL), sealed with 9 layers of gauze, and cultured with shaking at 37°C and 200 r / min. During the fermentation process, the pH was maintained at 7.0-7.2 by adding ammonia water, and fermentation was maintained by adding 60% (m / v) glucose solution. The fermentation cycle was set to 26-30 hours.

[0178] The above bacterial strains were subjected to shaking fermentation culture, and the L-arginine concentration in the fermentation supernatant was measured. The method for measuring L-arginine concentration followed industry standard GB 36897-2018.

[0179] TIFF2026525966000012.tif73170

[0180] The results are shown in Table 10. After 26-30 hours of fermentation, enhancing the expression level of the cynT gene did not significantly affect the growth of the genetically modified strain ARG10. In ARG-cynT, the L-arginine concentration increased from 26.3 g / L to 30.2 g / L, and the L-arginine yield improved by 14.8%. In ARG-pZ8-cynT, the L-arginine concentration increased from 26.3 g / L to 30.9 g / L, and the L-arginine yield improved by 17.5%. In ARG:ΔcynT, the L-arginine concentration decreased from 26.3 g / L to 24.4 g / L, and the L-arginine yield decreased by 7.2%. The L-arginine production by W3110 with enhanced cynT expression was 0.40 g / L.

[0181] The results showed that enhancing the expression level of the cynT gene in E. coli significantly improved the L-arginine production capacity of genetically modified strains. Conversely, knocking out the cynT gene did not affect the normal growth of the strains, but it significantly reduced the L-arginine production capacity of the genetically modified strains, further suggesting that the cynT gene contributes to the improvement of L-arginine production capacity. [Industrial applicability]

[0182] This invention replaces the endogenous promoter of the E. coli cynX gene or cynT gene with Ptrc, integrates it into the ygaY pseudolocus of the genome, and drives the cynX gene or cynT gene with the strong promoter Ptrc to enhance its expression intensity, thereby achieving more cost-effective L-arginine fermentation production. Using the L-arginine gene-modified strain ARG10 (Patent: ZL201911211097.X) as the starting strain, enhancing the expression intensity of the cynX gene increased the L-arginine accumulation concentration in the strain from 26.3 g / L to 29.4 g / L, and the L-arginine yield improved by 11.8%. Enhancing the expression intensity of the cynT gene increased the L-arginine accumulation concentration in the strain from 26.3 g / L to 30.2 g / L, and the L-arginine yield improved by 14.8%.

Claims

1. Recombinant enterobacteria, wherein the recombinant enterobacteria include recombinant bacteria obtained by overexpressing a gene encoding a cyanate transporter and / or a gene encoding carbonic anhydrase in a receptor enterobacteria.

2. The recombinant enterobacteria according to claim 1, characterized in that the gene for the cyanate transporter is derived from Escherichia coli, and the gene for the carbonic anhydrase is derived from Escherichia coli.

3. The aforementioned cyanate transporter is A1) A protein having the amino acid sequence shown in SEQ ID No. 4, encoded by a gene encoding a cyanate transporter. A2) A protein having 80% or more identity with the protein described in A1) and having cyanate transporter activity, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence of A1), A3) A fusion protein having cyanate transporter activity obtained by ligating a tag to the N-terminus and / or C-terminus of A1) or A2), Contains one of the following proteins: The carbonic anhydrase described above is B1) A protein having the amino acid sequence shown in SEQ ID No. 5, encoded by the gene encoding carbonic anhydrase. A protein having 80% or more identity with the protein described in B1) and possessing carbonic anhydrase activity, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence of B2) and B1), A fusion protein having carbonic anhydrase activity, obtained by ligating a tag to the N-terminus and / or C-terminus of B3) B1) or B2), Recombinant enterobacteria according to claim 1 or 2, characterized by containing any one of the proteins.

4. The gene encoding the aforementioned cyanate transporter is, C1) A DNA molecule whose nucleotide sequence is SEQ ID No. 2, A DNA molecule having 80% or more identity with the nucleotide sequence defined in C2) and C1), and encoding the cyanate transporter, C3) A DNA molecule that hybridizes with a nucleotide sequence defined by any one of C1) to C2) under stringent conditions, and which encodes the cyanate transporter. Includes any one of the following: The gene encoding the carbonic anhydrase is D1) A DNA molecule whose nucleotide sequence is SEQ ID No. 3, A DNA molecule having 80% or more identity with the nucleotide sequence defined in D2) and D1), and which encodes the carbonic anhydrase, D3) A DNA molecule that hybridizes with a nucleotide sequence defined in any one of D1) to D2) under stringent conditions, and which encodes the carbonic anhydrase, Recombinant enterobacteria according to claim 1, characterized by containing one of the following.

5. A method for constructing recombinant intestinal bacteria according to claim 1, Q1) To control the arginine production of a microorganism by controlling the expression of the gene encoding the cyanate transporter described in claim 1, or by controlling the activity or content of the cyanate transporter, and to obtain a microorganism in which the arginine production has been altered. Q2) A method comprising controlling the arginine production of a microorganism by controlling the expression of a gene encoding the carbonic anhydrase described in claim 1, or by controlling the activity or content of the carbonic anhydrase, thereby obtaining a microorganism in which the arginine production has been altered.

6. A method for controlling the expression of a gene encoding the cyanate transporter, or for controlling the activity or content of the cyanate transporter, is: M1) A method for introducing the gene encoding the cyanate transporter into a target microorganism, M2) A method for introducing a gene encoding the amino acid sequence shown in SEQ ID No. 4 into a target microorganism. Includes any one of the following: A method for controlling the expression of the gene encoding the carbonic anhydrase, or for controlling the activity or content of the carbonic anhydrase, is: N1) A method for introducing the gene encoding the carbonic anhydrase into a target microorganism. N2) A method for introducing a gene encoding the amino acid sequence shown in SEQ ID No. 5 into a target microorganism. The method according to claim 5, characterized by including any one of the following.

7. Use of the intestinal bacteria described in claim 1 in any one of the following: E1) Use in controlling arginine production by microorganisms, E2) Use in the manufacture of arginine, E3) Use of arginine in the construction of genetically engineered microorganisms.

8. Use of the intestinal bacteria described in claim 1 in the production of arginine or foods, pharmaceuticals and / or feeds containing arginine.

9. Use of the method according to claim 5 or 6 in any one of the following: E1) Use in controlling arginine production by microorganisms, E2) Use in the manufacture of arginine, E3) Use of arginine in the construction of genetically engineered microorganisms.

10. Use of the method according to claim 5 or 6 in the production of arginine or a food, pharmaceutical and / or feed containing arginine.

11. The use of protein, The aforementioned protein includes protein A or protein B. The aforementioned protein A includes a cyanate transporter having the amino acid sequence shown in SEQ ID No.

4. The aforementioned protein B contains a carbonic anhydrase having the amino acid sequence shown in SEQ ID No.

5. A usage characterized by including one of the following: F1) Use in controlling arginine production by microorganisms, F2) Use in the manufacture of arginine, F3) Use of arginine in the construction of genetically engineered microorganisms.

12. A nucleic acid molecule comprising nucleic acid molecule A or nucleic acid molecule B, wherein nucleic acid molecule A encodes the cyanate transporter in the intestinal bacteria described in claim 1, and nucleic acid molecule B encodes the carbonic anhydrase in the intestinal bacteria described in claim 1.

13. An expression cassette comprising expression cassette A or expression cassette B, wherein expression cassette A comprises nucleic acid molecule A as described in claim 12, and expression cassette B comprises nucleic acid molecule B as described in claim 12.

14. A recombinant vector comprising recombinant vector A or recombinant vector B, wherein recombinant vector A comprises nucleic acid molecule A as described in claim 12 or expression cassette A as described in claim 13, and recombinant vector B comprises nucleic acid molecule B as described in claim 12 or expression cassette B as described in claim 13.

15. A whole cell catalyst comprising whole cell catalyst A or whole cell catalyst B, wherein whole cell catalyst A comprises nucleic acid molecule A as described in claim 12, and whole cell catalyst B comprises nucleic acid molecule B as described in claim 12.