CN118207191A

CN118207191A - A methionine aminopeptidase mutant, Escherichia coli engineering bacteria and application

Info

Publication number: CN118207191A
Application number: CN202211628640.8A
Authority: CN
Inventors: 宗剑飞; 李庆刚; 李玉; 肖卫华; 路福平
Original assignee: Shandong Synthetic Vision Biotechnology Co ltd
Current assignee: Shandong Synthetic Vision Biotechnology Co ltd
Priority date: 2022-12-18
Filing date: 2022-12-18
Publication date: 2024-06-18
Also published as: WO2024130899A1

Abstract

本发明属于基因工程技术领域，涉及一种甲硫氨酸氨肽酶突变体、大肠杆菌工程菌及应用。本发明提供的甲硫氨酸氨肽酶突变体对野生型甲硫氨酸氨肽酶进行了点突变，表达该甲硫氨酸氨肽酶突变体的大肠杆菌工程菌的2'‑岩藻糖基乳糖和3‑岩藻糖基乳糖的发酵产率得到出乎预料地提高。进一步对基因工程菌的wcaG、gmd、lacY、manA、manB、setA基因进行过表达，敲除lacZ、wcaj、nudd基因，质粒上多拷贝过表达futC 或futA及manC基因更有利于提高2'‑岩藻糖基乳糖和3‑岩藻糖基乳糖的产率。The present invention belongs to the field of genetic engineering technology, and relates to a methionine aminopeptidase mutant, an Escherichia coli engineered bacterium and an application. The methionine aminopeptidase mutant provided by the present invention carries out a point mutation on the wild-type methionine aminopeptidase, and the fermentation yield of 2'-fucosyllactose and 3-fucosyllactose of the Escherichia coli engineered bacterium expressing the methionine aminopeptidase mutant is unexpectedly improved. Further, the wcaG , gmd , lacY , manA , manB , and setA genes of the genetically engineered bacterium are overexpressed, the lacZ , wcaj , and nudd genes are knocked out , and multiple copies of futC or futA and manC genes on the plasmid are overexpressed, which is more conducive to improving the yield of 2'-fucosyllactose and 3-fucosyllactose.

Description

Methionine aminopeptidase mutant, escherichia coli engineering bacteria and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a methionine aminopeptidase mutant, escherichia coli engineering bacteria and application thereof.

Background

Breast milk oligosaccharides (human milk oligosaccharides, HMOs) are a nutritional component in breast milk. The composition is complex, and the known composition is up to 200. 2' -fucosyllactose (2 ' -fucosyllactose,2' -FL) and 3-fucosyllactose (3-fucosyllactose, 3-FL) are main components of breast milk oligosaccharide, play important physiological functions in aspects of regulating intestinal flora, regulating immunity and the like, and have wide market prospect. The European Commission has approved 2' -fucosyllactose and 3-fucosyllactose as new resource foods for sale in 2019 and 2021, respectively.

Microbial fermentation processes are currently the predominant production processes for 2' -fucosyllactose and 3-fucosyllactose. The commonly used strains are E.coli of various genetic backgrounds. The preparation of various human milk oligosaccharides by E.coli fermentation involves a number of common enzymes and transporters (e.g.sugar efflux transporter SetA as shown in WO2010142305A 1). Wherein the biosynthetic pathways of 2' -fucosyllactose and 3-fucosyllactose are very similar; to increase the fermentation yields of 2' -fucosyllactose and 3-fucosyllactose, the strategies for editing the genes involved in the biosynthetic pathways of both (e.g., knock-out of specific genes, overexpression of specific genes, insertion of specific genes, etc.) are also very similar （Huang D, Yang KX, Liu J, et al. Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement[J]. Metab Eng, 2017, 41: 23-38; Xu Zheng, li Na, chen Yingli, et al.

The study of 2 '-fucosyllactose production by E.coli fermentation has seen an in-depth knowledge in the art of key enzymes of the de novo synthesis and salvage pathways of 2' -fucosyllactose, including related enzymes involved in the degradation of synthetic precursors, sugar efflux transporters, etc. And the influence of the genes on the fermentation yield of 2' -fucosyllactose and 3-fucosyllactose is examined by editing the genes of related enzymes or transporter coding genes. According to the report, one or more of the following genes were knocked out: beta-galactosidase encoding gene lacZ, UDP-glucose lipid carrier transferase encoding gene wcaj, GDP-mannose hydrolase encoding gene nudd, regulatory genes in lactose lac operon sequence lacI, L-fucose isomerase encoding gene fucI, L-fucokinase encoding gene fucK, L-fuco-1-phosphate aldolase encoding gene fucA, D-arabinose isomerase encoding gene ara A, L-rhamnose isomerase encoding gene rhaA; and/or overexpressing or inserting one or more of the following genes: GDP-fucose synthase-encoding gene wcaG, GDP-mannose-4, 6-dehydratase-encoding gene gmd, beta-galactosidase-encoding gene lacY, phosphomannose isomerase-encoding gene manA, phosphomannose mutase-encoding gene manB, sugar efflux transporter A-encoding gene setA, mannose-1-phosphoguanine transferase-encoding gene manC, 2' -fucosyllactose-encoding gene futC, L-fucoskinase/GDP-L-fucose pyrophosphorylase-encoding gene fkp contributes to the improvement of fermentation yields of 2' -fucosyllactose and 3-fucosyllactose, wherein fucI, fucK, fucA, ara A, rhaA, fkp and the like participate in the salvage synthesis pathway of 2' -fucosyllactose. Due to the low activity and insoluble expression (insoluble expression) of α - (1, 3) -fucosyltransferase (FutA), the fermentation yield of 3-fucosyllactose is lower compared to 2' -fucosyllactose. Thus, further overexpression or insertion of the α - (1, 3) -fucosyltransferase encoding gene futA or beneficial mutation of the α - (1, 3) -fucosyltransferase encoding gene futA is more advantageous for improving fermentation yield of 3-fucosyllactose （Yun HC , Park BS , Seo J, et al. Biosynthesis of the human milk oligosaccharide 3-fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and β-galactosidase modification[J]. Biotechnology and Bioengineering, 2019.）.

In the prior publications, the construction of engineering bacteria for the fermentative production of 2' -fucosyllactose and 3-fucosyllactose has mostly involved the editing of genes encoding the above enzymes and transporters (for example, CN112662604A, CN112501106A, CN114276971A, CN113195509A, CN113151211a, etc.). However, the yields of 2' -fucosyllactose and 3-fucosyllactose produced by fermentation in industrial production are still low, resulting in high costs which affect the downstream industrial development. And there is currently no study on the effect of methionine aminopeptidase on the fermentative production of 2' -fucosyllactose and 3-fucosyllactose by E.coli.

Disclosure of Invention

In order to solve the technical problems, the invention adopts a gene editing technology to edit coding genes of various enzymes or transporters of the escherichia coli. The methionine aminopeptidase mutant capable of improving the yields of 2' -fucosyllactose and 3-fucosyllactose is obtained by a gene editing technology, and corresponding escherichia coli engineering bacteria are constructed.

According to one of the technical schemes, the invention provides a methionine aminopeptidase mutant, the amino acid sequence of which corresponds to SEQ ID NO:24, the amino acid position 44 of the amino acid sequence of the wild-type methionine aminopeptidase (Map) has a mutation: i > S, i.e., isoleucine (Ile) at position 44, is mutated to serine (Ser). The mutation is beneficial to improving the fermentation yield of 2' -fucosyllactose and 3-fucosyllactose.

It is well known to those skilled in the art that mutations at key sites of enzymes cause alterations in enzyme function. In other words, mutations to non-critical sites of the enzyme do not result in a change in the function of the enzyme. Thus, in addition to the mutation at amino acid position 44 corresponding to the wild-type methionine aminopeptidase, mutations at other non-critical positions are acceptable without affecting the function of the enzyme. Thus, the methionine aminopeptidase mutant hybridizes to SEQ ID NO:24 > 90% homology of the amino acid sequence of wild-type methionine aminopeptidase is acceptable.

Further preferably, the amino acid sequence of the methionine aminopeptidase mutant is as set forth in SEQ ID NO: 15. SEQ ID NO:15, the methionine aminopeptidase mutant shown in fig. 15 was mutated from isoleucine to serine at only amino acid position 44 relative to the wild-type methionine aminopeptidase. Fermentation verification using E.coli shows that SEQ ID NO:15 is advantageous for improving the fermentation yield of 2' -fucosyllactose and 3-fucosyllactose.

On the basis of the second technical scheme, the invention provides a coding gene which expresses the methionine aminopeptidase mutant in cells.

Preferably, the coding gene expresses the sequence of SEQ ID NO:15, and a methionine aminopeptidase mutant as shown in FIG. 15. Due to the degeneracy of the codons, an amino acid sequence can be expressed by translation of a myriad of different nucleic acid sequences. Thus, the present invention further provides a coding gene carrying at least the sequence of SEQ ID NO:22 or a nucleotide sequence fragment encoding the same amino acid sequence. SEQ ID NO:22 can encode in a cell a nucleotide fragment that expresses SEQ ID NO:15, and a methionine aminopeptidase mutant as shown in FIG. 15.

According to the third technical scheme, the escherichia coli genetic engineering bacteria which can be used for producing 2 '-fucosyllactose and/or 3-fucosyllactose through fermentation and are beneficial to improving the fermentation yield of the 2' -fucosyllactose and/or 3-fucosyllactose are provided. The genetically engineered bacterium can synthesize 2' -fucosyllactose and/or 3-fucosyllactose in vivo and express the methionine aminopeptidase mutant. Preferably, the genetically engineered bacterium expresses SEQ ID NO:15, and a methionine aminopeptidase mutant as shown in FIG. 15. Further preferably, the genome of the genetically engineered bacterium carries at least the sequence of SEQ ID NO:22 or a nucleotide sequence fragment encoding the same amino acid sequence, wherein the nucleotide sequence set forth in SEQ ID NO:22, and the nucleotide fragment can express SEQ ID NO after being introduced into genetically engineered bacteria by site-directed mutagenesis of a gene encoding wild methionine aminopeptidase: 15, and a methionine aminopeptidase mutant as shown in FIG. 15.

Still more preferably, the target gene (nucleotide fragment shown as SEQ ID NO: 22) is introduced into the genetically engineered bacterium by a plasmid transformation method or the like.

Still more preferably, the starting strain of the genetically engineered bacterium is selected from any one of E.coli K12 MG1655, E.coli BL21 (DE 3), E.coli JM109, and E.coli BW 25113. These strains have been used in a large number of ways, with E.coli K12 MG1655 (ESCHERICHIA COLI STRAIN K MG 1655) being one of the most well known and studied organisms in biology. Wherein E.coli K12 MG1655 has been deposited with the American type culture Collection (accession numbers ATCC 53103, ATCC 47076, ATCC 700926); coli BL21 (DE 3) has been deposited at BCCM genecorner (accession number LMBP 1455); coli JM109 has been deposited in the U.S. model culture collection library (accession numbers ATCC68635, ATCC 68868); coli BW25113 has been deposited at the E.coli genetics inventory center (Coli Genetics Stock Center) (accession number CGSC#7636). As starting strains commonly used by those skilled in the art, those skilled in the art are able to know the sources and purchase channels of the above strains.

Obviously, in order to further increase the fermentation yield of 2 '-fucosyllactose and/or 3-fucosyllactose, genes encoding enzymes or transporters involved in the 2' -fucosyllactose and/or 3-fucosyllactose synthesis pathway in the genetically engineered bacteria may be subjected to further gene editing. These gene edits may be the knock-out of at least one of the following genes on the E.coli genome: beta-galactosidase encoding gene lacZ, UDP-glucose lipid carrier transferase encoding gene wcaj, GDP-mannose hydrolase encoding gene nudd, regulatory genes lacI, L-fucose isomerase encoding gene fucI, L-fucokinase encoding gene fucK, L-fuco-1-phosphate aldolase encoding gene fucA, D-arabinose isomerase encoding gene ara A, L-rhamnose isomerase encoding gene rhaA in lactose lac operon sequence. It is also possible to overexpress or insert at least one of the following genes: GDP-fucose synthase-encoding gene wcaG, GDP-mannose-4, 6-dehydratase-encoding gene gmd, beta-galactosidase-encoding gene lacY, phosphomannose isomerase-encoding gene manA, phosphomannose mutase-encoding gene manB, sugar efflux transporter A-encoding gene setA, mannose-1-phosphoguanine transferase-encoding gene manC, 2' -fucosyllactose-encoding gene futC, alpha- (1, 3) -fucosyltransferase-encoding gene futA, L-fucose kinase/GDP-L-fucose pyrophosphorylase-encoding gene fkp. Combinations of the above-described various (gene knockout, gene insertion, gene overexpression) gene editing means, for example, knockout of lacZ and overexpression of manC, are also possible.

Still further preferably, in order to further increase the fermentation yield of 2 '-fucosyllactose and/or 3-fucosyllactose, the gene editing of genes encoding related enzymes or transporters involved in the 2' -fucosyllactose and/or 3-fucosyllactose synthesis pathway in the genetically engineered bacterium comprises:

knocking out a beta-galactosidase coding gene lacZ on the genome of the escherichia coli;

inserting GDP-fucose synthase encoding gene wcaG into colibacillus genome;

inserting GDP-mannose-4, 6-dehydratase coding gene gmd on the escherichia coli genome;

in situ over-expression of the beta-galactosidase encoding gene lacY;

inserting a phosphomannose isomerase encoding gene manA into the genome of the escherichia coli;

inserting a phosphomannose mutase encoding gene manB into the genome of the escherichia coli;

Knocking out UDP-glucose lipid carrier transferase coding gene wcaj on the escherichia coli genome;

knocking out GDP-mannose hydrolase coding gene nudd on the genome of the escherichia coli;

in situ overexpression of sugar efflux transporter A encoding gene setA;

overexpressing mannose-1-phosphate guanine transferase encoding gene manC on the plasmid;

The 2' -fucosyllactose encoding gene futC is overexpressed on the plasmid or the α - (1, 3) -fucosyltransferase encoding gene futA is overexpressed on the plasmid.

Wherein the above gene editing of lacZ, wcaG, gmd, lacY, manA, manB, wcaj, nudd, setA, manC is advantageous for improving the fermentation yield of 2' -fucosyllactose and 3-fucosyllactose; the above-described gene editing for futC is advantageous for improving the fermentation yield of 2 '-fucosyllactose, and the above-described gene editing for futA is advantageous for improving the fermentation yield of 2' -fucosyllactose.

Further, in the foregoing gene editing, wcaG, gmd, manA, manB is a single copy insert genome over-expression; manC, futA, futC is multicopy overexpression.

Further, in the foregoing gene editing, manA, manB, wcaG, gmd and lacY were overexpressed using the P _trc promoter.

Further, the plasmid is selected from any one of pTrc99a, pSB4K5, pET28a or pET22 b.

Further, the nucleotide sequence of the P _trc promoter is shown as SEQ ID NO: 4.

Further, in the aforementioned gene editing, the nucleotide sequence of lacZ is shown in SEQ ID NO:3 is shown in the figure; the nucleotide sequence of wcaG is shown as SEQ ID NO:5 is shown in the figure; the nucleotide sequence of gmd is shown in SEQ ID NO:6 is shown in the figure; lacY has the nucleotide sequence shown in SEQ ID NO: shown in figure 7; the nucleotide sequence of manA is shown in SEQ ID NO: shown as 9; manB has the nucleotide sequence shown in SEQ ID NO:10 is shown in the figure; wcaj has the nucleotide sequence shown in SEQ ID NO: 11. shown; nudd has the nucleotide sequence shown in SEQ ID NO: 13; setA has the nucleotide sequence shown in SEQ ID NO: 14; the nucleotide sequence of manC is shown in SEQ ID NO: shown at 17; futC has the nucleotide sequence shown in SEQ ID NO: shown at 18.

Further, in the foregoing gene editing, the nucleotide sequence of futA is as set forth in SEQ ID NO: 21. The nucleotide sequence templates that can be employed in plasmid construction are diverse and variable. Preferably, the sequence of SEQ ID NO:20 is a futA template.

Further, in the aforementioned gene editing, setA is preceded by in-situ overexpression by inserting a constitutive promoter and/or in-situ overexpression by inserting a promoter of a chloramphenicol resistance gene. Preferably, the nucleotide sequence of the promoter of the chloramphenicol resistance gene is as set forth in SEQ ID NO: 23. Preferably, the constitutive promoter is selected from any one of P_J23102、P_J23104、P_J23105、P_J23108、P_J23100、P_J23110、P_J23111、P_J23113、P_J23119、P₆₃₇、P₆₉₉.

The term "in situ overexpression" is a term opposite to the expression on a plasmid, and is also a term colloquially accepted in the art, and refers to the expression in situ (in situ), that is, the expression level of a target gene located in the chromosome in situ (in situ) is regulated and controlled by molecular biological means, such as promoter, ribosome binding site, transcription regulatory factor modification or codon optimization, etc. In situ overexpression of genes, i.e., overexpression in situ on a chromosome, can be increased by inserting additional promoters, or by other means that will be readily apparent to those skilled in the art, such as: by way of example, the setA gene may also be used to modify the promoter activity of the small molecule regulatory RNA gene sgrS in front of the setA gene, or to insert an additional promoter in front of the sgrS gene. Promoters for additional insertion for gene overexpression (including in situ overexpression and overexpression on plasmids) may be constitutive promoters and/or inducible promoters. The constitutive and inducible promoters described herein may be promoters suitable for use in prokaryotic expression systems, and particularly suitable for use in E.coli expression systems, including natural promoters and artificially constructed promoters.

The above-mentioned genetic engineering bacteria, mutants, coding genes, and gene editing techniques related to construction, and promoters (e.g., constitutive promoters P406, P479, P535, etc., and inducible expression promoters Ptac, etc.) for realizing in-situ overexpression or over-expression of the above-mentioned genes are known in the art, and can be found in, for example, peng Xiuling et al, the "genetic engineering experiment technique" (Changsha: 1998 2 th edition of Hunan science and technology Press), yuan Wuzhou, the "genetic engineering" (Beijing: chemical industry Press, 2019 2 nd edition), wei Yuta, the "genetic engineering principles and techniques" (Beijing: beijing university press, 2017 1 st edition), cao Weijun, the "microbiological engineering" (2007 2 nd edition of science and Press), etc. There are also sufficient disclosures and reports in the technical literature previously disclosed, such as, but not limited to, the following:

(1) Chen D, et al. Development of a DNA double-strand break-free base editing tool in Corynebacterium glutamicum for genome editing and metabolic engineering - ScienceDirect. Metabolic Engineering Communications, 2020, 11, e00135.

(2) Liu Yang, et al, metabolic control of microbial cell factories, journal of bioengineering, 2021, 37 (5): 1541-1563.

(3) Liang ST, et al. Activities of constitutive promoters inEscherichia coli. Journal of Molecular Biology, 1999, 292(1):19-37.

(4) Zhou L, et al. Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A. Microbial Cell Factories, 2017, 16(1): 84.

(5) Zhao, D, et al. CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. Scientific Reports, 2017, 7(1):16624.

(6) Guo, M, et al. Using the promoters of MerR family proteins as "rheostats" to engineer whole-cell heavy metal biosensors with adjustable sensitivity[J]. Journal of Biological Engineering, 2019, 13(1):1-9(PMID: 31452678).

The fourth technical scheme of the invention provides application of the genetically engineered bacterium in fermentation production of 2' -fucosyllactose and/or 3-fucosyllactose. Wherein the genetically engineered bacterium over-expressing futA on the plasmid is especially suitable for improving the fermentation yield of 3-fucosyllactose, and the genetically engineered bacterium over-expressing futC on the plasmid is especially suitable for improving the fermentation yield of 2' -fucosyllactose.

The invention has the beneficial effects that:

The invention obtains the corresponding methionine aminopeptidase by carrying out site-directed mutagenesis on the escherichia coli methionine aminopeptidase. It was found that the yield of E.coli 2' -fucosyllactose and 3-fucosyllactose expressing the methionine aminopeptidase was unexpectedly improved. When the E.coli setA gene is additionally subjected to in-situ overexpression and beneficial gene editing such as in-situ overexpression, plasmid overexpression and the like are performed on E.coli lacZ, wcaG, gmd, lacY, manA, manB, wcaj, nudd, futC, manC, futA, the yields of 2' -fucosyllactose and 3-fucosyllactose are higher.

Detailed Description

The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

The primer sequences in the following examples were written in order of 5 'to 3'.

EXAMPLE 1 construction of Strain TKYW2-2

Based on Escherichia coli W2 (E.coli K12 MG1655 delta lacIZ:: P _trc-wcaG-gmd-lacy, △adhE::P_trc -manB-manA) described in patent document CN112501106A, UDP-glucose lipid carrier transferase coding gene wcaj and GDP-mannose hydrolase coding gene nudd on the genome are knocked out, and further sugar outflow transporter gene setA is overexpressed in situ on the genome, so as to construct strain TKYW2-2.

The construction of E.coli W2 is briefly described based on the content of patent document CN112501106A, and the construction method thereof is introduced into this example. The escherichia coli W2 in CN112501106A is constructed by taking escherichia coli K12 MG1655 (ESCHERICHIA COLI K MG 1655) as an initial strain, a P _lac promoter sequence and regulatory genes lacI and lacZ in a lactose lac operator sequence of the initial strain are knocked out, a wcaG, gmd and lacY are overexpressed by a P _trc promoter after the original lacZ site to obtain a W1 strain, and manA and manB are overexpressed by a P _trc promoter at a adhe site of an alcohol dehydrogenase coding gene to obtain the W2 strain. The nucleotide sequence of the P _lac promoter involved in the construction of the escherichia coli W2 is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of lacI is shown in SEQ ID NO:2 is shown in the figure; the nucleotide sequence of lacZ is shown in SEQ ID NO:3 is shown in the figure; the nucleotide sequence of the P _trc promoter is shown in SEQ ID NO:4 is shown in the figure; the nucleotide sequence of wcaG is shown as SEQ ID NO:5 is shown in the figure; the nucleotide sequence of gmd is shown in SEQ ID NO:6 is shown in the figure; lacY has the nucleotide sequence shown in SEQ ID NO: shown in figure 7; the nucleotide sequence of adhE is shown in SEQ ID NO: shown as 8; the nucleotide sequence of manA is shown in SEQ ID NO: shown as 9; manB has the nucleotide sequence shown in SEQ ID NO: shown at 10.

Construction of Strain W2 delta wcaj

Using strain W2 as the starting strain, wcaj (nucleotide sequence shown as SEQ ID No. 11) was knocked out using CRISPR/Cas9 technology. The CRISPR/Cas9 technology used in the experiment refers to earlier study report 【Zhao D, et al. CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. Sci Rep 7, 16624】. by first constructing a first step homologous recombination fragment comprising an upstream and downstream homology arm, a chloramphenicol resistance gene cat and a general n20+ngg sequence (TAGTCCATCGAACCGAAGTAAGG), introducing the first step homologous recombination fragment into a W2 strain containing a pCAGO plasmid by electrotransformation, performing the first step recombination, pCAGO plasmid containing a recombinase gene, and 【Zhao D, et al. CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. Sci Rep 7, 16624】. selection of Cas9 and gRNA genes, etc., for the second homologous recombination. And selecting the correct clone after the second homologous recombination, and passaging to lose pCAGO plasmids, thereby obtaining the W2 delta wcaj strain knocked out of wcaj genes.

The specific method is described in detail below:

(1) And constructing homologous recombination fragments in the first step. The E.coli strain E.coli K12 MG1655 genome (GeneBank accession NO. NC-000913) was used as a template, and primers up-1 and up-2, and primers down-1 and down-2 in Table 1 were used for PCR amplification to obtain the upstream and downstream homology arms of homologous recombination, respectively. A strain genome with a chloramphenicol resistance gene cat (the nucleotide sequence is shown as SEQ ID NO:12, the SEQ ID NO:12 is the nucleotide sequence of a cat gene and a promoter thereof) stored in a laboratory is used as a template, and PCR amplification is carried out by using primers cat-1 and cat20-2 to obtain a fragment with a cat-N20 sequence. The upper and downstream homology arms, the fragments with cat-N20 sequence, the 3 fragments are used as templates, and the primers up-1 and down-2 are used for overlapping PCR amplification to obtain the first step homologous recombination fragments.

(2) The first step is homologous recombination. The pCAGO plasmid was transformed into strain W2 using a conventional plasmid transformation method to obtain strain W2 (pCAGO). W2 (pCAGO) was prepared using LB medium containing 1% glucose and IPTG (isopropyl-. Beta. -D-thiogalactoside) at a concentration of 0.1 mM, the first homologous recombination fragment was introduced by electrotransformation, the transformed bacterial liquid was spread on LB plates containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, and 1% glucose, and cultured at 30 ℃. And selecting the transformant for colony PCR identification to obtain the correct homologous recombination strain in the first step.

(3) And a second step of homologous recombination. The first homologous recombinant strain was inoculated into LB liquid medium containing 100 mg/L ampicillin, 0.1 mM IPTG and 2g/L arabinose, cultured at 30℃for 6 hours or more, streaked on plates to isolate single colonies, and clones capable of growing on LB plates containing 100 mg/L ampicillin but incapable of growing on LB plates containing 25 mg/L chloramphenicol were selected. Sequencing confirmed the correct clone in which the second homologous recombination occurred, and further culturing it at 37℃to lose pCAGO of the plasmid therein, thereby obtaining strain W2Δ wcaj.

TABLE 1 primers used to knock out wcaj Gene

2. Construction of Strain W2 Delta wcaj Delta nudd

Based on the Escherichia coli strain W2 delta wcaj, the nudd gene (the nucleotide sequence of which is shown as SEQ ID NO: 13) on the genome is knocked out by using the same method as the CRISPR/Cas9 technology, and the strain W2 delta wcaj delta nudd is constructed and named ZKYW1. The specific method is described in detail below:

(1) And constructing homologous recombination fragments in the first step. The genome of the strain E, the coll K12 MG1655 is used as a template, and the primer pairs Nup-1 and Nup-2 in the table 2 are respectively utilized, and the primer pairs Ndown-1 and Ndown-2 are amplified by PCR to obtain an upstream homology arm and a downstream homology arm of homologous recombination. The fragment with the cat-N20 sequence obtained when the strain W2 delta wcaj was constructed was used as a template, and PCR amplification was performed using primers Ncat-1 and Ncat-2 in Table 2, to obtain a new fragment with the cat-N20 sequence. The upper and downstream homology arms, new fragments with cat-N20 sequence, the 3 fragments as templates, and the primers Nup-1 and Ndown-2 are used for overlapping PCR amplification to obtain the first step homologous recombination fragments.

(2) The first step is homologous recombination. The pCAGO plasmid was transformed into the strain W2.DELTA. wcaj by a conventional plasmid transformation method to obtain the strain W2.DELTA.wcaj (pCAGO). W2.DELTA.wcaj (pCAGO) was competent in preparation of LB medium containing 1% glucose and IPTG at a concentration of 0.1 mM, the first homologous recombination fragment was introduced by electrotransformation, and the transformed bacterial liquid was spread on LB plate containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, and 1% glucose, and cultured at 30 ℃. And selecting the transformant for colony PCR identification to obtain the correct homologous recombination strain in the first step.

(3) And a second step of homologous recombination. The procedure for the second homologous recombination is the same as that described above for the knockdown wcaj gene. Sequencing confirmed that the correct clone for the second homologous recombination was obtained, which was further cultured at 37℃to lose pCAGO of the plasmids, thereby obtaining strain W2Δ wcaj Δ nudd, designated TKYW1.

TABLE 2 primers used to knock out nudd Gene

3. Construction of Strain TKYW2-2

Based on the strain TKYW1, by using the same method as the CRISPR/Cas9 technology, a constitutive promoter P _J23110 (http:// parts. Ig/Part: BBa_J 23100) is inserted in front of the sugar efflux transporter setA gene (nucleotide sequence shown in SEQ ID NO: 14), and the sequence of the promoter P _J23110 is: TTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGC; meanwhile, when CRISPR/Cas9 is utilized for the second recombination, a promoter of a chloramphenicol resistance gene (the nucleotide sequence is shown as SEQ ID NO: 23) is reserved so as to realize in-situ over-expression of setA by a double promoter, and the obtained strain is named TKYW-2.

(1) And constructing homologous recombination fragments in the first step. The genome of the strain E, the coll K12 MG1655 is used as a template, and primer pairs Sup-1 and Sup-2 in the table 3 are respectively used as primers, and the primer pairs SDown-1 and SDown-2 are respectively subjected to PCR amplification to obtain an upstream homology arm and a downstream homology arm of homologous recombination. The fragment with the cat-N20 sequence obtained when the strain W2 delta wcaj was constructed was used as a template, and PCR amplification was performed using the primers Scm-1 and Scm-2 in Table 3, to obtain a fragment with the cat gene sequence. PCR amplification was performed using N20-1 as the upstream primer and 110-2 as the downstream primer without using a template to obtain a gene fragment with the P _J23110 promoter. And (3) using Sup-1 and SDown-2 as primers, performing overlapping PCR amplification by using 4 fragments as templates, wherein the fragments comprise an upstream homology arm, a fragment with a cat gene sequence, a gene fragment with a P _J23110 promoter and a downstream homology arm obtained by the PCR amplification.

TABLE 3 construction of primers for strains that overexpress setA Gene on the genome

(2) The first step is homologous recombination. The pCAGO plasmid was transformed into strain TKYW1 using conventional plasmid transformation methods to obtain strain TKYW (pCAGO). TKYW1 (pCAGO) was prepared using LB medium containing 1% glucose and IPTG at a concentration of 0.1 mM, the fragments for the first homologous recombination were introduced by the electrotransformation method, and the transformed bacterial liquids were spread on LB plates containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, and 1% glucose, respectively, and cultured at 30 ℃. And selecting the transformant for colony PCR identification to obtain the correct strain of the homologous recombination in the first step.

(3) And a second step of homologous recombination. The procedure for the second homologous recombination is the same as that described above for the knockdown wcaj gene. Sequencing and verification to obtain the correct clone with the second homologous recombination, further culturing the clone at 37 ℃ to lose pCAGO plasmids therein, thereby obtaining a strain with a P _J23110 promoter, which is named TKYW2-2.

EXAMPLE 2 construction of Strain TKYW3

The amino acid sequence of the wild methionine aminopeptidase of Escherichia coli K12 MG1655 is shown in SEQ ID NO: shown at 24. Based on strain TKYW2-2, point mutations were made to the methionine aminopeptidase gene map on the genome using the same method as the CRISPR/Cas9 technique described above. The 44 th isoleucine of the translation protein is mutated into serine (Map ^Ile44Ser), and the amino acid sequence corresponding to the mutation of methionine aminopeptidase is shown as SEQ ID NO:15, the constructed strain was designated TKYW.

1. Construction of homologous recombination fragments in the first step

The nucleotide sequence of MG1655map ^T131G（map^T131G mutant with map gene point mutation stored in laboratory is shown as SEQ ID NO: 22) are used as templates, and primer pairs Mup-1 and Mup-2 in Table 4 are used as primers, and primer pairs Mdown-1 and Mdown-2 are used as primers, respectively, to obtain the upstream and downstream homology arms of homologous recombination by PCR amplification. The PCR amplification is carried out by using the fragment with the cat-N20 sequence obtained by PCR when the strain W2 delta wcaj is constructed as a template and utilizing the primers Mcat-1 and Mcat20-2 to obtain a new fragment with the cat-N20 sequence. The upper and downstream homology arms, the new fragment with cat-N20 sequence, the 3 fragments are used as templates, and the primers Mup-1 and Mdown-2 are used for carrying out overlapping PCR to obtain a first homologous recombination fragment, wherein the fragment contains the point mutation of the map gene, namely the 131 th base of the wild map gene is changed from T to G.

2. First step homologous recombination

The pCAGO plasmid was transformed into strain TKYW2-2 using conventional plasmid transformation methods to obtain strain TKYW2-2 (pCAGO). TKYW2-2 (pCAGO) was prepared using LB medium containing 1% glucose and IPTG at a concentration of 0.1 mM, the first homologous recombination fragment was introduced by electrotransformation, and the transformed bacterial liquid was spread on LB plates containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, and 1% glucose, and cultured at 30 ℃. And selecting the transformant for colony PCR identification to obtain the correct homologous recombination strain in the first step.

3. Second step homologous recombination

The procedure for the second homologous recombination is the same as that described above for the knockdown wcaj gene. Sequencing verifies that the correct clone for the second homologous recombination is obtained, and further cultures it at 37 ℃ to lose pCAGO plasmid therein, thus obtaining strain TKYW2-2 map ^T131G, named TKYW.

TABLE 4 primers used to construct map Gene Point mutant strains

Example 3 construction of plasmid pTrc99a-P _trc -futC-manC

The plasmid pTrc99a-futC-manC described in the patent document CN112501106A is used as a template (the nucleotide sequence of the P _trc promoter involved in construction of the plasmid pTrc99a-futC-manC is shown as SEQ ID NO: 4; the nucleotide sequence of the arabinose inducible promoter P _ara promoter is shown as SEQ ID NO: 16; the nucleotide sequence of the mannose-1-guanyl phosphate transferase coding gene manC is shown as SEQ ID NO: 17; the nucleotide sequence of the 2' -fucosyl lactose synthase coding gene futC is shown as SEQ ID NO: 18), PCR amplification is carried out by using the primers Darac-F and Darac-R in Table 5, the PCR product is purified and recovered, and then the PCR product is self-spliced, transformed into competent cells of E.coli JM109 by using the enzyme (pEASY-Uni SeamlessCloning and Assembly Kit, beijing 34343456/L ampicillin), and the recombinant plasmid pTrc 99-plasmid with the nucleotide sequence of 100-mg/L ampicillin is obtained by sequencing and verification of the transformant on LB plate, and the plasmid pTrc99a plasmid which has the correct nucleotide sequence is named as SEQ ID NO: 28-76. 19.

TABLE 5 construction of primers for plasmid pTrc99a-P _trc -futC-manC

Example 4 construction of plasmid pTrc99a-P _trc -futA-manC

Plasmid pTrc99a-P _trc -futA-manC was constructed by replacing futC in plasmid pTrc99a-P _trc -futC-manC with the alpha- (1, 3) -fucosyltransferase gene futA derived from helicobacter pylori (Helicobacter pylori) NCTC 11637. FutA can catalyze and produce 3-fucosyl lactose by using GDP-fucose and lactose as substrates.

PCR amplification was performed using plasmid pTrc99a-P _trc -futA-manC as template and FUTA-ZT-F and FUTA-ZT-R as primers in Table 6, to give a linear vector fragment with futC removed. The synthetic vector containing futA gene fragment (nucleotide sequence shown as SEQ ID NO: 20) is used as a template (the gene fragment contains futA gene shown as SEQ ID NO: 21), and FUTA-F and FUTA-R are used as primers for PCR amplification to obtain the fragment with futA gene. After the two PCR products are purified and recovered, the two PCR products are connected by using a seamless cloning enzyme, and are transformed into E.coli JM109 competent cells, and are cultured on an LB plate containing 100 mg/L ampicillin, and transformant sequencing verification is carried out, so that the correct recombinant plasmid named pTrc99a-Ptrc-futA-manC is obtained.

TABLE 6 construction of primers for plasmid pTrc99a-Ptrc-futA-manC

Example 52 construction of fucosyllactose and 3-fucosyllactose producing Strain and fermentation test

Plasmids pTrc99a-P _trc -futC-manC and pTrc99a-P _trc -futA-manC were introduced into TKYW-2 and TKYW3, respectively, by means of electrotransformation, to construct 2' -fucosyllactose-producing strains TKYW-2 (pTrc 99a-P _trc -futC-manC) and TKYW (pTrc 99a-P _trc -futC-manC), and 3-fucosyllactose-producing strains TKYW-2 (pTrc 99a-P _trc -futA-manC) and TKYW (pTrc 99a-P _trc -futA-pTmanC), respectively. The fermentation production level of the strain is tested, and the culture medium is as follows: LB medium: naCl 10 g/L, yeast powder 5 g/L, peptone 10 g/L and pH 7.0. Fermentation medium: KH ₂PO₄/g/L, yeast powder 8 g/L, (NH ₄)₂SO₄ 4 g/L, citric acid 1.7/g/L, mgSO ₄·7H₂ O2 g/L, thiamine 10 mg/L, glycerol 10 g/L, lactose 5 g/L,1 ml/L microelements （FeCl₃·6H₂O 25 g/L,MnCl₂·4H₂O 9.8 g/L,CoCl₂·6H₂O 1.6 g/L,CuCl₂·H₂O 1 g/L,H₃BO₃ 1.9 g/L, ZnCl₂ 2.6 g/L,Na₂MOO₄·2H₂O 1.1 g/L,Na₂SeO₃ 1.5 g/L,NiSO₄·6H₂O 1.5 g/l）, were pH adjusted to 7.2 with ammonia.

The fermentation test process comprises the following steps:

And (3) respectively picking single colonies of the 2' -fucosyllactose production strain and the 3-fucosyllactose production strain, transferring the single colonies into an LB liquid medium containing 50 mg/L ampicillin, and culturing in a shaking flask at 37 ℃ and 220 rpm on a shaking table for overnight. 10 microliters of culture solution is taken as seeds and transferred into a 24-deep pore plate containing 1mL fermentation culture medium in each pore, 50 mg/L ampicillin and 0.1 mmol/L IPTG are contained in the fermentation culture medium, and the culture is carried out in a pore plate shake incubator at 37 ℃ and the rotating speed of 800 revolutions per minute. 3 samples were grown in parallel for each strain. After culturing 48 h, 0.5 ml was sampled, cells were broken by an ultrasonic breaker, the supernatant was collected by centrifugation, boiled for ten minutes, an equal volume of acetonitrile was added, the supernatant was collected by centrifugation again, and then filtered with a 0.22 μm filter membrane. The concentration of 2' -fucosyllactose or 3-fucosyllactose in the sample is detected by HPLC, the chromatographic column used in the HPLC analysis is Carbohydrate ES 5u 250mm 4.6mm, the detector is an evaporative light detector, the mobile phase is 70% acetonitrile (acetonitrile: water), the flow rate is 0.8mL/min, the column temperature is set to 30 ℃, and the sample injection amount is 5 [ mu ] L. The sample concentration was quantified using a 2' -fucosyllactose or 3-fucosyllactose standard.

TABLE 72 results of fermentation test of fucosyllactose producing strains

TABLE 83 fermentation test results of fucosyllactose producing strains

As can be seen from tables 7 and 8, after the 44 th isoleucine of Map protein is mutated into serine, the yield of 2' -fucosyllactose or 3-fucosyllactose of the strain can be greatly improved.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A methionine aminopeptidase mutant, characterized in that the amino acid sequence of the methionine aminopeptidase mutant corresponds to the 44th amino acid position of the wild-type methionine aminopeptidase shown in SEQ ID NO: 24, and has a mutation: I>S; and the methionine aminopeptidase mutant has a homology of > 90% with the amino acid sequence of the wild-type methionine aminopeptidase shown in SEQ ID NO: 24.

2 . The methionine aminopeptidase mutant according to claim 1 , wherein the amino acid sequence of the methionine aminopeptidase mutant is as shown in SEQ ID NO: 15. 3 .

3. A coding gene, characterized in that the coding gene carries at least a nucleotide sequence fragment encoding the methionine aminopeptidase mutant according to claim 1 or 2.

4. The coding gene according to claim 3, characterized in that the coding gene carries at least a nucleotide sequence fragment encoding the methionine aminopeptidase mutant according to claim 2.

5 . The coding gene according to claim 4 , characterized in that the coding gene carries at least the nucleotide fragment shown in SEQ ID NO: 22.

6. A genetically engineered Escherichia coli, characterized in that the genetically engineered bacterium expresses the methionine aminopeptidase mutant according to claim 1 or 2, and the genetically engineered bacterium is an Escherichia coli capable of synthesizing 2'-fucosyllactose and/or 3-fucosyllactose in vivo.

7. The genetically engineered bacterium according to claim 6, characterized in that the genetically engineered bacterium expresses the methionine aminopeptidase mutant according to claim 2.

8. The genetically engineered bacterium according to claim 7, characterized in that the genetically engineered bacterium expresses the methionine aminopeptidase mutant through the coding gene shown in SEQ ID NO: 22.

9. The large genetically engineered bacterium according to claim 6, characterized in that the starting strain of the genetically engineered bacterium is selected from any one of Escherichia coli K12 MG1655, Escherichia coli BL21 (DE3), Escherichia coli JM109, and Escherichia coli BW25113.

10. The genetically engineered bacterium according to claim 6, characterized in that the genetically engineered bacterium comprises gene editing of genes encoding enzymes or transporters related to the synthesis pathway of 2'-fucosyllactose and/or 3-fucosyllactose;

Preferably, it comprises knocking out at least one of the following genes on the Escherichia coli genome: a β-galactosidase encoding gene, a UDP-glucose lipid carrier transferase encoding gene, a GDP-mannose hydrolase encoding gene, a regulatory gene in the lactose lac operon sequence, an L-fucose isomerase encoding gene, an L-fucokinase encoding gene , an L-fucose-1-phosphate aldolase encoding gene, a D-arabinose isomerase encoding gene , an L-rhamnose isomerase encoding gene; and/or it comprises overexpressing or inserting at least one of the following genes: a GDP-fucose synthase encoding gene , a GDP-mannose-4,6-dehydratase encoding gene , a β -galactosidase permease encoding gene , a phosphomannose isomerase encoding gene, a phosphomannose mutase encoding gene , a sugar efflux transporter A encoding gene , a mannose-1-phosphate guanine transferase encoding gene , a 2'-fucosyllactose synthase encoding gene , an α-(1,3)-fucosyltransferase encoding gene , L-fucokinase/GDP-L-fucose pyrophosphorylase encoding gene;

More preferably, the genetically engineered bacteria include the following gene editing:

Knock out the β -galactosidase encoding gene in the Escherichia coli genome;

Insert the GDP-fucose synthase encoding gene into the Escherichia coli genome;

Inserting the GDP-mannose-4,6-dehydratase encoding gene into the Escherichia coli genome;

Overexpression of the gene encoding β -galactosidase in situ;

Inserting a phosphomannose isomerase encoding gene into the Escherichia coli genome;

Inserting a phosphomannose mutase encoding gene into the Escherichia coli genome;

Knock out the UDP-glucose lipid carrier transferase encoding gene in the Escherichia coli genome;

Knock out the GDP-mannose hydrolase encoding gene in the Escherichia coli genome;

In situ overexpression of the gene encoding sugar efflux transporter A;

The gene encoding mannose-1-phosphate guanylyltransferase was overexpressed on a plasmid;

The gene encoding 2'-fucosyllactose synthase is overexpressed on the plasmid or the gene encoding α- (1,3)-fucosyltransferase is overexpressed on the plasmid.

11. The genetically engineered bacterium according to claim 10, characterized in that the GDP-fucose synthase encoding gene, GDP-mannose-4,6-dehydratase encoding gene, phosphomannose isomerase encoding gene, and phosphomannose mutase encoding gene are single-copy inserted into the genome for overexpression; and the mannose-1-phosphate guanylyltransferase encoding gene, α- (1,3)-fucosyltransferase encoding gene, and 2'-fucosyllactose synthase encoding gene are multiple-copy overexpressed.

12. The genetically engineered bacterium according to claim 10, characterized in that the gene encoding phosphomannose isomerase, the gene encoding phosphomannose mutase, the gene encoding GDP-fucose synthase, the gene encoding GDP-mannose-4,6-dehydratase and the gene encoding β- galactosidase are overexpressed using a Ptrc promoter; the plasmid is selected from any one of pTrc99a, pSB4K5, pET28a or pET22b; the nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO: 4.

13. The genetically engineered bacterium according to claim 10, characterized in that the nucleotide sequence of the β- galactosidase encoding gene is shown in SEQ ID NO: 3; the nucleotide sequence of the GDP-fucose synthase encoding gene is shown in SEQ ID NO: 5; the nucleotide sequence of the GDP-mannose-4,6-dehydratase encoding gene is shown in SEQ ID NO: 6; the nucleotide sequence of the β -galactosidase encoding gene is shown in SEQ ID NO: 7; the nucleotide sequence of the phosphomannose isomerase encoding gene is shown in SEQ ID NO: 9; the nucleotide sequence of the phosphomannose mutase encoding gene is shown in SEQ ID NO: 10; the nucleotide sequence of the UDP-glucose lipid carrier transferase encoding gene is shown in SEQ ID NO: 11; the nucleotide sequence of the GDP-mannose hydrolase encoding gene is shown in SEQ ID NO: 13; the nucleotide sequence of the sugar efflux transporter A encoding gene is shown in SEQ ID NO: 14; the nucleotide sequence of the mannose-1-phosphate guanine transferase encoding gene is shown in SEQ ID NO: The nucleotide sequence of the gene encoding 2'-fucosyllactose synthase is shown in SEQ ID NO: 17; the nucleotide sequence of the gene encoding 2'-fucosyllactose synthase is shown in SEQ ID NO: 18; the nucleotide sequence of the gene encoding α- (1,3)-fucosyltransferase is shown in SEQ ID NO: 21.

14. The genetically engineered bacterium according to claim 10, characterized in that a constitutive promoter is inserted in front of the sugar efflux transporter A encoding gene for in situ overexpression and/or a promoter of a chloramphenicol resistance gene is inserted in front of the gene for in situ overexpression; the nucleotide sequence of the promoter of the chloramphenicol resistance gene is shown in SEQ ID NO: 23.

15. The genetically engineered bacterium according to claim 14, characterized in that the constitutive promoter is selected from any one of P _J23102 , P _J23104 , P _J23105 , P _J23108 , P _J23100 , P _J23110 , P _J23111 , P _J23113 , P _J23119 , P ₆₃₇ , and P ₆₉₉ .

16. Use of the genetically engineered bacterium according to any one of claims 10 to 15 in the fermentation production of 2'-fucosyllactose, characterized in that the genetically engineered bacterium overexpresses a 2'-fucosyllactose synthase encoding gene on a plasmid .

17. Use of the genetically engineered bacteria according to any one of claims 10 to 15 in the fermentation production of 3-fucosyllactose, characterized in that the genetically engineered bacteria overexpresses an α- (1,3)-fucosyltransferase encoding gene on a plasmid .