LgtA mutant and application thereof in production of human milk oligosaccharide

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an LgtA mutant and application thereof in production of human milk oligosaccharide. According to the present invention, a gene editing technology is adopted to mutate a key enzyme beta-1, 3-N-acetyl glucosamine transferase for synthesizing an intermediate product LNT II so as to obtain a mutant having at least one of R13H mutation, L24M mutation and R205C mutation on the basis of a wild type LgtA represented by SEQ ID NO.1; further, the mutant is applied to the construction process of production plasmids of the LNTII, the LNnT and the LNT, and the yield of the LNTII, the LNnT and the LNT is further increased.

Description

Technical field: The invention belongs to the technical field of genetic engineering, and specifically relates to an LgtA mutant and application thereof in the production of human milk oligosaccharides. Background technology: Breast milk is rich in six nutrients needed by the human body and is the most ideal and high-quality natural food for newborns. Human milk oligosaccharides (HMOs) are important active factors in breast milk, second only to lactose and lipids, and play a positive role in regulating infant intestinal flora and protecting infant health. There are more than 200 types of HMOs, including lactoyl- N -trisaccharide (lacto- N -triose, LNT Ⅱ) is the basic structural unit of human milk oligosaccharides with complex structures. However, due to the cumbersome synthesis steps and high production costs of HMOs with complex structures, industrial production cannot be achieved. How to effectively synthesize HMOs with complex structures on a large scale is still a hot topic in current research.

[0003] LNT II as a synthetic lactoyl- N -New tetrasaccharide (Lacto- N -neotetraose, LNnT) and lactose- N -Tetrasaccharide (Lacto- N -tetraose, LNT) and other complex structure HMOs core units, its large-scale synthesis helps to increase the production of complex structure HMOs. When E. coli synthesizes LNT Ⅱ, glucose or glycerol is used as the carbon source and lactose is used as the substrate. Glucose-6-phosphate (Glc-6-P) pgi The encoded glucose-6-phosphate isomerase is converted into fructose-6-phosphate (F6P), which is then glmS Encoded glutamine-fructose-6-phosphate aminotransferase, glmM Encoded by phosphoglucosamine mutase, glmU The UDP-GlcNAc is converted into uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) under the catalysis of the encoded N-acetylglucosamine-1-phosphate uridine transferase / glucosamine-1-phosphate acetyltransferase. Subsequently, UDP-GlcNAc undergoes a glycosyl transfer reaction with lactose under the action of β-1,3-N-acetylglucosamine transferase (LgtA) to produce lactose-N-triose (LNT II). LNT II is lgtB Under the catalysis of the encoded β-1,4-galactosyltransferase, it combines with UDP-Gal to generate LNnT. w.b.g.O. LNT is produced under the catalysis of the encoded β-1,3-galactosyltransferase.

[0004] LgtA is a key enzyme in the synthesis of LNT Ⅱ. Improving its catalytic efficiency is beneficial to the synthesis of LNT Ⅱ. However, there is no report on the crystal structure and enzymatic properties of LgtA. Increasing the performance of the enzyme through enzyme modification can increase the yield of the target product. Therefore, obtaining mutants with high catalytic efficiency by modifying LgtA is of great significance to promote the industrial production of HMOs with complex structures such as LNT Ⅱ, LNnT and LNT. Summary of the invention: The present invention utilizes gene editing technology to mutate the key enzyme β-1,3-N-acetylglucosamine transferase for the synthesis of the intermediate product LNT Ⅱ. The mutant is then applied to the construction of production plasmids for LNT Ⅱ, LNnT and LNT, further increasing the yields of LNT Ⅱ, LNnT and LNT.

[0006] One of the technical solutions provided by the present invention is an LgtA mutant, wherein the mutant is obtained by causing at least one of the mutations R13H, L24M, and R205C to occur on the basis of the wild-type LgtA shown in SEQ ID NO.1; Further, the mutant is a R13H mutant, and the amino acid sequence is shown in SEQ ID NO.2; Further, the mutant is a L24M mutant, and the amino acid sequence is shown in SEQ ID NO.3; Further, the mutant is a R205C mutant, and the amino acid sequence is shown in SEQ ID NO.4; Further, the mutant is a R13H / L24M mutant, and the amino acid sequence is shown in SEQ ID NO.5; Further, the mutant is a R13H / R205C mutant, and the amino acid sequence is shown in SEQ ID NO.6; Further, the mutant is a L24M / R205C mutant, and the amino acid sequence is shown in SEQ ID NO.7; Furthermore, the mutant is a R13H / L24M / R205C mutant, and the amino acid sequence is shown in SEQ ID NO.8.

[0007] The second technical solution provided by the present invention is the application of the LgtA mutant described in the first technical solution, especially in the production of LNT Ⅱ, LNnT and LNT; Further, the gene encoding the LgtA mutant described in one of the technical solutions was used to replace the production plasmid pTrc99a-P trc - lgtA、 pTrc99a-P trc - lgtB- lgtA、 pTrc99a-P trc - w.b.g.O. - lgtA In lgtA The genes were then introduced into the E. coli host for the production of LNT Ⅱ, LNnT and LNT; Furthermore, the E. coli host is E. coli K12 MG1655 knocked out of the lactose operon sequence lacZ , in the original lacZ P trc Promoter overexpression lacY Obtained later.

[0008] Beneficial effects: The present invention obtains seven LgtA mutants by gene editing technology, and constructs the production plasmid pTrc99a-P trc - lgtA、 pTrc99a-P trc - lgtB - lgtA、 pTrc99a-P trc - w.b.g.O. - lgtA, The results showed that the recombinant strain containing the LgtA mutant could increase the production of LNT Ⅱ, LNnT and LNT compared with the wild-type LgtA. It can be seen that the mutations of LgtA including R13H, L24M and R205C are beneficial to increase the production of LNT Ⅱ, LNnT and LNT. Description of the drawings: Figure 1 The first step of homologous recombination colony PCR verification of strain H0.

[0010] Figure 2 This is the second step homologous recombination colony PCR verification of strain H0. Specific implementation method: The present invention is further described below by specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments may be well known to those skilled in the art, and suitable ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications of the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0012] The following definitions are used in the present invention: 1. Nomenclature of amino acid and DNA sequences The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0013] 2. Identification of LgtA mutants The mutated amino acid in the LgtA mutant is represented by "original amino acid + position + replaced amino acid". For example, R13H means that the amino acid at position 13 is replaced by H from the R of the wild-type LgtA, and the position number corresponds to the amino acid sequence number of the wild-type LgtA in SEQ ID NO.1. The specific information is as follows:

[0014] The wild-type LgtA involved in the present invention has an amino acid sequence as shown in SEQ ID NO.1: MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIA DFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The amino acid sequence of the R13H mutant involved in the present invention is shown in SEQ ID NO.2: MPSEAFRRHRAYHENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The L24M mutant involved in the present invention has an amino acid sequence as shown in SEQ ID NO.3: MPSEAFRRHRAYRENKLQPLVSVMICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The R205C mutant involved in the present invention has an amino acid sequence as shown in SEQ ID NO.4: MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The R13H / L24M mutant involved in the present invention has an amino acid sequence as shown in SEQ ID NO.5: MPSEAFRRHRAYHENKLQPLVSVMICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The R13H / R205C mutant involved in the present invention has an amino acid sequence as shown in SEQ ID NO.6: MPSEAFRRHRAYHENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The L24M / R205C mutant involved in the present invention has the amino acid sequence shown in SEQ ID NO.7: MPSEAFRRHRAYRENKLQPLVSVMICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The R13H / L24M / R205C mutant involved in the present invention has the amino acid sequence shown in SEQ ID NO.8: MPSEAFRRHRAYHENKLQPLVSVMICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIA DFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR The present invention will be further explained below through specific implementation modes.

[0015] Example 1 Construction of strain H0 Escherichia coli K12 MG1655 ( Escherichia coli K12 MG1655) was constructed as the starting strain, and CRISPR / Cas9 technology (Zhao D, et al. CRISPR / Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. SciRep 7, 16624) was used to knock out the lactose operon sequence of the starting strain. lacZ , in the original lacZ P trc Promoter overexpression lacY , strain H0 was obtained.

[0016] The specific construction method of strain H0 is as follows: 1. Construction of homologous recombination fragments Using the wild-type strain MG1655 preserved in the laboratory as a template, the primers in Table 1 were used to construct homologous recombination fragments. Using lac-up-f / r and lac-down-f / r as primers, PCR amplification was performed to obtain the upstream and downstream homology arms of homologous recombination. Using the pTrc99a plasmid (GeneBank: U13872) as a template, the promoter P was obtained using primers lac-trc-f / r as a template. trc Sequence fragment. Artificially synthesized chloramphenicol resistance gene cat , cat The promoter and N20 sequence (SEQ ID NO.9) vector were used as templates, and PCR amplification was performed using the primer pair lac-cat-f / r to obtain a cat- The fragment of N20 sequence. The homology arms above and downstream, P trc Promoter fragment with cat- The four fragments of N20 sequence were used as templates, and overlapping PCR was performed using primers lac-up-f and lac-down-r to obtain homologous recombinant fragments.

[0017] 2. First step: homologous recombination The pCAGO plasmid was transformed into Escherichia coli K12 MG1655 using conventional plasmid transformation methods to obtain strain Escherichia coli K12 MG1655 (pCAGO). Escherichia coli K12 MG1655 (pCAGO) competent cells were prepared using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG. The homologous recombination fragment obtained in step 1 was introduced using the electroporation method. The transformed bacterial solution was spread on an LB plate containing 100 mg / L ampicillin and 25 mg / L chloramphenicol, as well as 1% glucose, and cultured at 30°C. Transformants were picked for colony PCR verification (verification primers: lac-yz-f / r). If the recombination was successful, the band size was about 2732 bp, and the verification results were as follows: Figure 1 As shown, the bands are correct, that is, the first homologous recombination is successful, and the correct transformants are picked for the second step of homologous recombination.

[0018] 3. Second step homologous recombination The strain that has been successfully verified for the first recombination was inoculated into an LB test tube containing 100 μg / mL AMP and 0.1 mM IPTG, and cultured on a shaker at 30°C for more than 6 hours to induce the expression of the CRISPR / Cas9 system and λ-red protein to complete the second recombination. Three zones were drawn on the LB plate containing ampicillin to separate single colonies, and the isolated single colonies were picked and spotted on the chloramphenicol-resistant LB plate and the ampicillin-resistant LB plate respectively. The single colonies that did not grow on the chloramphenicol medium but grew on the ampicillin medium were selected and verified by colony PCR (the verification primers are: lac-yz-f / r). If the recombination is correct, the band size is about 1797 bp, and the verification result is as follows Figure 2 As shown, the band is correct, and the PCR product of the band is sequenced. The sequencing result is correct, and the second-step homologous recombination strain is obtained. The second-step homologous recombination strain is further cultured at 37°C, and the pCAGO plasmid is lost, thereby obtaining a knockout strain. lacZ And in the original lacZ Ptrc Promoter overexpression lacY The strain was named H0.

[0019] Table 1 Knockout constructs lacZ Use P trc Overexpression lacY Primers used for strains

[0020] Example 2 Construction of LNTⅡ production plasmid (1) Construction of plasmid pTrc99a-P trc - lgtA lgtA Genes encoding β-1,3- N -acetyl glucosamine transferase; using plasmid pTrc99a as a template and 99a-F / R in Table 2 as primers for PCR amplification, a linear vector pTrc99a-P trc ; artificially synthesized and codon-optimized lgtA The sequence was used as a template and lgtA-F / R in Table 2 was used as primers for PCR amplification to obtain a linear gene fragment. lgtA (shown in SEQID NO.10). The linear vector and linear gene fragment obtained by the above PCR were purified and recovered, connected using the ClonExpressⅡ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA .

[0021] (2) Construction of plasmid pTrc99a-P trc - lgtA R13H (i.e., pTrc99a-P trc - lgtA Plasmid lgtA The 13th amino acid of the encoded β-1,3-N-acetylglucosamine transferase undergoes a mutation from R to H) Plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using fp-13-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P containing the gene encoding the LgtA R13H mutation. trc - lgtA-13. The linear vector obtained by the above PCR was purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into E. coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA R13H .

[0022] (3) Construction of plasmid pTrc99a-P trc - lgtA L24M (i.e., pTrc99a-P trc - lgtA Plasmid lgtA The 24th amino acid of the encoded β-1,3-N-acetylglucosamine transferase undergoes a mutation from L to M) Plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using fp-24-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P containing the gene encoding the LgtA L24M mutation. trc - lgtA -24. The linear vector obtained by the above PCR was purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA L24M .

[0023] (4) Construction of plasmid pTrc99a-P trc - lgtA R205C (i.e., pTrc99a-P trc - lgtA Plasmid lgtA The 205th amino acid of the encoded β-1,3-N-acetylglucosamine transferase undergoes a mutation from R to C) Plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P containing the gene encoding the LgtA R205C mutation. trc- lgtA -205. The linear vector obtained by the above PCR was purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into E. coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA R205C .

[0024] (5) Construction of plasmid pTrc99a-P trc - lgtA R13H,L24M (i.e., pTrc99a-P trc - lgtA Plasmid lgtA The 13th amino acid of the encoded β-1,3-N-acetylglucosamine transferase undergoes a mutation from R to H, and the 24th amino acid undergoes a mutation from L to M) Plasmid pTrc99a-P trc - lgtA R13H As a template, PCR amplification was performed using fp-24-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P containing the gene encoding the LgtA mutations R13H and L24M. trc - lgtA -13 / 24. The linear vector obtained by the above PCR was purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA R13H,L24M .

[0025] (6) Construction of plasmid pTrc99a-P trc - lgtA R13H,R205C (i.e., pTrc99a-P trc - lgtA Plasmid lgtA The 13th amino acid of the encoded β-1,3-N-acetylglucosamine transferase undergoes a mutation from R to H, and the 205th amino acid undergoes a mutation from R to C) Plasmid pTrc99a-P trc - lgtA R13HAs a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P containing the gene encoding the LgtA mutations R13H and R205C. trc - lgtA -13 / 205. The linear vector obtained by the above PCR was purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA R13H,R205C .

[0026] (7) Construction of plasmid pTrc99a-P trc - lgtA L24M,R205C (i.e., pTrc99a-P trc - lgtA Plasmid lgtA The 24th amino acid of the encoded β-1,3-N-acetylglucosamine transferase has a mutation from L to M, and the 205th amino acid has a mutation from R to C) Plasmid pTrc99a-P trc - lgtA L24M As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P containing the gene encoding the LgtA mutations L24M and R205C. trc - lgtA -24 / 205. The linear vector obtained by the above PCR was purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA L24M,R205C .

[0027] (8) Construction of plasmid pTrc99a-P trc - lgtA R13H,L24M,R205C (i.e., pTrc99a-P trc - lgtA Plasmid lgtAThe 13th amino acid of the encoded β-1,3-N-acetylglucosamine transferase mutated from R to H, the 24th amino acid mutated from L to M, and the 205th amino acid mutated from R to C) Plasmid pTrc99a-P trc - lgtA R13H,L24M As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P containing the gene encoding the LgtA mutations R13H, L24M and R205C. trc - lgtA -13 / 24 / 205. The linear vector obtained by the above PCR was purified and recovered, ligated using the ClonExpress Ⅱ Recombination Ligation Kit (Novozyme Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA R13H,L24M,R205C .

[0028] Table 2 Construction of plasmid pTrc99a-P trc - lgtA All primers for mutants

[0029] Example 3 Construction of LNnT production plasmid (1) Construction of plasmid pTrc99a-P trc - lgtB - lgtA lgtB The gene encodes the lipooligosaccharide biosynthesis protein lex-1, and its nucleotide sequence is shown in SEQ ID NO.11. trc - lgtA Plasmid pTrc99a-P was constructed based on trc - lgtB - lgtA . With plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using lgtA-F and 99a-R in Table 3 as primers to obtain the linear vector pTrc99a-P trc -lgtA; artificially synthesized and codon-optimized lgtB The sequence was used as a template and lgtB-F / R in Table 3 was used as primers for PCR amplification to obtain a linear gene fragment. lgtBThe linear vector and linear gene fragment obtained by the above PCR were purified and recovered, connected using the ClonExpress Ⅱ recombination ligation kit, transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtB - lgtA .

[0030] (2) Construction of plasmid pTrc99a-P trc - lgtB - lgtA R13H Plasmid pTrc99a-P trc - lgtB - lgtA As a template, PCR amplification was performed using fp-13-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - lgtB - lgtA -13. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - lgtB - lgtA R13H .

[0031] (3) Construction of plasmid pTrc99a-P trc - lgtB - lgtA L24M Plasmid pTrc99a-P trc - lgtB - lgtA As a template, PCR amplification was performed using fp-24-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - lgtB - lgtA -24. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - lgtB - lgtA L24M .

[0032] (4) Construction of plasmid pTrc99a-P trc - lgtB - lgtA R205C Plasmid pTrc99a-P trc - lgtB - lgtA As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - lgtB - lgtA -205. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - lgtB - lgtA R205C .

[0033] (5) Construction of plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M Plasmid pTrc99a-P trc - lgtB - lgtA R13H As a template, PCR amplification was performed using fp-24-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - lgtB - lgtA -13 / 24. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M .

[0034] (6) Construction of plasmid pTrc99a-P trc - lgtB-lgtA R13H,R205C Plasmid pTrc99a-P trc - lgtB - lgtA R13H As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - lgtB - lgtA -13 / 205. The specific construction process refers to Example 2 of the present application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - lgtB-lgtA R13H,R205C .

[0035] (7) Construction of plasmid pTrc99a-P trc - lgtB - lgtA L24M,R205C Plasmid pTrc99a-P trc - lgtB - lgtA L24M As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - lgtB - lgtA -24 / 205. The specific construction process refers to Example 2 of the present application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - lgtB - lgtA L24M,R205C .

[0036] (8) Construction of plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M,R205C Plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - lgtB - lgtA -13 / 24 / 205. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M,R205C .

[0037] Table 3 Construction of plasmid pTrc99a-P trc - lgtB - lgtA Primers used

[0038] Example 4 Construction of LNT production plasmid (1) Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA w.b.g.O. The gene encodes β-1,3-galactosyltransferase, and its nucleotide sequence is shown in SEQ ID NO.12. trc - lgtAPlasmid pTrc99a-P was constructed based on trc - w.b.g.O. - lgtA . With plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using lgtA-F and 99a-R in Table 4 as primers to obtain the linear vector pTrc99a-P trc -lgtA; w.b.g.O. The sequence in Table 4 was used as a template. w.b.g.O. -F / R as primers for PCR amplification to obtain linear gene fragments w.b.g.O. The linear vector and linear gene fragment obtained by the above PCR were purified and recovered, connected using the ClonExpress Ⅱ recombination ligation kit, transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - w.b.g.O. - lgtA .

[0039] (2) Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H Plasmid pTrc99a-P trc - w.b.g.O. - lgtA As a template, PCR amplification was performed using fp-13-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - w.b.g.O. - lgtA -13. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H .

[0040] (3) Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA L24M Plasmid pTrc99a-P trc - w.b.g.O. - lgtA As a template, PCR amplification was performed using fp-24-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - w.b.g.O. - lgtA -24. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - w.b.g.O. - lgtA L24M .

[0041] (4) Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA R205C Plasmid pTrc99a-P trc - w.b.g.O. - lgtA As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - w.b.g.O. - lgtA -205. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - w.b.g.O. - lgtA R205C .

[0042] (5) Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H,L24M Plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H As a template, PCR amplification was performed using fp-24-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - w.b.g.O. - lgtA -13 / 24. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H,L24M .

[0043] (6) Construction of plasmid pTrc99a-P trc - w-b-g-O-lg-T-A R13H,R205C Plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13HAs a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - w.b.g.O. - lgtA -13 / 205. The specific construction process refers to Example 2 of the present application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - w-b-g-O-lg-T-A R13H,R205C .

[0044] (7) Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA L24M,R205C Plasmid pTrc99a-P trc - w.b.g.O. - lgtA L24M As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - w.b.g.O. - lgtA -24 / 205. The specific construction process refers to Example 2 of the present application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - w.b.g.O. - lgtA L24M,R205C .

[0045] (8) Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H,L24M,R205C Plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H,L24M As a template, PCR amplification was performed using fp-205-f / r in Table 2 as primers to obtain the linear vector pTrc99a-P trc - w.b.g.O. - lgtA -13 / 24 / 205. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained, which is named plasmid pTrc99a-P trc - w.b.g.O. - lgtA R13H,L24M,R205C .

[0046] Table 4 Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA Primers used

[0047] Example 5 Construction and fermentation test of LNTⅡ, LNnT and LNT production strains By using the electroporation method, the LNTⅡ, LNnT, and LNT production plasmids described in Examples 2, 3, and 4 of the present application were introduced into H0, respectively, to construct the strains shown in Table 5: Table 5 Strains and plasmids used in this patent

[0048] The production levels of the above strains were tested by fermentation. The culture medium used was: LB medium (1 L): NaCl 10 g, yeast powder 5 g, peptone 10 g.

[0049] Fermentation medium (1 L): KH2PO4 3 g, yeast powder 8 g, (NH4)2SO4 4 g, citric acid 1.7 g, MgSO4·7H2O2 g, thiamine 10 mg, MOPS 60 g, glycerol 10 g, lactose 5 g, 1 mL trace elements, ammonia water to adjust pH to 7.0.

[0050] Trace elements (1 L): FeCl3·6H2O 25 g, MnCl2·4H2O 9.8 g, CoCl2·6H2O 1.6 g, CuCl2·H2O 1 g, H3BO31.9 g, ZnCl22.6 g, Na2M O O4·2H2O 1.1 g, Na2SeO31.5 g, NiSO4·6H2O 1.5 g.

[0051] The fermentation test process is: Single colonies of LNT Ⅱ, LNnT and LNT production strains were picked and cultured overnight in LB liquid medium containing 50 mg / L ampicillin at 37°C and 220 rpm. The overnight culture solution was used as seed solution and transferred to a 24-well plate containing 1 mL fermentation medium at a 2% (v / v) inoculation volume. The fermentation medium contained 50 mg / L ampicillin and 0.1mM IPTG at 37°C and 800 rpm. Three parallel cultures were cultured for each strain.

[0052] During the fermentation process, the growth of the bacteria (OD 600), LNT Ⅱ, LNnT and LNT yields, the concentrations of LNT Ⅱ, LNnT and LNT in the samples were detected by high performance liquid chromatography, and the sample concentrations were quantified using the standard curves of LNT Ⅱ, LNnT and LNT. The high performance liquid chromatography detection conditions of LNT Ⅱ, LNnT and LNT refer to Example 4 of patent CN117736280A. The results are shown in Tables 6 to 8: Table 6 Test results of LNT Ⅱ produced by different strains

[0053] Table 7 Test results of LNnT produced by different strains

[0054] Table 8 Test results of LNT production by different strains

[0055] It can be seen from Tables 6 to 8 that after expressing the LgtA mutants separately or in combination, the yields of LNT Ⅱ, LNnT and LNT were all improved to a certain extent compared with the expression of wild-type LgtA.

[0056] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. An LgtA mutant, characterized in that: The mutant is obtained by causing at least one of the mutations R13H, L24M and R205C to occur on the wild-type LgtA shown in SEQ ID NO.

1.

2. The LgtA mutant according to claim 1, characterized in that The mutant is any one of the following (1)-(7): (1) The mutant is an R13H mutant, and the amino acid sequence is shown in SEQ ID NO.2; (2) The mutant is the L24M mutant, and the amino acid sequence is shown in SEQ ID NO.3; (3) The mutant is the R205C mutant, and the amino acid sequence is shown in SEQ ID NO.4; (4) The mutant is a R13H / L24M mutant, and the amino acid sequence is shown in SEQ ID NO.5; (5) The mutant is a R13H / R205C mutant, and the amino acid sequence is shown in SEQ ID NO.6; (6) The mutant is L24M / R205C mutant, and the amino acid sequence is shown in SEQ ID NO.7; (7) The mutant is a R13H / L24M / R205C mutant, and the amino acid sequence is shown in SEQ ID NO.

8. The gene encoding the LgtA mutant according to claim 1 .

4. Use of the LgtA mutant according to claim 1 or the encoding gene according to claim 3 in the production of LNT Ⅱ, LNnT and LNT.

5. The use according to claim 4, characterized in that The gene encoding the LgtA mutant of claim 1 is replaced with the production plasmid pTrc99a-P trc - lgtA, pTrc99a-P trc - ikB - lgtA, pTrc99a-P trc - wxya - oeLh In oeLh The gene was cloned and introduced into the E. coli host for the production of LNTⅡ, LNnT and LNT.

6. The use according to claim 5, characterized in that The E. coli host is a knockout of the lactose operon sequence lacZ Genes and overexpression lacY acquired after the gene.

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