LgtA mutants and their use in human milk oligosaccharide production

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a LgtA mutant and application thereof in human milk oligosaccharide production. By means of gene editing technology, the key enzyme beta-1, 3-N-acetylglucosamine transferase of a synthesis intermediate LNT II is mutated, and a mutant with at least one of R13H, L24M and R205C mutations on the basis of the wild-type LgtA shown in SEQ ID NO. 1 is obtained. Further, the mutant is applied to the production plasmid construction process of LNT II, LNnT and LNT, and the yield of LNT II, LNnT and LNT is further improved.

Description

Technical field:

[0002] The present invention belongs to the technical field of genetic engineering, and particularly relates to an LgtA mutant and application thereof in the production of human milk oligosaccharides. Background technology:

[0004] Breast milk is rich in the six nutrients needed by the human body and is the most ideal and highest quality natural food for newborns. Human milk oligosaccharides (HMOs) are important active factors in breast milk, second only to lactose and lipids. They play a positive role in regulating the intestinal flora of infants and protecting their health. There are more than 200 types of HMOs, among which lacto- N -trisaccharide (lacto- N L-triose (LNT II) is the fundamental building block of complex human milk oligosaccharides. However, due to the complex synthesis steps and high production costs, industrial production of complex HMOs has been hampered. The efficient and large-scale synthesis of complex HMOs remains a research hotspot.

[0005] LNT II as a synthetic lacto- N -New tetrasaccharide (Lacto- N -neotetraose, LNnT) and lactose- N -tetrasaccharide (Lacto- N -tetraose, LNT) and other complex structure HMOs core unit, 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) is synthesized in pgi Glucose-6-phosphate isomerase is catalyzed by the gene encoding glucose-6-phosphate to convert it into fructose-6-phosphate (F6P), which is then converted into glmS Encoded glutamine-fructose-6-phosphate aminotransferase, glmM Encoded phosphoglucosamine mutase, glmU The enzyme N-acetylglucosamine-1-phosphate uridine transferase / glucosamine-1-phosphate acetyltransferase encodes the UDP-GlcNAc, which is then converted to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). Subsequently, UDP-GlcNAc undergoes a transglycosylation reaction with lactose under the action of β-1,3-N-acetylglucosamine transferase (LgtA) to form lactose-N-triose (LNT II). lgtB Under the catalysis of the encoded β-1,4-galactosyltransferase, it combines with UDP-Gal to generate LNnT. wbgOThe catalysis of the encoded beta-1, 3-galactosyltransferase generates LNT.

[0006] LgtA is a key enzyme for synthesizing LNT II, and improving the catalytic efficiency of LgtA is conducive to the synthesis of LNT II. However, there is no report on the crystal structure and enzymatic properties of LgtA. By modifying the enzyme to increase the performance of the enzyme, the yield of the target product can be improved. Therefore, it is of great significance to promote the industrial production of LNT II, LNnT and LNT and other complex structure HMOs by obtaining a mutant with high catalytic efficiency through modification of LgtA. SUMMARY

[0008] The present application utilizes gene editing technology to mutate the key enzyme beta-1, 3-N-acetylglucosamine transferase for synthesizing intermediate product LNT II. Further, the mutant is applied to the production plasmid construction process of LNT II, LNnT and LNT, further improving the yield of LNT II, LNnT and LNT.

[0009] One of the technical solutions provided by the present application is an LgtA mutant, which is obtained by at least one of R13H, L24M and R205C mutations based on the wild type LgtA shown in SEQ ID NO. 1.

[0010] Further, the mutant is an R13H mutant, and the amino acid sequence is shown in SEQ ID NO. 2.

[0011] Further, the mutant is an L24M mutant, and the amino acid sequence is shown in SEQ ID NO. 3.

[0012] Further, the mutant is an R205C mutant, and the amino acid sequence is shown in SEQ ID NO. 4.

[0013] Further, the mutant is an R13H / L24M mutant, and the amino acid sequence is shown in SEQ ID NO. 5.

[0014] Further, the mutant is an R13H / R205C mutant, and the amino acid sequence is shown in SEQ ID NO. 6.

[0015] Further, the mutant is an L24M / R205C mutant, and the amino acid sequence is shown in SEQ ID NO. 7.

[0016] Further, the mutant is an R13H / L24M / R205C mutant, and the amino acid sequence is shown in SEQ ID NO. 8.

[0017] 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;

[0018] Furthermore, 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 - wbgO - lgtA in lgtA The gene was then introduced into the E. coli host for the production of LNT II, ​​LNnT, and LNT;

[0019] Furthermore, the E. coli host is E. coli K12 MG1655 knocked out the lactose operon sequence lacZ , in the original lacZ P trc Promoter overexpression lacY Obtained after.

[0020] Beneficial effects:

[0021] The present invention obtains seven LgtA mutants by gene editing technology and constructs them into production plasmid pTrc99a-P trc - lgtA、 pTrc99a-P trc - lgtB - lgtA、 pTrc99a-P trc - wbgO - lgtA, The recombinant strains containing the LgtA mutants were introduced into E. coli strains producing LNT II, ​​LNnT, and LNT, and found that the recombinant strains containing the LgtA mutants increased the production of LNT II, ​​LNnT, and LNT compared to those containing wild-type LgtA. This indicates that LgtA mutations containing R13H, L24M, and R205C are beneficial for increasing the production of LNT II, ​​LNnT, and LNT. Description of the drawings:

[0023] Figure 1 This is the first step of homologous recombination colony PCR verification of strain H0.

[0024] Figure 2 This is the second-step homologous recombination colony PCR verification of strain H0. Specific implementation method:

[0026] The present application is further described in the following specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments can be known to those skilled in the art, and appropriate ones can be selected from the means and materials known to solve the corresponding technical problems. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present application, and the essence and scope of the present application are only limited by the claims. For those skilled in the art, various changes or modifications to the composition and amount of the materials in these embodiments without departing from the essence and scope of the present application also fall within the protection scope of the present application.

[0027] The following definitions are used in the present application:

[0028] 1. Nomenclature of amino acid and DNA nucleic acid sequences

[0029] The recognized IUPAC nomenclature of amino acid residues is used, in either single-letter or three-letter code form. The DNA nucleic acid sequence uses the recognized IUPAC nomenclature.

[0030] 2. Identification of LgtA mutants

[0031] The "original amino acid + position + substituted amino acid" is used to represent the mutated amino acid in the LgtA mutant. For example, R13H, indicates that the amino acid at position 13 is replaced by H from R of wild-type LgtA, and the position number corresponds to the amino acid sequence number of wild-type LgtA in SEQ ID NO. 1. The specific information is as follows:

[0032]

[0033] The wild-type LgtA involved in the present application has an amino acid sequence as shown in SEQ ID NO. 1:

[0034] MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0035] The R13H mutant involved in the application has an amino acid sequence as shown in SEQ ID NO. 2:

[0036] MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0037] The L24M mutant involved in the application has an amino acid sequence as shown in SEQ ID NO. 3:

[0038] MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0039] The R205C mutant involved in the application has an amino acid sequence as shown in SEQ ID NO. 4:

[0040] MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0041] The R13H / L24M mutant involved in the application has an amino acid sequence as shown in SEQ ID NO. 5:

[0042] MPSEAFRRHRAYHENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0043] The R13H / R205C mutant involved in the application has an amino acid sequence as shown in SEQ ID NO. 6:

[0044] MPSEAFRRHRAYHENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0045] The L24M / R205C mutant involved in the application has an amino acid sequence as shown in SEQ ID NO. 7:

[0046] MPSEAFRRHRAYRENKLQPLVSVMICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0047] The R13H / L24M / R205C mutant according to the present application has an amino acid sequence as shown in SEQ ID NO. 8:

[0048] MPSEAFRRHRAYRENKLQPLVSVMICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTDGTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTECDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR

[0049] The present application will be further explained by the specific embodiments below.

[0050] Example 1 Construction of strain H0

[0051] Escherichia coli K12 MG1655 (ATCC 47076) was used as the host strain. Escherichia coliK12 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 lac operon sequence of the starting strain. lacZ , in the original lacZ P trc Promoter overexpression lacY , strain H0 was obtained.

[0052] The specific construction method of strain H0 is as follows:

[0053] 1. Construction of homologous recombination fragments

[0054] Using the wild-type strain MG1655 preserved in the laboratory as a template, the primers listed in Table 1 were used to construct homologous recombination fragments. PCR amplification was performed using primers lac-up-f / r and lac-down-f / r 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. 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 primer pair lac-cat-f / r to obtain a fragment with 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.

[0055] 2. First step: homologous recombination

[0056] The pCAGO plasmid was transformed into Escherichia coli K12 MG1655 using conventional plasmid transformation methods to obtain the strain Escherichia coli K12 MG1655 (pCAGO). Escherichia coli K12 MG1655 (pCAGO) was prepared using LB medium containing 1% (m / v) glucose and 0.1 mM IPTG. The homologous recombinant fragment obtained in step 1 was introduced by electroporation. The transformed bacterial liquid was spread on LB plates 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 result was 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.

[0057] 3. Second step homologous recombination

[0058] The strain that has been successfully recombined once 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 areas were drawn on the LB plate containing ampicillin to isolate single colonies. 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 (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 to lose the pCAGO plasmid, thereby obtaining the knockout lacZ And in the original lacZ P trc Promoter overexpression lacY The strain was named H0.

[0059] Table 1 Knockout Construction lacZ Use P trc Overexpression lacY Primers used for strains

[0060]

[0061] Example 2 Construction of LNT II production plasmid

[0062] (1) Construction of plasmid pTrc99a-Ptrc - lgtA

[0063] lgtA Gene 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 SEQ ID NO. 10). The linear vector and linear gene fragment obtained by PCR were purified and recovered, ligated using the ClonExpress II Recombination Ligation Kit (Novozymes Biotech Co., Ltd.), transformed into Escherichia coli DH5α competent cells, and cultured on LB plates containing 100 mg / L ampicillin. Transformants were selected for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA .

[0064] (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 an R to H mutation.

[0065] Plasmid pTrc99a-P trc - 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 containing the gene encoding the LgtA R13H mutation. trc - lgtA The linear vector obtained by PCR was purified and recovered, ligated using the ClonExpress II Recombination Ligation Kit (Novozymes Biotech Co., Ltd.), and transformed into Escherichia coli DH5α competent cells. The cells were cultured on LB plates containing 100 mg / L ampicillin. Transformants were selected for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - lgtA R13H .

[0066] (3) Construction of plasmid pTrc99a-Ptrc - lgtA L24M (i.e. pTrc99a-P trc - lgtA in the plasmid lgtA encoding β-1,3-N-acetylglucosamine transferase with a mutation of the 24th amino acid from L to M)

[0067] PCR amplification was performed with the plasmid pTrc99a-P trc - lgtA as the template and fp-24-f / r in Table 2 as the primers to obtain a linear vector pTrc99a-P trc - lgtA -24 containing the gene encoding LgtA with L24M mutation. The linear vector obtained by PCR above was purified and recovered, and was ligated using a ClonExpress II recombination ligation kit (Novagen Biotechnology Co., Ltd.) and transformed into E. coli DH5α competent cells, which were cultured on an LB plate containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification. The correct recombinant plasmid was obtained and named as plasmid pTrc99a-P trc - lgtA L24M .

[0068] (4) Construction of plasmid pTrc99a-P trc - lgtA R205C (i.e. pTrc99a-P trc - lgtA in the plasmid lgtA encoding β-1,3-N-acetylglucosamine transferase with a mutation of the 205th amino acid from R to C)

[0069] PCR amplification was performed with the plasmid pTrc99a-P trc - lgtA as the template and fp-205-f / r in Table 2 as the primers to obtain a linear vector pTrc99a-P trc - lgtA -205 containing the gene encoding LgtA with R205C mutation. The linear vector obtained by PCR above was purified and recovered, and was ligated using a ClonExpress II recombination ligation kit (Novagen Biotechnology Co., Ltd.) and transformed into E. coli DH5α competent cells, which were cultured on an LB plate containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification. The correct recombinant plasmid was obtained and named as plasmid pTrc99a-P trc - lgtA R205C .

[0070] (5) Constructing plasmid pTrc99a-P trc - lgtA R13H,L24M (i.e. pTrc99a-P trc - lgtA in the plasmid lgtA The 13th amino acid of the encoded β-1, 3-N-acetylglucosamine transferase is mutated from R to H, and the 24th amino acid is mutated from L to M

[0071] The plasmid pTrc99a-P trc - lgtA R13H is used as the template, and the fp-24-f / r in Table 2 is used as the primer to perform PCR amplification, to obtain a linear carrier pTrc99a-P trc - lgtA -13 / 24. The linear carrier obtained by the above-mentioned PCR is purified and recovered, and is connected using a ClonExpress II recombination connection kit (Novozyme Biotech Co., Ltd.), and is transformed into E. coli DH5α competent cells, and is cultured on an LB plate containing 100 mg / L ampicillin, and the transformants are picked for colony PCR and sequencing verification, to obtain a correct recombinant plasmid, which is named as plasmid pTrc99a-P trc - lgtA R13H,L24M .

[0072] (6) Constructing plasmid pTrc99a-P trc - lgtA R13H,R205C (i.e. pTrc99a-P trc - lgtA in the plasmid lgtA The 13th amino acid of the encoded β-1, 3-N-acetylglucosamine transferase is mutated from R to H, and the 205th amino acid is mutated from R to C

[0073] The plasmid pTrc99a-P trc - lgtA R13H is used as the template, and the fp-205-f / r in Table 2 is used as the primer to perform PCR amplification, to obtain a linear carrier pTrc99a-P trc - lgtA-13 / 205. The linear vector obtained by the above PCR was purified, recovered, and ligated using a ClonExpress II recombination ligation kit (Novagen Biotechnology 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. The correct recombinant plasmid was named plasmid pTrc99a-P trc - lgtA R13H,R205C .

[0074] (7) Construction of plasmid pTrc99a-P trc - lgtA L24M,R205C (i.e., pTrc99a-P trc - lgtA In the plasmid lgtA encoding β-1, 3-N-acetylglucosamine transferase, the 24th amino acid was mutated from L to M, and the 205th amino acid was mutated from R to C

[0075] PCR amplification was performed using plasmid pTrc99a-P trc - lgtA L24M as the template and fp-205-f / r in Table 2 as the primers, to obtain a linear vector pTrc99a-P trc - lgtA -24 / 205. The linear vector obtained by the above PCR was purified, recovered, and ligated using a ClonExpress II recombination ligation kit (Novagen Biotechnology 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. The correct recombinant plasmid was named plasmid pTrc99a-P trc - lgtA L24M,R205C .

[0076] (8) Construction of plasmid pTrc99a-P trc - lgtA R13H,L24M,R205C (i.e., pTrc99a-P trc - lgtA In the plasmid lgtA encoding β-1, 3-N-acetylglucosamine transferase, the 13th amino acid was mutated from R to H, the 24th amino acid was mutated from L to M, and the 205th amino acid was mutated from R to C

[0077] PCR amplification was performed using plasmid pTrc99a-Ptrc - lgtA R13H,L24M As a template, PCR amplification was performed with fp-205-f / r in Table 2 as primers to obtain a linear vector pTrc99a-P trc - lgtA -13 / 24 / 205 containing the coding gene of LgtA with R13H, L24M and R205C mutations. The linear vector obtained by PCR above was purified and recovered, and ligated using ClonExpress II recombination ligation kit (Novagen Biotechnology Co., Ltd.) and 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, named plasmid pTrc99a-P trc - lgtA R13H,L24M,R205C .

[0078] Table 2 Construction of plasmid pTrc99a-P trc - lgtA and all primers of the mutant

[0079]

[0080] Example 3 Construction of LNnT production plasmid

[0081] (1) Construction of plasmid pTrc99a-P trc - lgtB - lgtA

[0082] lgtB The gene encodes a lipid oligosaccharide biosynthesis protein lex-1, and its nucleotide sequence is shown as SEQ ID NO. 11. Based on the plasmid pTrc99a-P trc - lgtA , the plasmid pTrc99a-P trc - lgtB - lgtA is constructed. As a template, PCR amplification is performed with plasmid pTrc99a-P trc - lgtA as a template, and lgtA-F and 99a-R in Table 3 as primers to obtain a linear vector pTrc99a-P trc -lgtA; as a template, PCR amplification is performed with artificially synthesized and codon-optimized lgtB sequence as primers in Table 3 to obtain a linear gene fragment lgtB(SEQ ID NO. 11); the linear vector and the linear gene fragment obtained by PCR above were purified and recovered, and ligated using a ClonExpress II recombination ligation kit, transformed into E. coli DH5a competent cells, cultured on LB plates containing 100 mg / L ampicillin, and the transformants were picked for colony PCR and sequencing verification, to obtain a correct recombinant plasmid, designated as plasmid pTrc99a-P trc - lgtB - lgtA .

[0083] (2) Construction of plasmid pTrc99a-P trc - lgtB - lgtA R13H

[0084] The plasmid pTrc99a-P trc - lgtB - lgtA was used as a template for PCR amplification with the primers fp-13-f / r in Table 2, to obtain a linear vector pTrc99a-P trc - lgtB - lgtA -13. The specific construction process is referred to in Example 2 of the present application, to obtain a correct recombinant plasmid, designated as plasmid pTrc99a-P trc - lgtB - lgtA R13H .

[0085] (3) Construction of plasmid pTrc99a-P trc - lgtB - lgtA L24M

[0086] The plasmid pTrc99a-P trc - lgtB - lgtA was used as a template for PCR amplification with the primers fp-24-f / r in Table 2, to obtain a linear vector pTrc99a-P trc - lgtB - lgtA -24. The specific construction process is referred to in Example 2 of the present application, to obtain a correct recombinant plasmid, designated as plasmid pTrc99a-P trc - lgtB - lgtA L24M .

[0087] (4) Construction of plasmid pTrc99a-P trc - lgtB - lgtAR205C

[0088] Plasmid pTrc99a-P trc - lgtB - lgtA fp-24-f / r in Table 2 as primers to obtain linear vector pTrc99a-P trc - lgtB - lgtA -24. The specific construction process is referred to Example 2 of the present application, and the correct recombinant plasmid is named as plasmid pTrc99a-P trc - lgtB - lgtA R205C .

[0089] (5) Construction of plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M

[0090] Plasmid pTrc99a-P trc - lgtB - lgtA R13H as primers to obtain linear vector pTrc99a-P trc - lgtB - lgtA -13 / 24. The specific construction process is referred to Example 2 of the present application, and the correct recombinant plasmid is named as plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M .

[0091] (6) Construction of plasmid pTrc99a-P trc - lgtB-lgtA R13H,R205C

[0092] Plasmid pTrc99a-P trc - lgtB - lgtA R13H as primers to obtain linear vector pTrc99a-P trc - lgtB - lgtA -13 / 205. The specific construction process is referred to Example 2 of the present application, and the correct recombinant plasmid is named as plasmid pTrc99a-P trc - lgtB-lgtA R13H,R205C .

[0093] (7) Construct plasmid pTrc99a-P trc - lgtB - lgtA L24M,R205C

[0094] Take plasmid pTrc99a-P trc - lgtB - lgtA L24M as template, and take fp-205-f / r in Table 2 as primers to perform PCR amplification to obtain linear carrier pTrc99a-P trc - lgtB - lgtA -24 / 205. Refer to the specific construction process in Example 2 of the present application to obtain the correct recombinant plasmid, which is named as plasmid pTrc99a-P trc - lgtB - lgtA L24M,R205C .

[0095] (8) Construct plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M,R205C

[0096] Take plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M as template, and take fp-205-f / r in Table 2 as primers to perform PCR amplification to obtain linear carrier pTrc99a-P trc - lgtB - lgtA -13 / 24 / 205. Refer to the specific construction process in Example 2 of the present application to obtain the correct recombinant plasmid, which is named as plasmid pTrc99a-P trc - lgtB - lgtA R13H,L24M,R205C .

[0097] Table 3 Construct plasmid pTrc99a-P trc - lgtB - lgtA Primer used

[0098]

[0099] Example 4 Construct LNT production plasmid

[0100] (1) Construct plasmid pTrc99a-P trc - wbgO - lgtA

[0101] wbgO The gene encodes β-1,3-galactosyltransferase, and its nucleotide sequence is shown in SEQ ID NO.12. trc - lgtA The plasmid pTrc99a-P was constructed based on trc - wbgO - lgtA . Using plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using primers lgtA-F and 99a-R in Table 4 to obtain the linear vector pTrc99a-P trc -lgtA; wbgO The sequence in Table 4 was used as a template. wbgO -F / R as primers for PCR amplification to obtain linear gene fragments wbgO The linear vector and linear gene fragment obtained by PCR were purified and recovered, ligated using the ClonExpress II recombination ligation kit, transformed into Escherichia coli DH5α competent cells, cultured on LB plates containing 100 mg / L ampicillin, and transformants were selected for colony PCR and sequencing verification to obtain the correct recombinant plasmid, which was named plasmid pTrc99a-P trc - wbgO - lgtA .

[0102] (2) Construction of plasmid pTrc99a-P trc - wbgO - lgtA R13H

[0103] Plasmid pTrc99a-P trc - wbgO - 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 - wbgO - lgtA -13. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained and named as plasmid pTrc99a-P trc - wbgO - lgtA R13H .

[0104] (3) Construction of plasmid pTrc99a-P trc - wbgO - lgtA L24M

[0105] The plasmid pTrc99a-P trc - wbgO - lgtA The linear vector pTrc99a-P trc - wbgO - lgtA -24. The specific construction process is referred to Example 2 of the present application, and the correct recombinant plasmid is named as plasmid pTrc99a-P trc - wbgO - lgtA L24M .

[0106] (4) Construction of plasmid pTrc99a-P trc - wbgO - lgtA R205C

[0107] The plasmid pTrc99a-P trc - wbgO - lgtA The linear vector pTrc99a-P trc - wbgO - lgtA -205. The specific construction process is referred to Example 2 of the present application, and the correct recombinant plasmid is named as plasmid pTrc99a-P trc - wbgO - lgtA R205C .

[0108] (5) Construction of plasmid pTrc99a-P trc - wbgO - lgtA R13H,L24M

[0109] The plasmid pTrc99a-P trc - wbgO - lgtA R13H The linear vector pTrc99a-P trc - wbgO - lgtA -13 / 24. The specific construction process is referred to Example 2 of the present application, and the correct recombinant plasmid is named as plasmid pTrc99a-P trc - wbgO - lgtA R13H,L24M .

[0110] (6) Constructing plasmid pTrc99a-P trc - wbgO-lgtA R13H,R205C

[0111] Take plasmid pTrc99a-P trc - wbgO - lgtA R13H as a template, and perform PCR amplification with fp-205-f / r in Table 2 as primers to obtain linear vector pTrc99a-P trc - wbgO - lgtA -13 / 205. For the specific construction process, refer to Example 2 of the present application, and obtain the correct recombinant plasmid, which is named as plasmid pTrc99a-P trc - wbgO-lgtA R13H,R205C .

[0112] (7) Constructing plasmid pTrc99a-P trc - wbgO - lgtA L24M,R205C

[0113] Take plasmid pTrc99a-P trc - wbgO - lgtA L24M as a template, and perform PCR amplification with fp-205-f / r in Table 2 as primers to obtain linear vector pTrc99a-P trc - wbgO - lgtA -24 / 205. For the specific construction process, refer to Example 2 of the present application, and obtain the correct recombinant plasmid, which is named as plasmid pTrc99a-P trc - wbgO - lgtA L24M,R205C .

[0114] (8) Constructing plasmid pTrc99a-P trc - wbgO - lgtA R13H,L24M,R205C

[0115] Take plasmid pTrc99a-P trc - wbgO - lgtA R13H,L24M as a template, and perform PCR amplification with fp-205-f / r in Table 2 as primers to obtain linear vector pTrc99a-P trc - wbgO - lgtA -13 / 24 / 205. The specific construction process refers to Example 2 of this application, and the correct recombinant plasmid is obtained and named as plasmid pTrc99a-P trc - wbgO - lgtA R13H,L24M,R205C .

[0116] Table 4 Construction of plasmid pTrc99a-P trc - wbgO - lgtA Primers used

[0117]

[0118] Example 5 Construction and fermentation test of LNT II, ​​LNnT, and LNT production strains

[0119] By electroporation, the LNT II, ​​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:

[0120] Table 5 Strains and plasmids used in this patent

[0121]

[0122] The production levels of the above strains were tested by fermentation. The culture medium used was:

[0123] LB medium (1 L): NaCl 10 g, yeast powder 5 g, peptone 10 g.

[0124] Fermentation medium (1 L): KH2PO4 3 g, yeast powder 8 g, (NH4)2SO4 4 g, citric acid 1.7 g, MgSO4·7H2O 2 g, thiamine 10 mg, MOPS 60 g, glycerol 10 g, lactose 5 g, 1 mL trace elements, ammonia water to adjust the pH to 7.0.

[0125] Trace elements (1 L): FeCl3·6H2O 25 g, MnCl2·4H2O 9.8 g, CoCl2·6H2O 1.6 g, CuCl2·H2O 1 g, H3BO3 1.9 g, ZnCl2 2.6 g, Na2M O O4·2H2O 1.1 g, Na2SeO31.5 g, NiSO4·6H2O 1.5 g.

[0126] The fermentation test process is:

[0127] Single colonies of LNT II, LNnT and LNT production strains were picked respectively, and cultured in LB liquid medium containing 50 mg / L ampicillin at 37℃, 220 rpm, overnight. The bacterial liquid cultured overnight was used as seed liquid, and the bacterial liquid was transferred to a 24-well plate containing 1 mL fermentation medium at a 2% (v / v) inoculation amount, the fermentation medium contained 50 mg / L ampicillin and 0.1 mM IPTG, and the fermentation was carried out at 37℃, 800 rpm. Each strain was cultured in parallel for 3 times.

[0128] During the fermentation process, the growth (OD 600 ) of the bacterial cells, the yields of LNT II, LNnT and LNT were determined, the concentrations of LNT II, LNnT and LNT in the samples were detected by high performance liquid chromatography, and the sample concentrations were quantified by using the standard curves of LNT II, LNnT and LNT. The high performance liquid chromatography detection conditions of LNT II, LNnT and LNT are referred to the example 4 of the patent CN117736280A. The results are shown in Tables 6 to 8:

[0129] Table 6 Test results of different strains producing LNT II

[0130]

[0131] Table 7 Test results of different strains producing LNnT

[0132]

[0133] Table 8 Test results of different strains producing LNT

[0134]

[0135] It can be seen from Tables 6 to 8 that after expressing the LgtA mutant and the combined expression of the LgtA mutant respectively, the yields of LNT II, LNnT and LNT are improved to a certain extent compared with the expression of wild type LgtA.

[0136] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various forms and details of changes, modifications, replacements and modifications to these embodiments without departing from the spirit and principles of the present application, and the scope of the present application 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 basis of the wild-type LgtA shown in SEQ ID NO.

1. The mutant is any one of the following (1) to (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 the R13H / L24M mutant, and the amino acid sequence is shown in SEQ ID NO.5; (5) The mutant is the R13H / R205C mutant, and the amino acid sequence is shown in SEQ ID NO.6; (6) The mutant is the 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.

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

3. Use of the LgtA mutant according to claim 1 in the production of LNT II, ​​LNnT and LNT, characterized in that: The coding gene of the mutant is expressed in an E. coli host, wherein the E. coli host is a host in which the lactose operon sequence is deleted. lacZ Genes and overexpression lacY acquired after the gene.

4. The use according to claim 3, wherein The gene encoding the LgtA mutant of claim 1 is replaced with the production plasmid pTrc99a-P trc - lgtA, pTrc99a-P trc - lgtB - lgtA or pTrc99a-P trc - wxya - lgtA in lgtA The gene was cloned and introduced into the E. coli host for the production of LNTⅡ, LNnT and LNT.

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