A transcriptional regulator MarR mutant and its application in human milk oligosaccharide production

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a MarR mutant and its application in the production of human milk oligosaccharides. The mutant is obtained by causing R77S and / or L127P mutations on the basis of wild-type MarR. The coding gene of the above mutant is applied to strains producing HMOs such as 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL, and the growth ability and production performance of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production strains may be improved by enhancing the stress resistance of the strains.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a transcriptional regulatory factor MarR mutant and its application in the production of human milk oligosaccharides. Background Art

[0002] Human milk oligosaccharides (HMOs) are a type of carbohydrate in breast milk, second only to lactose and lipids. HMOs can inhibit the binding of pathogens to the intestinal mucosa and reduce the risk of infection; they can also promote the growth of beneficial bacteria and maintain the balance of intestinal microecology. In addition, HMOs also have the effect of promoting early brain development in newborns and improving infant immunity. There are more than 200 types of HMOs, which can be divided into fucosylated lactose, such as 2'-fucosyllactose (2'-fucosyllactose, 2'-FL) and 3-fucosyllactose (3-fucosyllactose, 3-FL), sialylated lactose, such as 3'-sialyllactose (3'-sialyllactose, 3'-SL) and 6'-sialyllactose (6'-sialyllactose, 6'-SL), and neutral non-fucosylated lactose, such as lactoyl- N -trisaccharide (lacto- N -triose, LNTII), lactoyl- N -New tetrasaccharide (Lacto- N -neotetraose, LNnT) and lactose- N -tetraose (Lacto-N-tetraose, LNT), accounting for 35%~50%, 12%~14% and 42%~55% respectively. In recent years, 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL have been produced by microbial synthesis, enzymatic method and chemical synthesis. Among them, microbial synthesis is more suitable for large-scale industrial production because of its relatively low cost and easy to expand culture.

[0003] Escherichia coli is used as a chassis strain for the microbial synthesis of various HMOs because of its advantages such as clear genetic background, simple culture method, short growth cycle and mature gene manipulation technology. The metabolic pathway for the de novo synthesis of 2'-FL and 3-FL by microorganisms is as follows: intracellular fructose 6-phosphate is the initial substrate, and GDP-fucose is finally generated through the action of a series of specific enzymes such as ManA (phosphomannose isomerase), ManB (phosphomannose mutase), ManC (mannose-1-phosphate guanyltransferase), GmD (GDP-mannose dehydrogenase) and WcaG (GDP-fucose synthase). Finally, GDP-fucose reacts with lactose to generate 2'-FL under the catalysis of α-(1,2)-fucosyltransferase FutC, and 3-FL under the catalysis of α-(1,3)-fucosyltransferase FutA. In Escherichia coli, glucose or glycerol is used as the carbon source and lactose is used as the substrate when synthesizing LNnT. lgtA Encoded β-1,3- N Under the catalysis of -acetyl glucosamine aminotransferase, lactose reacts with UDP-GlcNAc to generate the intermediate product LNT II. lgtB Under the catalysis of the encoded β-1,4-galactosyltransferase, it combines with UDP-Gal to generate LNnT. w.b.g.O. In the synthesis of sialylated human milk oligosaccharides, Escherichia coli uses the UDP-GlcNAc synthesis pathway as the basis, and in the presence of UDP- N -acetylglucosamine-2-epimerase NeuC, N -Cytidine monophosphate is synthesized by acetylneuraminic acid synthase NeuB and N-acetylneuraminic acid cytidylyltransferase NeuA N -Acetylneuraminic acid CMP-Neu5Ac. CMP-Neu5Ac combines with lactose under the catalysis of α2,3-sialyltransferase α2,3-SiaT and α2,6-sialyltransferase α2,6-SiaT to synthesize 3'-SL and 6'-SL respectively.

[0004] In the process of producing various HMOs using microbial cell factories, it is often necessary to introduce exogenous synthesis pathways, which leads to an imbalance in intracellular metabolism. As the fermentation process proceeds, the cells produce a large number of byproducts, such as lactic acid and other secondary metabolites. These products not only accumulate in the cells, but also are excreted into the fermentation broth, causing the cells to be stressed by an unfavorable environment, which has a negative impact on cell growth and thus affects the production of HMOs. Therefore, the problem of poor tolerance of cells to environmental stress has always been a long-standing key issue in microbial cell factories. MarR (multiple antibiotic resistance regulator) is widely present in bacteria and can regulate a variety of metabolic processes. For example, MarR can sense a variety of antibiotics, detergents, and oxidants and confer bacterial resistance (He Z, Ke Z, Wang W, et al. Biophysical and Biochemical Characterization of the Binding ofthe MarR-like Transcriptional Regulator Saro_0803 to the nov1 Promotor and Its Inhibition by Resveratrol[J]. Biomolecules, 2023, 13(3): 541.).

[0005] At present, a lot of work has been done on the construction of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL synthesis pathways, and relevant production strains have been obtained. However, during the fermentation process, the growth of the production strains was poor. It is speculated that the reason may be the large accumulation of metabolites, which led to the growth restriction of the strains and affected the synthesis efficiency of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL. How to improve the strain's resistance to stress during the fermentation process, enhance its tolerance to metabolites, and then increase the yield of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL has become an urgent problem to be solved. Summary of the invention

[0006] In order to solve the above technical problems, the present invention uses gene editing technology to modify the wild type Escherichia coli marR Gene editing has been carried out to obtain marRThe mutant genetically engineered chassis strain was applied to the construction of production strains of HMOs such as 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL, and it was found that the strain's stress resistance during the fermentation process was improved, and the yields of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL were further improved.

[0007] One of the technical solutions provided by the present invention is a MarR mutant, which is obtained by causing R77S and / or L127P mutations on the basis of the wild-type MarR shown in SEQ ID NO.1, and is named R77S mutant, L127P mutant and R77S / L127P mutant respectively;

[0008] Furthermore, the amino acid sequence of the R77S mutant is shown in SEQ ID NO.3;

[0009] Furthermore, the amino acid sequence of the L127P mutant is shown in SEQ ID NO.5;

[0010] Furthermore, the amino acid sequence of the R77S / L127P mutant is shown in SEQ ID NO.7.

[0011] The present invention also provides a gene encoding the MarR mutant described in one of the technical solutions;

[0012] Furthermore, the nucleotide sequence of the gene encoding the R77S mutant is shown in SEQ ID NO.4;

[0013] Furthermore, the nucleotide sequence of the gene encoding the L127P mutant is shown in SEQ ID NO.6;

[0014] Furthermore, the nucleotide sequence of the gene encoding the R77S / L127P mutant is shown in SEQ ID NO.8.

[0015] The third technical solution provided by the present invention is the application of the MarR mutant described in the first technical solution, especially in enhancing the stress resistance of the strain; more particularly, in producing human milk oligosaccharides, including but not limited to: 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL;

[0016] Furthermore, by mutating the MarR encoding gene in the 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production strains into the encoding gene of the MarR mutant described in one of the technical solutions, the yield of 2'-FL, 3-FL, LNTII, LNnT, LNT, 3'-SL and 6'-SL can be increased.

[0017] Beneficial effects:

[0018] The present invention obtains MarR mutants, namely R77S mutants, L127P mutants and R77S / L127P mutants, by gene editing technology. The coding genes of the above mutants are applied to Escherichia coli producing HMOs such as 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL, which may improve the growth ability and production performance of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL producing strains by enhancing the stress resistance of the strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1. Colony PCR verification of homologous recombination in the first step of strain D0.

[0020] Fig. 2 Colony PCR verification of the second step homologous recombination of strain D0.

[0021] Fig. 3 Colony PCR verification of homologous recombination in the first step of strain D1.

[0022] Fig. 4 Colony PCR verification of the second step homologous recombination of strain D1.

[0023] Figure 5 Strain D1△ wcaJ The first step is to verify homologous recombination by colony PCR.

[0024] Figure 6 Strain D1△ wcaJ The second step is to verify the homologous recombination by colony PCR.

[0025] Figure 7 The first step of homologous recombination colony PCR verification of strain Q1.

[0026] Fig. 8 Colony PCR verification of the second step homologous recombination of strain Q1.

[0027] Fig. 9 Colony PCR verification of homologous recombination in the first step of strain Q2.

[0028] Fig. 10 Colony PCR verification of the second step homologous recombination of strain Q2.

[0029] Figure 11 Colony PCR verification of homologous recombination in the first step of strain Q4.

[0030] Figure 12 Colony PCR verification of the second step homologous recombination of strain Q4.

[0031] Figure 13 Colony PCR verification of homologous recombination in the first step of strain Q5.

[0032] Figure 14 Colony PCR verification of the second step homologous recombination of strain Q5. DETAILED DESCRIPTION

[0033] 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.

[0034] The following definitions are used in the present invention:

[0035] 1. Nomenclature of amino acid and DNA sequences

[0036] 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.

[0037] 2. Identification of MarR mutants

[0038] The mutated amino acid in the MarR mutant is represented by "original amino acid + position + replaced amino acid". For example, R77S means that the amino acid at position 77 is replaced by S from the wild-type MarR R, and the position number corresponds to the amino acid sequence number of the wild-type MarR in SEQ ID NO.1. The specific information is as follows:

[0039]

[0040] The wild-type MarR involved in the present invention has an amino acid sequence as shown in SEQ ID NO.1:

[0041] MKSTSDLFNEIIPLGRLIHMVNQKKDRLLNEYLSPLDITAAQFKVLCIRCAACITPVELKKVLSVDLGALTRMLDRLVCKGWVERLPNPNDKRGVLVKLTTGGAAICEQCHQLVGQDLHQELTKNLTADEVATLEYLLKKVLP.

[0042] The amino acid sequence of the R77S mutant involved in the present invention is shown in SEQ ID NO.3:

[0043] MKSTSDLFNEIIPLGRLIHMVNQKKDRLLNEYLSPLDITAAQFKVLCIRCAACITPVELKKVLSVDLGALTRMLDSLVCKGWVERLPNPNDKRGVLVKLTTGGAAICEQCHQLVGQDLHQELTKNLTADEVATLEYLLKKVLP.

[0044] The amino acid sequence of the L127P mutant involved in the present invention is shown in SEQ ID NO.5:

[0045] MKSTSDLFNEIIPLGRLIHMVNQKKDRLLNEYLSPLDITAAQFKVLCIRCAACITPVELKKVLSVDLGALTRMLDRLVCKGWVERLPNPNDKRGVLVKLTTGGAAICEQCHQLVGQDLHQELTKNPTADEVATLEYLLKKVLP.

[0046] The amino acid sequence of the R77S / L127P mutant involved in the present invention is shown in SEQ ID NO.7:

[0047] MKSTSDLFNEIIPLGRLIHMVNQKKDRLLNEYLSPLDITAAQFKVLCIRCAACITPVELKKVLSVDLGALTRMLDSLVCKGWVERLPNPNDKRGVLVKLTTGGAAICEQCHQLVGQDLHQELTKNPTADEVATLEYLLKKVLP.

[0048] The present invention will be further explained below through specific implementation modes.

[0049] Example 1 Construction of strain D0

[0050] Escherichia coli ( Escherichia coli) K12 MG1655 was used 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. Sci Rep 7, 16624) was used to knock out the lactose operon sequence of the starting strain. lacZ , in the original lacZ P trc Promoter overexpression wcaG、gmd and lacY , strain D0 was obtained.

[0051] The specific construction method of strain D0 is as follows:

[0052] The homologous recombination fragments were constructed using the wild-type strain MG1655 preserved in the laboratory as a template and the primers in Table 1 were used to construct the homologous recombination fragments. PCR amplification was performed using lac-up-f / r and lac-down-f / r as primers to obtain lacZ Using pTrc99a plasmid (GeneBank: U13872) as template and primers lac-trc-f / r as template, promoter P was obtained. trc Sequence fragment. Artificially synthesized chloramphenicol resistance gene cat , cat The promoter and N20 sequence vector were used as templates (SEQ ID NO.9), and PCR amplification was performed using primer pair lac-cat-f / r to obtain a cat- Using the wild-type strain MG1655 as a template, PCR amplification was performed using primer pairs wcaG-f / r and gmd-f / r to obtain fragments with N20 sequences. wcAg and gmd Sequence fragment. The upstream and downstream homology arms, P trc Promoter fragment with cat- A fragment of the N20 sequence, wcAg and gmd These six sequence fragments were used as templates, and overlapping PCR was performed using primers lac-up-f and lac-down-r to obtain homologous recombinant fragments.

[0053] In the first step, homologous recombination was carried out by transforming the pCAGO plasmid into Escherichia coli K12 MG1655 using conventional plasmid transformation methods to obtain the strain Escherichia coli K12 MG1655 (pCAGO). The competent Escherichia coli K12 MG1655 (pCAGO) was 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. The transformants were picked for colony PCR verification (verification primers: lac-yz-f / r). If the recombination was successful, the band size was about 5148 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.

[0054] In the second step, homologous recombination, the strain that has been successfully verified for the first recombination is 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 are drawn on the LB plate containing ampicillin to separate single colonies, and the separated single colonies are picked and spotted on the chloramphenicol-resistant LB plate and the ampicillin-resistant LB plate respectively. The single colonies that do not grow on the chloramphenicol medium but grow on the ampicillin medium are selected and verified by colony PCR (the verification primers are: lac-yz-f / r). If the recombination is correct, the band size is about 4213 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, and the sequencing result is correct, and the second-step homologous recombination strain is obtained. lacZ And in the original lacZ P trc Promoter overexpression wcaG、gmd and lacY , named D0.

[0055] Table 1 Knockout constructs lacZ Use P trc Overexpression wcaG、gmd and lacY Primers used for strains

[0056]

[0057] Example 2 Construction of strain D1

[0058] Using D0 as the starting strain, the alcohol dehydrogenase encoding gene was cloned using CRISPR / Cas9 technology. adhe Replace with Ptrc Promoter overexpression manA and manB , and obtained the D1 strain.

[0059] The specific construction method of strain D1 is as follows:

[0060] Using the wild-type strain MG1655 preserved in the laboratory as a template, the primers in Table 2 were used to construct homologous recombination fragments. Using manAB-up-f / r and manAB-down-f / r as primers, PCR amplification was performed to obtain adhe Using pTrc99a plasmid (GeneBank: U13872) as template and primers manAB-trc-f / r as template, promoter P was obtained. trc Sequence fragment. Artificially synthesized chloramphenicol resistance gene cat , cat The promoter and N20 sequence vector were used as templates (SEQID NO.9), and PCR amplification was performed using the primer pair manAB-cat-f / r to obtain a cat- Using the wild-type strain MG1655 as a template, PCR amplification was performed using primer pairs manB-f / r and manA-f / r to obtain fragments with N20 sequence. manB and manA Sequence fragment. Homology arms above and downstream, P trc Promoter fragment with cat- A fragment of the N20 sequence, manB and manA These six sequence fragments were used as templates, and overlapping PCR was performed using primers manAB-up-f and manAB-down-r to obtain homologous recombinant fragments.

[0061] The obtained homologous recombination fragments were transformed into D0 by electroporation. The specific operation process of gene editing is referred to Example 1 of this application. Figure 3 and Figure 4 As shown, the PCR band size of the colony with successful first recombination was 6075 bp (validation primers: manAB-yz-f / r), and the PCR band size of the colony with successful second recombination was 5140 bp (validation primers: manAB-yz-f / r), thus obtaining the knockout adhe And in the original adhe P trc Promoter overexpression manA and manB , named D1.

[0062] Table 2 Knockout constructs adhe Use P trc Overexpression manA and manB Primers used for strains

[0063]

[0064] Example 3 Construction of strain D1Δ wcaJ

[0065] Based on Escherichia coli D1, the UDP-glucose lipid carrier transferase encoding gene was knocked out using CRISPR / Cas9 technology. wcaJ , strain D1△ was constructed wcaJ .

[0066] Strain D1△ wcaJ The specific construction method is as follows:

[0067] Using the wild-type strain MG1655 preserved in the laboratory as a template, the primers in Table 3 were used to construct homologous recombination fragments. Using wcaJ-up-f / r and wcaJ-down-f / r as primers, PCR amplification was performed to obtain wcaJ The upstream and downstream homology arms of cat , cat The promoter and N20 sequence vector were used as templates (SEQ ID NO.9), and PCR amplification was performed using primer pair wcaJ-cat-f / r to obtain a cat- The fragment of N20 sequence. The homology arms above and downstream, with cat- The three fragments of N20 sequence were used as templates, and overlapping PCR was performed using primers wcaJ-up-f and wcaJ-down-r to obtain homologous recombinant fragments.

[0068] The obtained recombinant fragment was transformed into the D1 strain by electroporation. The specific operation process of gene editing is referred to Example 1 of this application. Figure 5 and Figure 6 As shown, the PCR band size of the colony with successful first recombination was 2119 bp (validation primers: wcaJ-yz-f / r), and the PCR band size of the colony with successful second recombination was 1184 bp (validation primers: wcaJ-yz-f / r), thereby obtaining the UDP-glucose lipid carrier transferase encoding gene on the knockout genome. wcaJ , named D1△ wcaJ .

[0069] Table 3 Construction of D1△ wcaJ Primers used for strains

[0070]

[0071] Example 4 Construction of strain Q1

[0072] With D1△ wcaJ The strain was used as the starting strain, and the CRISPR / Cas9 technology was used to mutate the coding genes of multiple antibiotic resistance regulators on the genome marR ,Will marR The 229th nucleotide cytosine of the gene was mutated to adenine (C to A), and strain Q1 was obtained.

[0073] The specific construction method of strain Q1 is as follows:

[0074] (1) Construction of homologous recombination fragments

[0075] Using the wild-type strain MG1655 preserved in the laboratory as a template, the primer pairs marr-up-f1 / r1 and marr-down-f1 / r1 in Table 4 were used as primers to amplify the upstream and downstream homology arms of homologous recombination by PCR. wcaJ The PCR-derived cat- The fragment of N20 sequence was used as template (SEQ ID NO.9), and PCR amplification was performed using the primer pair marr-cat-f1 / r1 to obtain a new cat- The fragment of N20 sequence. The homology arms above and downstream, the new cat- The three fragments were used as templates to perform overlapping PCR using primers marr-up-f1 and marr-down-r1 to obtain homologous recombinant fragments containing marR Gene mutation, i.e. wild type marR The 229th nucleotide of the gene, cytosine, mutated to adenine (C mutated to A).

[0076] (2) First step: homologous recombination

[0077] The homologous recombination fragments were transformed into D1△ wcaJ For the specific operation process of gene editing in the strain, refer to Example 1 of this application. Select the transformant for colony PCR verification. If the recombination is correct, the band size is about 2629 bp. The verification result is as follows Figure 7 As shown, the bands were correct and the first step homologous recombination strain was obtained.

[0078] (3) Second step: homologous recombination

[0079] The grown single clones were verified by colony PCR. If the recombination was correct, the band size would be 1694 bp. Figure 8The band was correct, and the PCR product of the band was sequenced. The sequencing result was correct, and the second-step homologous recombination strain was obtained. The second-step homologous recombination strain was further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining a strain with marR The strain with the mutation (SEQ ID NO.4) was named Q1.

[0080] Table 4 Primers used to construct Q1 strain

[0081]

[0082] Example 5 Construction of strains Q2 and Q3

[0083] In strain D1△ wcaJ Based on the Q1, CRISPR / Cas9 technology was used to marR The thymine at position 380 of the gene was mutated to cytosine (T to C), and strains Q2 and Q3 were obtained.

[0084] The specific construction methods of strains Q2 and Q3 are as follows:

[0085] Using the wild-type strain MG1655 preserved in the laboratory as a template, the primers in Table 5 were used to construct homologous recombination fragments. Using marr-up-f2 / r2 and marr-down-f2 / r2 as primers, PCR amplification was performed to obtain the upstream and downstream homology arms of homologous recombination. cat , cat The promoter and N20 sequence vector were used as templates (SEQ ID NO.9), and PCR amplification was performed using the primer pair marr-cat-f2 / r2 to obtain a cat- The fragment of N20 sequence. The upper and downstream homology arms, cat- The three fragments were used as templates to perform overlapping PCR using primers marr-up-f2 and marr-down-r2 to obtain homologous recombination fragments. marR The mutation site of the gene, i.e. the wild type marR The thymine at position 380 of the gene mutated to cytosine (T mutated to C).

[0086] The obtained recombinant fragments were transformed into D1△ wcaJ For the specific operation process of gene editing in the Q1 strain, please refer to Example 1 of this patent. Figure 9 and Figure 10As shown, the PCR band size of the colony with successful primary recombination was about 2809 bp, and the PCR band size of the colony with successful secondary recombination was about 1874 bp. The homologous recombination strain obtained in the second step was further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining strains Q2 and Q3.

[0087] Table 5 Primers used to construct Q2 and Q3 strains

[0088]

[0089] Example 6 Construction of strains H0, Q4, Q5 and Q6

[0090] 1. Construction of strain H0:

[0091] 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.

[0092] The specific construction method of strain H0 is as follows:

[0093] (1) Construction of homologous recombination fragments

[0094] Using the wild-type strain MG1655 preserved in the laboratory as a template, the primers in Table 6 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 vector were used as templates (SEQ ID NO.9), 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, Ptrc 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.

[0095] (2) First step: homologous recombination

[0096] 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, and 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 band was correct, that is, the first homologous recombination was successful. The correct transformant was picked for the second step of homologous recombination.

[0097] (3) Second step: homologous recombination

[0098] The strain that successfully verified the 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 h 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 separated 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 were: lac-yz-f / r). If the recombination is correct, the band size is about 1797 bp, the band is verified to be 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 was further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining the knockout strain. lacZ And in the original lacZ P trc Promoter overexpression lacY The strain was named H0.

[0099] Table 6 Knockout Construction lacZ Use P trc Overexpression lacY Primers used for strains

[0100]

[0101] 2. Construction of strains Q4, Q5 and Q6

[0102] Using the H0 strain as the starting strain, the CRISPR / Cas9 technology was used to mutate the genes encoding multiple antibiotic resistance regulators on the genome marR ,Will marR The 229th nucleotide cytosine of the gene was mutated to adenine (C was mutated to A), and strain Q4 was obtained. The specific construction method of strain Q4 is referred to Example 4 of the present application. Figure 11 and Figure 12 As shown, the PCR band size of the colony with successful first recombination is about 2629 bp, and the PCR band size of the colony with successful second recombination is about 1694 bp.

[0103] Based on strains H0 and Q4, CRISPR / Cas9 technology was used to marR The thymine at position 380 of the gene was mutated to cytosine (T was mutated to C), and strains Q5 and Q6 were obtained. The specific construction method is referred to Example 5 of the present application. Figure 13 and Figure 14 As shown, the PCR band size of the colony with successful first recombination is about 2809 bp, and the PCR band size of the colony with successful second recombination is about 1874 bp.

[0104] Example 7 Construction of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production plasmids

[0105] pTrc99a-P trc - futC - manC (for production of 2'-FL), pTrc99a-P trc - futA - manC (for the production of 3-FL) are disclosed in Example 3 and Example 4 of CN116334025A respectively;

[0106] pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist (for production of 3'-SL) and pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ST6 For the specific construction process (for producing 6'-SL), please refer to Example 2 and Example 3 of patent CN117736280A.

[0107] pTrc99a-P trc - lgtA (For the production of LNT II) 、 pTrc99a-P trc - lgtB - lgtA (For the production of LNnT) 、 pTrc99a-P trc - w.b.g.O. - lgtA (For producing LNT); the specific construction method is as follows:

[0108] lgtA Genes encoding β-1,3- N -acetyl glucosamine transferase (Protein ID: AAF42258.1), and plasmid pTrc99a was used as a template to construct plasmid pTrc99a-P trc - lgtA .

[0109] Using plasmid pTrc99a as template and 99a-F / R in Table 7 as primers, PCR amplification was performed to obtain the linear vector pTrc99a-P trc ; artificially synthesized and codon-optimized lgtA The sequence was used as a template and lgtA-F / R in Table 7 was used as primers for PCR amplification to obtain a linear gene fragment. lgtA The linear vector and linear gene fragment obtained by the above PCR were purified and recovered, connected 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 .

[0110] Table 7 Construction of plasmid pTrc99a-P trc - lgtA Primers used

[0111]

[0112] lgtB The gene encoding the lipooligosaccharide biosynthesis protein lex-1 (Protein ID: BAS48030.1) is located in the plasmid pTrc99a-P trc - lgtA The plasmid pTrc99a-P was constructed based on trc - lgtB - lgtA . Using plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using lgtA-F and 99a-R in Table 8 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 8 was used as primers for PCR amplification to obtain a linear gene fragment. lgtB 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 - lgtB - lgtA .

[0113] Table 8 Construction of plasmid pTrc99a-P trc - lgtB - lgtA Primers used

[0114]

[0115] w.b.g.O. The gene encoding β-1,3-galactosyltransferase is located in the plasmid pTrc99a-P trc - lgtA The plasmid pTrc99a-P was constructed based on trc - w.b.g.O. - lgtA . Using plasmid pTrc99a-P trc - lgtA As a template, PCR amplification was performed using lgtA-F and 99a-R in Table 9 as primers to obtain the linear vector pTrc99a- trc -lgtA; w.b.g.O. The sequence in Table 9 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-Ptrc - w.b.g.O. - lgtA .

[0116] Table 9 Construction of plasmid pTrc99a-P trc - w.b.g.O. - lgtA Primers used

[0117]

[0118] Example 8 Construction of 2'-FL and 3-FL production strains

[0119] The plasmid pTrc99a-P trc - futC - manC and pTrc99a-P trc - futA - manC Import D1△ separately wcaJ , Q1, Q2 and Q3 strains, respectively constructed:

[0120] (1) 2'-fucosyllactose producing strain: P1[D1△ wcaJ (pTrc99a-P trc - futC - manC )], P2[Q1(pTrc99a-P trc - futC - manC )], P3[Q2 (pTrc99a-P trc - futC - manC )] and P4[Q3 (pTrc99a-P trc - futC - manC )];

[0121] (2) 3-fucosyllactose producing strain: P5[D1△ wcaJ (pTrc99a-P trc - futA - manC )], P6[Q1(pTrc99a-P trc - futA - manC )], P7[Q2 (pTrc99a-P trc - futA - manC )] and P8[Q3 (pTrc99a-P trc - futA - manC )].

[0122] Example 9 Construction of LNT II, ​​LNnT, and LNT production strains

[0123] The plasmid pTrc99a-P trc - lgtA , pTrc99a-P trc - lgtB - lgtA、 pTrc99a-P trc - w.b.g.O. - lgtA Import them into H0, Q4, Q5 and Q6 respectively, and construct:

[0124] (1) LNT II production strain:

[0125] P9[H0 (pTrc99a-P trc - lgtA )]; P10[Q4 (pTrc99a-P trc - lgtA )]; P11[Q5 (pTrc99a-P trc - lgtA )]; P12[Q6 (pTrc99a-P trc - lgtA )];

[0126] (2) LNnT production strains:

[0127] P13[H0 (pTrc99a-P trc - lgtB - lgtA )]; P14[Q4 (pTrc99a-P trc - lgtB - lgtA )]; P15[Q5 (pTrc99a-P trc - lgtB - lgtA )]; P16[Q6 (pTrc99a-P trc - lgtB - lgtA )];

[0128] (3) LNT production strains:

[0129] P17[H0 (pTrc99a-P trc - w.b.g.O. - lgtA )]; P18[Q4 (pTrc99a-P trc - w.b.g.O. - lgtA )]; P19[Q5 (pTrc99a-Ptrc - w.b.g.O. - lgtA )]; P20[Q6 (pTrc99a-P trc - w.b.g.O. - lgtA )].

[0130] Example 10 Construction of 3'-SL and 6'-SL producing strains

[0131] The plasmid pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist and pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ST6 Import them into H0, Q4, Q5 and Q6 respectively, and construct:

[0132] (1) 3'-SL producing strains:

[0133] P21[H0 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )];

[0134] P22[Q4 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )];

[0135] P23[Q5 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )];

[0136] P24[Q6 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA-ist )];

[0137] (2) 6'-SL producing strains:

[0138] P25[H0 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )];

[0139] P26[Q4 (pTrc99a-P J23119- neuB–neuC -P trc - neuA - ST6 )];

[0140] P27[Q5 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )];

[0141] P28[Q6 (pTrc99a-P J23119 - neuB–neuC -P trc - neuA - ST6 )].

[0142] The strains and plasmids involved in this application are shown in Table 10.

[0143] Table 10 Strains and plasmids used in this patent

[0144]

[0145] Example 11 Fermentation test of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL producing strains

[0146] The culture medium used was:

[0147] LB medium: NaCl 10 g / L, yeast powder 5 g / L, peptone 10 g / L, pH 7.0.

[0148] Fermentation medium: KH 2 PO 4 3 g / L, yeast powder 8 g / L, (NH 4 ) 2 SO 4 4 g / L, citric acid 1.7 g / L, MgSO 4 7H 2 O2 g / L, thiamine 10 mg / L, glycerol 10 g / L, lactose 5 g / L, 1 ml / L trace elements (FeCl 3 6H 2 O25 g / L, MnCl 2 ·4H 2 O 9.8 g / L, CoCl 2 6H 2 O 1.6 g / L, CuCl 2 ·H 2 O 1 g / L, H 3 BO3 1.9 g / L, ZnCl 2 2.6 g / L, Na 2 MOO 4 ·2H 2 O 1.1 g / L, Na 2 SeO 3 1.5 g / L, NiSO 4 6H 2 O 1.5 g / L, and the pH value was adjusted to 7.2 with ammonia water.

[0149] The fermentation test process is:

[0150] Single colonies of the 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production strains shown in Table 10 were selected and cultured overnight in LB liquid medium containing 50 mg / L ampicillin at 37°C and 220 rpm. The overnight cultured bacterial liquid was used as seed liquid and transferred to a 24-well plate containing 1 mL of fermentation medium at a 1% inoculation volume. The fermentation medium contained 50 mg / L ampicillin and 0.1 mmol / L IPTG and fermented at 37°C and 800 rpm. Three samples were cultured in parallel for each strain.

[0151] During the fermentation process, the growth of the bacteria (OD 600 ), 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL content. HPLC was used to detect the concentrations of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL in the sample. The HPLC detection conditions of 2'-FL and 3-FL refer to Example 7 of patent CN116334025A. The HPLC detection conditions of LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL refer to Example 4 of patent CN117736280A. The sample concentrations were quantified using 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL standards. The results are shown in Tables 11 to 17:

[0152] Table 11 Fermentation test results of 2'-FL production strains

[0153]

[0154] Table 12 Fermentation test results of 3-FL production strains

[0155]

[0156] Table 13 LNT II production strain fermentation test results

[0157]

[0158] Table 14 LNnT production strain fermentation test results

[0159]

[0160] Table 15 LNT production strain fermentation test results

[0161]

[0162] Table 16 3'-SL production strain fermentation test results

[0163]

[0164] Table 17 6'-SL production strain fermentation test results

[0165]

[0166] It can be seen from Tables 11 to 17 that the growth ability of the strain at 48 h of fermentation was improved after the wild-type MarR was mutated with R77S and / or L127P. It is speculated that the strain's resistance to stress during the fermentation process was enhanced and the strain's tolerance to metabolites in the fermentation broth was improved. marR The presence of gene mutation points may improve the growth ability and production performance of 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL production strains by enhancing the stress resistance of the strains.

[0167] 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. Use of a MarR mutant in Escherichia coli for producing human milk oligosaccharides, characterized in that: The mutant is obtained by causing L127P mutation, or R77S and L127P mutation simultaneously on the basis of the wild-type MarR shown in SEQ ID NO.1; The human milk oligosaccharide is selected from the group consisting of: 2'-FL, 3-FL, LNT II, ​​LNnT, LNT, 3'-SL and 6'-SL; The production of human milk oligosaccharides can be increased by mutating the MarR encoding gene in the human milk oligosaccharide production strain into the encoding gene of the above-mentioned MarR mutant.

2. Use of a MarR mutant according to claim 1 in Escherichia coli for producing human milk oligosaccharides, characterized in that: The nucleotide sequence of the gene encoding the L127P mutant is shown in SEQ ID NO.6; the nucleotide sequence of the gene encoding the R77S / L127P mutant is shown in SEQ ID NO.8.

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