Sialyltransferase mutant with high yield of 3 '-sialyllactose as well as construction method and application of sialyltransferase mutant

Abstract

The invention relates to the technical field of microbial genetic engineering, in particular to a sialyltransferase mutant with high yield of 3 '-sialyllactose as well as a construction method and application of the sialyltransferase mutant. The sialyltransferase mutant is selected from at least one of an NST protein or a mutant and a 406 NST protein or a mutant, the NST protein comprises an amino acid residue site in a sequence as shown in SEQ ID NO.1, and the 406 NST protein comprises an amino acid sequence in a sequence as shown in SEQ ID NO.2. The sialyltransferase mutant has the advantages that the sialyltransferase mutant can be used for preparing sialyltransferase, and the sialyltransferase mutant can be used for preparing sialyltransferase. The invention also provides a nucleic acid molecule containing the sialyltransferase mutant, an expression cassette, a recombinant vector, a recombinant microorganism and a construction method and application thereof, and has great significance in large-scale industrial production of 3-sialyllactose.

Description

Technical Field

[0001] The invention relates to the technical field of microbial genetic engineering, and in particular to a sialyltransferase mutant with high 3'-sialyllactose production, and a construction method and application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in human milk and include a variety of unbound sugars. More than 200 HMO molecular structures have been identified, which can be roughly divided into three categories: neutral core HMOs, fucosylated HMOs, and sialylated HMOs. 3′-Sialyl lactose (3′-SL, Neu5Acα2,3Galβ1,4Glc) is a basic and representative sialylated HMO, which is linked to galactose in lactose by sialic acid residues with an α2,3-glycosidic bond. 3′-Sialyl lactose has various health benefits, including prebiotic effects, anti-adhesion and antibacterial abilities, antiviral efficacy, prevention of necrotizing enterocolitis (NEC), immunomodulatory properties, regulation of intestinal epithelial cell responses, and promotion of brain development and cognitive abilities. The U.S. Food and Drug Administration (FDA) has designated 3′-Sialyl lactose as a generally recognized as safe (GRAS) substance, which has promoted its commercial application in premium infant formula. In conclusion, 3′-sialyllactose has attracted much attention due to its various physiological properties.

[0003] However, the chemical synthesis of 3′-sialyllactose is cost-prohibitive for dairy and nutraceutical applications due to its inherent complexity, the total number of reaction steps, and the limited and high cost of raw materials. In addition, chemoenzymatic synthesis cannot achieve commercial production of 3′-sialyllactose. Therefore, biosynthesis using engineered strains for large-scale production has become an important research focus, which involves the expression of α2,3-sialyltransferase and the related enzyme NeuBCA involved in the CMP-Neu5Ac synthesis pathway. In previous studies, NST (NCBI sequence number: AAC44541) was a frequently used α2,3-sialyltransferase and a key factor for the efficient biosynthesis of 3′-sialyllactose. It is worth noting that in addition to the NST gene of N. meningitidis MC58L3, the 406NST gene (gene encoding α2,3-sialyltransferase 406NST, NCBI sequence number: AAC44543) was successfully cloned from N. meningitidis 406Y L3. Although the cap types of MC58 L3 and 406Y L3 strains are different, their lipooligosaccharides (LOS) are identical. Sequence analysis showed that there were 5 amino acid differences between the two genes, which can be attributed to 12 nucleotide differences. In the CAZy (Carbohydrate-Active enZymes) database (https: / / www.cazy.org / ), sialyltransferases are divided into six glycosyltransferase families (GT29, GT38, GT42, GT52, GT80, and GT97). Bacterial sialyltransferases usually belong to glycosyltransferase families GT42, GT52, or GT80, including regioselective α2,3-sialyltransferases (EC2.4.99.4), α2,6-sialyltransferases (EC2.4.99.1), and mixed α2,3 / α2,6-sialyltransferases. According to this classification, NST and 406NST, which are classified into the GT52 family, are monofunctional α2,3-sialyltransferases. The hydrolase activity of GT52 family enzymes is generally low (≤3-10% of native sialyltransferase activity). Unlike the extensive research on NST, there is little research on 406NST, and no reports have been found in the biosynthesis of 3′-sialyllactose, and the method of preparing 3′-sialyllactose by microbial fermentation of modified 406NST protein is even rarer. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a sialyltransferase mutant with high yield of 3'-sialyllactose and its construction method and application. The sialyltransferase mutant of the present invention is selected from at least one of NST protein or mutant, 406NST protein or mutant, the NST protein comprises the amino acid residue sites in the sequence shown in SEQ ID NO.1, and the 406NST protein comprises the amino acid sequence in the sequence shown in SEQ ID NO.2. The present invention also provides a nucleic acid molecule, an expression cassette, a recombinant vector, a recombinant microorganism construction method and application comprising the sialyltransferase mutant, which is of great significance to the large-scale industrial production of 3'-sialyllactose. The specific scheme of the present invention is as follows:

[0005] The first aspect of the present invention provides a sialyltransferase mutant, wherein the sialyltransferase mutant is selected from any one of the following:

[0006] A1) its amino acid sequence comprises a sequence obtained by mutation of an amino acid residue position in the sequence shown in SEQ ID NO.1, wherein the mutation position is selected from any one or more of the following positions: position 29, position 40, position 94, position 242, and position 273;

[0007] A2) is a protein having 98% or more identity with the protein described in A1) and having the same function as the protein described in A1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the mutant described in A1);

[0008] A3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in A1) and / or A2).

[0009] and / or

[0010] B1) Its amino acid sequence comprises a sequence obtained by mutation of an amino acid residue in the sequence shown in SEQ ID NO. 2, wherein the mutation site is selected from any one or more of the following sites: position 40, position 113, and position 120;

[0011] B2) is a protein having 98% or more identity with the protein described in B1) and having the same function as the protein described in B1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the mutant described in B1);

[0012] B3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in B1) and / or B2).

[0013] Furthermore, the mutation in A1) produced by the sequence shown in SEQ ID NO.1 is selected from any one or more of the following mutations:

[0014] Q at position 29 mutated to H; E at position 40 mutated to D; N at position 94 mutated to K; T at position 242 mutated to A; and K at position 273 mutated to N.

[0015] The mutations in B1) produced by the sequence shown in SEQ ID NO.2 are selected from any one or more of the following mutations:

[0016] The 40th position D mutates to any one of M, W, N, F, T, K, R, L, S, I, Q, H, P, C, V, A, Y, G;

[0017] No. 113N mutated to D;

[0018] The 120P mutated to H.

[0019] In a preferred embodiment of the present invention, the amino acid sequence of the sialyltransferase mutant comprises a sequence obtained by mutation of an amino acid residue site in the sequence shown in SEQ ID NO.2, wherein the mutation site is selected from any one of the following groups:

[0020] D40R-P120H, D40R-N113D, D40R-P120H-N113D.

[0021] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is selected from any one of the following:

[0022] C1) containing a nucleic acid molecule encoding the sialyltransferase mutant;

[0023] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0024] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0025] C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3).

[0026] In a specific embodiment of the present invention, the recombinant microorganism also includes the following operations: knocking out the genes lacZ, nanA and nanK, and integrating the exogenous genes neuB gene, neuC gene and neuA gene into the recombinant microorganism. Preferably, the recombinant microorganism is selected from at least one of Escherichia coli, yeast, Corynebacterium glutamicum, and Serratia. In a specific embodiment of the present invention, the recombinant microorganism is Escherichia coli.

[0027] The third aspect of the present invention provides a whole-cell catalyst, wherein the whole-cell catalyst comprises the sialyltransferase mutant and / or the biomaterial.

[0028] In a fourth aspect, the present invention provides the use of the sialyltransferase mutant and / or the biomaterial and / or the whole cell catalyst in any of the following situations:

[0029] 1. preparing 3'-sialyllactose;

[0030] II. preparing a product containing 3'-sialyllactose;

[0031] III. Improve the activity of α2,3-sialyltransferase;

[0032] IV. Construction of a recombinant microorganism for producing 3'-sialyllactose;

[0033] V. Regulating the production of 3'-sialyllactose by microorganisms;

[0034] VI. Improve the purity of 3'-sialyllactose produced by microorganisms.

[0035] In a fifth aspect, the present invention provides a method for producing 3'-sialyllactose comprising the following steps:

[0036] Step 1, constructing a recombinant cell or recombinant microorganism capable of expressing the sialyltransferase mutant according to any one of claims 1 to 5;

[0037] Step 2: culturing the recombinant cell or recombinant microorganism to obtain 3'-sialyllactose.

[0038] In a specific embodiment of the present invention, step one also includes the step of knocking out genes lacZ, nanA and nanK, and integrating exogenous genes neuB gene, neuC gene and neuA gene into the recombinant microorganism; the recombinant microorganism or recombinant cell in step two is selected from at least one of Escherichia coli, yeast, Corynebacterium glutamicum, and Serratia.

[0039] In a sixth aspect, the present invention provides a method for increasing the yield of 3-sialyllactose, which is at least one of the following D1) or D2):

[0040] The D1) comprises the following steps: inserting a sialyltransferase mutant gene into the bacterial genome to increase the production of 3'-sialyllactose in the bacteria;

[0041] Said D2) comprises the following steps: increasing the content and / or activity of sialyltransferase mutants in bacteria, or increasing the gene expression of sialyltransferase mutants in bacteria, so as to increase the production of 3'-sialyllactose in bacteria;

[0042] The sialyltransferase mutant includes any mutant described in the present invention;

[0043] The sialyltransferase mutant gene comprises a gene encoding any one of the sialyltransferase mutants of the present invention.

[0044] The beneficial effects of the present invention include but are not limited to:

[0045] 1. The present invention screened and obtained a series of sialyltransferase mutants by performing site-directed mutagenesis on the five amino acid differences between the amino acid sequences of α2,3-sialyltransferase encoded by the nst gene from Neisseria meningitidis MC58 and the 406nst gene from N. meningitidis 406Y. The enzyme activity of the series of enzyme mutants and the yield of 3'-sialyllactose prepared by the enzyme mutants were significantly improved compared with the wild type, and no by-products were generated;

[0046] 2. The present invention discloses for the first time that the D40 site of the 406NST protein significantly affects the yield of '-sialyllactose, and obtains the best recombinant microbial strain Z28 (406NST-D40R-N113D-P120H) by superimposing multi-point mutagenesis. The extracellular 3'-sialyllactose yield of the strain in a shake flask is 4.67g / L. In a 5L bioreactor, the extracellular 3'-sialyllactose yield is 29.54g / L, the total 3'-sialyllactose yield is 0.52g / L / h, and the lactose conversion rate is 0.62mol 3'-sialyllactose / mol. It is of great significance to the large-scale industrial production and application of 3'-sialyllactose. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The figures are a comparison of 3′-sialyllactose yields of various α2,3-sialyltransferases in the examples of the present invention (A), a comparison of HPLC products of Z1 and Z2 (B), and a comparison of amino acid sequences of NST, 406NST, and 126NST (C);

[0048] Figure 2 LC-MS diagram of 3′-sialyllactose standard product and Z2 in the embodiment of the present invention;

[0049] Figure 3 This is a comparison diagram of 3′-sialyllactose production of each group of NST site-directed mutagenesis mutants in the examples of the present invention;

[0050] Figure 4 In the embodiment of the present invention, the SWISS-MODEL server performs the following operations on 406NST (A) and

[0051] Homology modeling of 406NST-D40R-N113D-P120H (B);

[0052] Figure 5 This is a comparison diagram of 3′-sialyllactose production of mutants of 406NST at the D40 site in various groups according to the examples of the present invention;

[0053] Figure 6 This is a comparison diagram of 3′-sialyllactose production of mutants with multiple mutations superimposed on 406NST in each group in the examples of the present invention;

[0054] Figure 7 This is a diagram showing the fermentation yield of 3′-sialyllactose in a 5L fermenter in an example of the present invention. DETAILED DESCRIPTION

[0055] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0056] 1. The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are carried out in accordance with the kit instructions.

[0057] 2. Conventional procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0058] 3. The tandem target gene neuBCA involved in the following embodiments was constructed by Suzhou Anshengda Company on the vector of the pETDuet-1 plasmid MCS1 multiple cloning site to generate the plasmid pET-BCA; the target genes NST and 406NST were constructed by Suzhou Anshengda Company between the multiple cloning site 1 NcoI site and NotI site of the expression vector pCDFDuet-1 to generate plasmids pCD-NST and pCD-406NST. The mutant plasmids related to the target genes NST and 406NST involved were obtained by site-directed mutagenesis using plasmids pCD-NST and pCD-406NST as templates, respectively.

[0059] 4. Preparation of competent E. coli: TAKARA kit.

[0060] 5. 3′-sialyllactose fermentation process and detection:

[0061] (1) LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0062] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0063] (3) Define medium (DM): 20 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate. Adjust the pH to 6.8 with sodium hydroxide, sterilize at 115°C for 30 min, and add 4.5 mg / L thiamine before use.

[0064] (4) Antibiotic concentration: ampicillin 100 mg / L (liquid culture medium), streptomycin 50 mg / L.

[0065] (5) Fed-batch fermentation Feed solution: 600 g / L glycerol and 0.2 g / L thiamine, 200 g / L lactose, 50% ammonia water (w / v).

[0066] In the following examples, the specific steps of 3′-sialyllactose production and detection are as follows:

[0067] 1. 3′-sialyllactose shake flask fermentation process:

[0068] The constructed strain was inoculated into LB liquid medium containing corresponding resistance or no resistance, and cultured overnight for 12 hours to obtain seed liquid. The seed liquid was inoculated into DM fermentation medium and cultured at 37°C, 200 rpm until OD 600 The concentration of lactose was 0.6-0.8, and 3 g / L lactose and 0.5 mM IPTG were added. The induction culture was continued at 25°C and 200 rpm for 72 h.

[0069] 2. 3′-sialyllactose fed-batch fermentation process:

[0070] A single colony of the recombinant E. coli was picked from the plate and inoculated into a non-resistant LB medium for 12 h as the primary seed solution; the primary seed solution was inoculated into 150 mL of DM fermentation medium and cultured at 37 °C and 200 rpm until OD 600 =1.5 and then transferred to a 5L fermenter containing 1.5L DM fermentation medium. The initial temperature was maintained at 37°C. When OD600 At about 40°C, the temperature was lowered to 25°C, and lactose with a final concentration of 2g / L and 0.2mM IPTG were added. Subsequently, the fermentation feed solution was added to ensure that the final concentration of glycerol was maintained at 2-10g / L, the final concentration of lactose was maintained at 2-8g / L, the pH was maintained at 6.8±0.1 throughout the process, and the foam was controlled by adding a defoamer. The dissolved oxygen was controlled at 20%-30% by adjusting the stirring speed and ventilation volume.

[0071] 3. HPLC detection conditions: Take 1 mL of fermentation broth, centrifuge at 12,000 rpm for 5 min, and take the supernatant for HPLC determination.

[0072] HPLC detection conditions: through high performance liquid chromatography (HPLC) system (Agilent 1260InfinityⅡ); chromatographic column: Zorbax NH2; detector: Agilent 1260RID differential detector; mobile phase: 50% acetonitrile, 2mM MgCl2, 10mM H3PO4; flow rate: 0.8mL / min; column temperature: 30℃; injection volume: 10μL.

[0073] In the following examples, the CRISPR / Cas9 dual-plasmid gene editing system has been disclosed in the literature: Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S. Multigene Editing in the Escherichia Coli Genome via the CRISPR / Cas9 System[J]. Applied and Environmental Microbiology, 2015, 81(7), 2506-2514.

[0074] The recombinant strains, hosts including plasmids and genotypes involved in the following examples and the yield of 3′-sialyllactose obtained are shown in Table 1:

[0075] Table 1

[0076]

[0077]

[0078] In the following examples, the primer information involved is shown in Table 2:

[0079] Table 2

[0080]

[0081]

[0082] Example 1: Construction of recombinant expression vector

[0083] The specific steps for constructing the recombinant expression vector used to transform Escherichia coli are as follows (the primer sequences involved are shown in Table 2):

[0084] (1) Obtaining the neuBCA gene fragment and constructing the plasmid pET-BCA:

[0085] The MCS1 of the pETDuet-1 plasmid is used to insert neuB, neuC, and neuA to obtain a recombinant vector, and the recombinant vector is then transferred into a microorganism to obtain a recombinant microorganism, so as to realize the tandem expression of the gene sequence neuBCA.

[0086] The neuB gene, neuC gene and neuA gene are derived from Campylobacter jejuni or microorganisms that can express the same functional enzymes. The NCBI accession number of the neuB gene is AAK91726.1; the NCBI accession number of the neuC gene is AAK91727.1; and the NCBI accession number of the neuA gene is AAK91728.1. The nucleotide sequence of the neuB gene is shown in SEQ ID NO.3, the nucleotide sequence of the neuC gene is shown in SEQ ID NO.4, and the nucleotide sequence of the neuA gene is shown in SEQ ID NO.5.

[0087] The codon-optimized Campylobacter jejuni-derived gene neuBCA (nucleotide sequence shown in SEQ ID NO.6) was constructed into the NcoI and BamHI sites at the first position of the multiple cloning site of the expression vector pETDuet-1 to form a recombinant plasmid pET-BCA.

[0088] (2) Obtaining the α2,3-sialyltransferase gene fragment and constructing the plasmid pCD-α2,3-sialyltransferase:

[0089] The gene nst (nucleotide sequence is shown in SEQ ID NO.7) was constructed into MCS1 of the expression vector pCDFDuet-1 to form the recombinant plasmid pCDF-NST.

[0090] The gene 406nst (nucleotide sequence as shown in SEQ ID NO.8) was constructed into MCS1 of the expression vector pCDFDuet-1 to form the recombinant plasmid pCDF-406NST.

[0091] All NST mutant plasmids were obtained by PCR using pCDF-NST plasmid as a vector template by designing primers for each desired mutated amino acid site (the primer sequences involved are shown in Table 2 ).

[0092] All 406NST mutant plasmids were obtained by PCR using pCDF-406NST plasmid as a vector template by designing primers for each desired mutated amino acid site (the primer sequences involved are shown in Table 2 ).

[0093] Example 2: Fermentation production of 3'-sialyllactose by recombinant strains of different sialyltransferases

[0094] The genes lacZ, nanA and nanK in Escherichia coli BL21 (DE3) were knocked out. The specific steps refer to the patent publication number CN114874966A to obtain the recombinant bacterium EZAK.

[0095] (1) Production of 3'-sialyllactose by shake flask fermentation

[0096] The pET-BCA constructed in Example 1 and pCD-NST and pCD-406NST were respectively transferred into the above-constructed host EZAK to obtain recombinant strains Z1 and Z2.

[0097] The recombinant strains were inoculated into LB liquid medium containing the corresponding antibiotics, cultured overnight at 37°C, 200 rpm for 12 h to obtain seed solution, and the seed solution was inoculated into 25 mL DM fermentation medium at 37°C, 200 rpm to OD 600 Induce when the pH is 0.6-0.8, continue culturing for 72 hours, take 1 mL of fermentation liquid, and take the supernatant for HPLC determination.

[0098] (2) Screening of α2,3-sialyltransferase

[0099] like Figure 1 As shown in A, the 3′-sialyllactose production of strain Z2 carrying 406NST was 2.92 g / L, which was about 41% higher than that of strain Z1 carrying NST (2.07 g / L). Figure 1 B shows that the fermentation broth produced by strains Z1 and Z2 contains unknown byproducts different from Neu5Ac, ManNAc and 6′-sialyllactose. At the same magnification, HPLC results showed that the total yield of unknown byproducts of Z2 was still very low, and the 3′-sialyllactose yield of Z2 was 1.41 times that of Z1. Therefore, 406NST is a promising enzyme that can provide both high 3′-sialyllactose yield and low byproduct production. LC / MS analysis of the shake flask culture supernatant produced by strain Z2 further confirmed the synthesis of 3′-sialyllactose ( Figure 2 ).

[0100] Example 3: Site-directed mutagenesis and homology modeling

[0101] Compared with NST, the amino acid sequence similarity of 406NST is as high as 98.65% ( Figure 1C). There are only five amino acids (Q29H, E40D, N94K, T242A, K273N) that differ between NST and 406NST, but they lead to significant changes in 3′-sialyllactose production. Therefore, the five amino acids corresponding to the NST sequence were individually mutagenized, and each mutant was cloned into pCDF-Duet1 using pCD-NST as a template. Subsequently, the effects of each mutant on 3′-sialyllactose production were evaluated by simultaneously transferring pET-BCA and pCD-mutants into strain EZAK. The fermentation steps were the same as in Example 2 above, as Figure 3 As shown, both E40D and T242A had a positive effect on the yield of 3′-sialyllactose, among which the E40D mutation significantly increased the yield from 2.07 g / L to 2.92 g / L, which was comparable to the 3′-sialyllactose yield produced by strain Z2 (carrying 406NST). In contrast, strains Z3 (carrying Q29H), Z5 (carrying N94K) and Z7 (carrying K273N) had lower yields, with 3′-sialyllactose yields of 1.55 g / L, 1.93 g / L and 1.95 g / L, respectively, all lower than strain Z2; after N94K and K273N mutations at positions 94 and 273 of NST, respectively, the 3′-sialyllactose yields of strains Z5 and Z7 were lower than that of strain Z2, but strain Z6 constructed after T242A mutation had a slightly higher 3′-sialyllactose yield than strain Z2.

[0102] Example 4: Saturation mutagenesis and multiple point mutations

[0103] (1) Saturation mutagenesis

[0104] Strain Z4 (containing mutation NST-E40D) resulted in a significant increase in 3′-sialyllactose production, which means that position 40 may be a strong candidate for transferase activity in 406NST. Therefore, saturation mutation was performed on position D40 of 406NST to determine the effect of all potential amino acids at position D40 on the efficiency of 3′-sialyllactose synthesis. Each mutant was cloned into pCDF-Duet1 using pCD-406NST as a template and then transferred into EZAK simultaneously with pET-BCA. The fermentation steps were the same as in Example 3 above, as Figure 5As shown, the results showed that there were significant differences in the 3′-sialyllactose production of each mutant, highlighting the key role of the D40 position in 3′-sialyllactose production. The 3′-sialyllactose production of most mutants was lower than that of Z2 (containing 406NST), indicating that aspartic acid (D) was more efficient at the D40 position than most other amino acids. When the D40 position was changed to proline (P), strain Z20 (containing 406NST-D40P) lost the ability to produce 3′-sialyllactose. However, some amino acids are more efficient than aspartic acid (D). For example, strains Z10 (containing 406NST-D40N), Z14 (containing 406NST-D40R), and Z15 (containing 406NST-D40L) produced 3.29 g / L, 3.71 g / L, and 2.93 g / L of 3′-sialyllactose, respectively, which were all higher than Z2 (2.92 g / L). After the aspartic acid (D) at position D40 in 406NST was mutated to arginine (R), the production of 3′-sialyllactose increased 1.13 times compared with the original 406NST, thus identifying it as the best mutant and selecting it as the object for subsequent molecular modification.

[0105] (2) Multiple point mutations

[0106] This study explored the possibility of superimposing the P120H and N113D mutations on the mutant Z26 (406NST-D40R), either alone or in various combinations. The fermentation steps were the same as in Example 3 above. Figure 6 The results in Figure 2 showed that all mutants showed different degrees of increased 3′-sialyllactose production.

[0107] The 3′-sialyllactose production of (406NST-D40R-N113D-P120H) was 4.14, 4.06 and 4.67 g / L, respectively, which were 41.78%, 39.04% and 59.93% higher than that of strain Z2.

[0108] The mutant 406NST-D40R-N113D-P120H ( Figure 4 B), the nucleotide sequence is shown in SEQ ID NO.8. Position 120 is close to the donor, while position 113 is close to the acceptor. Both single-point mutations enhance the production of 3′-sialyllactose individually. However, due to the intricate interaction mechanism of these mutations, the double mutation cannot achieve the same yield increase rate as the single-point mutation. Ultimately, Z28 was determined to be the best strain, with the highest 3′-sialyllactose production among all strains, at 4.67 g / L.

[0109] Example 5: Highly efficient production of 3'-sialyllactose in an engineered bacterial fermenter

[0110] The production of 3′-sialyllactose was evaluated using the recombinant strain Z28 in a 5-L bioreactor containing DM and 20 g / L glycerol. After 15 h, when the OD 600 When the pH reached about 40, 0.2 mM IPTG and 2 g / L lactose were immediately added to initiate the production of 3′-sialyllactose in vivo. Figure 7 As shown, the maximum OD after 48 h 600 After 56 hours, the maximum production of extracellular 3′-sialyllactose reached 29.54 g / L, which was 6.42 times that of shake flask culture. After calculation, the total production of 3′-sialyllactose was 0.52 g / L / h, and the conversion rate of lactose was 0.62 mol 3′-sialyllactose / mol. At the end of fermentation, OD 600 Remaining at 102, this indicates that Z28 has excellent sustainable production and tolerance capabilities.

[0111] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A sialyltransferase mutant, characterized in that: Select from any of the following: A1) its amino acid sequence comprises a sequence obtained by mutation of an amino acid residue position in the sequence shown in SEQ ID NO.1, wherein the mutation position is selected from any one or more of the following positions: position 29, position 40, position 94, position 242, and position 273; A2) is a protein having 98% or more identity with the protein described in A1) and having the same function as the protein described in A1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the mutant described in A1); A3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in A1) and / or A2).

2. The sialyltransferase mutant according to claim 1, characterized in that The mutations in A1) produced by the sequence shown in SEQ ID NO.1 are selected from any one or more of the following mutations: The Q at position 29 mutated to H; The 40th position E mutated to D; The 94th position N mutated to K; T at position 242 mutated to A; The K at position 273 mutated to N.

3. A sialyltransferase mutant, characterized in that: Select from any of the following: B1) Its amino acid sequence comprises a sequence obtained by mutation of an amino acid residue in the sequence shown in SEQ ID NO. 2, wherein the mutation site is selected from any one or more of the following sites: position 40, position 113, and position 120; B2) is a protein having 98% or more identity with the protein described in B1) and having the same function as the protein described in B1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the mutant described in B1); B3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in B1) and / or B2).

4. The sialyltransferase mutant according to claim 3, characterized in that The mutations in B1) produced by the sequence shown in SEQ ID NO.2 are selected from any one or more of the following mutations: The 40th position D mutates to any one of M, W, N, F, T, K, R, L, S, I, Q, H, P, C, V, A, Y, G; No. 113N mutated to D; The 120P mutated to H.

5. The sialyltransferase mutant according to claim 4, characterized in that Its amino acid sequence comprises a sequence obtained by mutation of the amino acid residue sites in the sequence shown in SEQ ID NO. 2, wherein the mutation sites are selected from any one of the following groups: D40R-P120H, D40R-N113D, and D40R-P120H-N113D.

6. Biomaterial, characterized in that The biological material is selected from any one of the following: C1) contains a nucleic acid molecule encoding a sialyltransferase mutant according to any one of claims 1 to 5; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3).

7. A whole cell catalyst, characterized in that The whole cell catalyst comprises the sialyltransferase mutant according to any one of claims 1 to 5 and / or the biological material according to claim 6.

8. Use of the sialyltransferase mutants as claimed in claims 1 to 5 and / or the biomaterials as claimed in claim 6 and / or the whole cell catalysts as claimed in claim 7, characterized in that: The application is selected from any one of the following:

1. preparing 3'-sialyllactose; II. preparing a product containing 3'-sialyllactose; III. Improve the activity of α2,3-sialyltransferase; IV. Construction of a recombinant microorganism for producing 3'-sialyllactose; V. Regulating the production of 3'-sialyllactose by microorganisms; VI. Improve the purity of 3'-sialyllactose produced by microorganisms.

9. A method for producing 3'-sialyllactose, characterized in that: The steps include: Step 1, constructing a recombinant cell or recombinant microorganism capable of expressing the sialyltransferase mutant according to any one of claims 1 to 5; Step 2: culturing the recombinant cell or recombinant microorganism to obtain 3'-sialyllactose.

10. A method for increasing the yield of 3-sialyllactose, characterized in that: At least one of the following D1) or D2): The D1) comprises the following steps: inserting a sialyltransferase mutant gene into the bacterial genome to increase the production of 3'-sialyllactose in the bacteria; Said D2) comprises the following steps: increasing the content and / or activity of sialyltransferase mutants in bacteria, or increasing the gene expression of sialyltransferase mutants in bacteria, so as to increase the production of 3'-sialyllactose in bacteria; The sialyltransferase mutant comprises the mutant according to any one of claims 1-2; and / or The sialyltransferase mutant comprises the mutant according to any one of claims 3 to 5; The sialyltransferase mutant gene comprises a gene encoding the sialyltransferase mutant according to any one of claims 1-5.

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