Mutant of alpha-1, 2-fucosyltransferase and application thereof

Abstract

The invention relates to the technical field of fermentation engineering, in particular to a mutant of alpha-1, 2-fucosyltransferase and application of the mutant. On the basis of an amino acid sequence of alpha-1, 2-fucosyl transferase, the mutant comprises any one or more of the following mutations: K125D, A242C, Y269A, P284L or V292L. The invention provides various mutants of alpha-1, 2-fucosyltransferase, and the mutants can effectively improve the ability of escherichia coli to produce 2 '-fucosyllactose and inhibit the generation of impurities. The mutant of alpha-1, 2-fucosyltransferase provided by the invention can improve the yield of 2 '-fucosyllactose and solve the problem of generation of by-products in the fermentation process at the same time, which is of great significance in the field of production of 2'-fucosyllactose.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a mutant of alpha-1,2-fucosyltransferase and application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after lactose and fat, with a content of 2-5 g / L in human milk. HMOs play an important role in infant health. HMOs can be used as prebiotics, with many benefits such as regulating intestinal flora, enhancing intestinal barrier, promoting the development of infant immune system, and improving brain and cognitive development. At present, 200 human milk oligosaccharides have been identified, which can be divided into sialylated lactose, neutralized lactose and fucosylated lactose according to their constituent monomers.

[0003] 2'-Fucosyllactose (2'-FL) is a type of fucosylated lactose, with the highest content in human milk oligosaccharides, accounting for about 30% of human milk oligosaccharides. In addition, relevant studies have shown that 2'-FL has the effects of preventing intestinal diseases, improving immunity, and promoting brain development. The de novo synthesis of 2'-fucosyllactose is converted into GDP-L-fucose by mannose-6-phosphate isomerase ManA, phosphomannose mutase ManB, mannose-1-phosphate guanosyltransferase ManC, GDP-D-mannose-4, 6-dehydratase Gmd and GDP-fucose synthase WcaG in multiple steps. The substrates GDP-L-fucose and lactose are catalyzed by α-1,2-fucosyltransferase to synthesize 2'-fucosyllactose.

[0004] During the synthesis of 2'-FL, due to the non-specificity of α-1,2-fucosyltransferase, which has α-1,3-fucosyl transfer activity, it is easy to generate byproducts 3-fucosyllactose (3-FL) and lactose difucotetraose (DFL), which limits the production of 2'-FL and increases the difficulty of purifying the 2'-FL product. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a mutant of α-1,2-fucosyltransferase and application thereof.

[0006] In a first aspect, the present invention provides a mutant of α-1,2-fucosyltransferase, wherein the mutant comprises any one or more of the following mutations based on the amino acid sequence of α-1,2-fucosyltransferase: K125D, A242C, Y269A, P284L or V292L.

[0007] The expression of the mutations in the present invention as above is a commonly used expression in the art. Taking K125D as an example, the position of the mutation at position 125 on the uridine 5′-diphospho-N-acetylglucosamine 2 epimerase, K is the amino acid type before mutation (lysine), and D is the amino acid type after mutation (aspartic acid), that is, the lysine at position 125 is replaced by aspartic acid; A242C indicates that the alanine at position 242 is replaced by cysteine; Y269A indicates that the tyrosine at position 269 is replaced by alanine; P284L indicates that the proline at position 284 is replaced by leucine; V292L indicates that the valine at position 292 is replaced by leucine.

[0008] Furthermore, the mutant includes any one of the following based on the amino acid sequence of α-1,2-fucosyltransferase: i) mutations K125D and Y269A; ii) The method according to i) further comprising mutation A242C.

[0009] Furthermore, the α-1,2-fucosyltransferase is derived from Escherichia coli O126.

[0010] Furthermore, the α-1,2-fucosyltransferase comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids, and having the same functional protein.

[0011] Furthermore, the gene encoding the α-1,2-fucosyltransferase comprises any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

[0012] In a second aspect, the present invention provides the use of the aforementioned mutant in improving the ability of Escherichia coli to produce 2'-fucosyllactose.

[0013] The present invention further provides the use of the aforementioned mutant in reducing the level of impurities in the process of producing 2'-fucosyllactose in Escherichia coli; The impurities preferably include lactose, difucotetraose and 3-fucosyllactose.

[0014] Furthermore, the Escherichia coli is an Escherichia coli capable of producing 2'-fucosyllactose, and the mutant is introduced or integrated into the genome of the Escherichia coli; Preferably, the E. coli has reduced expression levels of lacZ, wcaJ and nudD genes.

[0015] Further preferably, the Escherichia coli also includes: manB, manC, gmd and wcaG genes. The above genes are integrated independently or in any combination into the genome of the genetically engineered bacteria, or exist in a recombinant plasmid carried by the genetically engineered bacteria, and the genes can be derived from exogenous genes or endogenous genes.

[0016] The lacZ of the present invention is a β-galactosidase gene, wcaJ is a UDP-glucose lipid carrier transferase gene, nudD is a GDP-mannosyl hydrolase gene, manB is a phosphomannose mutase gene, manC is a mannose-6-phosphate guanosine transferase gene, gmd is a GDP-mannose-1,4-dehydratase gene, and wcaG is a GDP-fucose synthase gene.

[0017] The present invention reduces the expression level of lacZ, wcaJ and nudD genes can be achieved by using the commonly used technical means of reducing gene expression levels in the art, such as gene knockout to cause the deletion of gene fragments or complete gene knockout, or RNA interference, etc. At the same time, reducing the gene expression level includes different degrees of gene level reduction, including complete loss of function.

[0018] In a third aspect, the present invention provides a method for improving the ability of Escherichia coli to produce 2'-fucosyllactose, comprising: using the aforementioned mutant to replace the α-1,2-fucosyltransferase in Escherichia coli, or introducing the aforementioned mutant into Escherichia coli.

[0019] Further, the mutant is introduced into Escherichia coli by one or more of plasmid transfection, CaCl2 transformation, electroporation or phage transformation.

[0020] In a fourth aspect, the present invention provides an Escherichia coli prepared by the aforementioned method.

[0021] The present invention has the following beneficial effects: The present invention provides a variety of mutants of α-1,2-fucosyltransferase, which have high catalytic activity and specificity. After being introduced into Escherichia coli, the yield of 2'-fucosyllactose can be significantly increased, and the yield of byproducts lactose difucotetraose and 3-fucosyllactose can be reduced, thereby solving the problem of improving the production performance of 2'-fucosyllactose and the generation of byproducts during the fermentation process, and having high economic value. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0024] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0025] Example 1 1. Obtaining chassis strains The chassis strain used in the present invention is an Escherichia coli capable of producing 2'-fucosyllactose (hereinafter referred to as 2'-FL), and any Escherichia coli with the corresponding ability in the prior art can be used. In this embodiment, the chassis strain obtained by the following method is preferably used: Using Escherichia coli BL21 (DE3) as the starting strain, metabolic genes related to 2'-FL synthesis precursors such as β-galactosidase gene (lacZ), UDP-glucose lipid carrier transferase gene (undecaprenyl-phosphate glucose-1-phosphate transferase, wcaJ) and GDP-mannose mannosyl hydrolase gene (nudD) were knocked out from the genome to obtain the chassis strain FL001.

[0026] 2. Obtaining mutants The α-1,2-fucosyltransferase (abbreviated as wbgL) involved in this example comes from Escherichia coliO126, whose GenBank accession number is ADN43847.1 (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2).

[0027] A total of 9 mutations are involved, including single mutations: L86K, K125D, V139P, A242C, Y269A, P284L and V292L. In addition, there are mutations of K125D and Y269A, and mutations of K125D, Y269A and A242C at the same time.

[0028] The nucleotide sequence mutations corresponding to these methods are as follows: Table 1 Nucleotide sequence changes

[0029] 3. Insert the nucleotide sequence of α-1,2-fucosyltransferase in step 2 into the restriction site (NcoⅠ / HindⅢ) of the expression vector pETDuet-1 to obtain the plasmid pET-wbgL. Because the synthesis of 2'-FL requires the construction of the precursor GDP-L-fucose pathway, insert the genes manC (AAC75110.1), manB (AAC75109.1), gmd (AAC75114.1) and wcaG (AAC75113.1) from Escherichia coli K-12 into pRSFDuet-1 in sequence to obtain the plasmid pRSF-CBGW. The plasmid pET-wbgL and pRSF-CBGW are co-transformed into the chassis strain FL001 to obtain the 2'-FL production strain named FL001-wbgL.

[0030] 4. Repeat step 3 for all mutants. The resulting strains are named: FL001-wbgL-L86K, FL001-wbgL-K125D, FL001-wbgL-V139P, FL001-wbgL- A242C, FL001-wbgL-Y269A, FL001-wbgL-P284L, FL001-wbgL-V292L, FL001-wbgL-K125D-Y269A, FL001-wbgL-K125D-Y269A-A242C. The names correspond to the mutation patterns.

[0031] 5. The production strains obtained in steps 3 and 4 were subjected to shake flask fermentation as follows: Shake flask fermentation medium: 30 g / L glycerol, 10 g / L anhydrous glucose, 17.9 g / L disodium hydrogen phosphate dodecahydrate, 3.1 g / L potassium dihydrogen phosphate, 2.0 g / L ammonium chloride, 1 g / L ammonium phosphate, 2.2 g / L trisodium citrate dihydrate, 2 g / L yeast powder, 15 g / L tryptone, 10 g / L magnesium sulfate heptahydrate, 0.015 g / L anhydrous calcium chloride, 0.01 g / L vitamin B1, plus 0.3 mL / L Triton X-100 and 10 mL / L trace elements.

[0032] Trace elements (g / L): nitrilotriacetic acid sodium salt 13.74g / L, ammonium ferric citrate 5.6g / L, zinc sulfate heptahydrate 0.9g / L, CoCl2·6H2O 0.2g / L, manganese chloride tetrahydrate 1.0g / L, CuCl2·2H2O 0.10g / L, boric acid 0.2g / L, Na2MoO4·2H2O 0.2g / L.

[0033] Fermentation method: Inoculate the seed liquid into the fermentation medium (containing 50µg / mL ampicillin and 50µg / mL kanamycin) at a 1% inoculum and culture at 37°C until OD 600 The pH value was 0.6-0.8, lactose and IPTG were added to make the final concentrations of 12 g / L and 0.4 mmol / L for induction, and the culture was carried out at 28°C and 230 r / min until the end of fermentation. The contents of 2'-FL, DFL and 3-FL in the fermentation broth were detected.

[0034] 6. The final fermentation results are as follows: Table 2 2'-FL, DFL and 3-FL yields of each production strain

[0035] From this we can see that: The present invention mutates the 125th residue K of the wbgL amino acid sequence to D, and the obtained strain has a 2'-FL yield of 8.06 g / L, which is increased by 5.36%, a DFL yield of 0.02 g / L, a 3-FL yield of 0.05 g / L, and greatly reduces byproducts.

[0036] By mutating residue A at position 242 of the wbgL amino acid sequence to C, the strain obtained had a 2'-FL yield of 9.24 g / L, an increase of 20.78%, a DFL yield of 0.75 g / L, and a 3-FL yield of 0.18 g / L.

[0037] The residue Y at position 269 of the wbgL amino acid sequence was mutated to A, and the obtained strain had a 2'-FL yield of 7.82 g / L and a DFL yield of 0.26 g / L, without the production of the byproduct 3-FL.

[0038] The combination of K125D and Y269A resulted in a strain with a 2'-FL yield of 8.35 g / L, which was 9.15% higher than that of the control strain FL001-wbgL, and no byproducts DFL and 3-FL were produced.

[0039] The strain K125D, Y269A and A242C were combined to obtain a 2'-FL yield of 10.43 g / L, an increase of 36.34%, showing a good effect of increasing 2'-FL yield. At the same time, no by-products DFL and 3-FL were detected in the fermentation broth.

[0040] From the above results, it can be seen that a variety of mutation methods can increase the strain's 2'-FL production and reduce DFL and 3-FL production. The two combinations of K125D and Y269A and K125D, Y269A and A242C can avoid the production of DFL and 3-FL, and the 2'-FL production is significantly improved.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant of α-1,2-fucosyltransferase, characterized in that: The mutant includes any one or more of the following mutations based on the amino acid sequence of α-1,2-fucosyltransferase: K125D, A242C, Y269A, P284L or V292L.

2. The mutant according to claim 1, characterized in that The mutant is based on the amino acid sequence of α-1,2-fucosyltransferase, including any one of the following: i) mutations K125D and Y269A; ii) The method according to i) further comprising mutation A242C.

3. The mutant according to claim 1 or 2, characterized in that The α-1,2-fucosyltransferase is derived from Escherichia coli O126.

4. The mutant according to claim 3, characterized in that The α-1,2-fucosyltransferase comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids, and having the same functional protein.

5. The mutant according to claim 4, characterized in that The coding gene of the α-1,2-fucosyltransferase comprises any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

6. Use of the mutant according to any one of claims 1 to 5 in improving the ability of Escherichia coli to produce 2'-fucosyllactose.

7. Use of the mutant according to any one of claims 1 to 5 in reducing the level of impurities in the process of producing 2'-fucosyllactose in Escherichia coli; The impurities preferably include lactose, difucotetraose and 3-fucosyllactose.

8. The use according to claim 6 or 7, characterized in that: The Escherichia coli is an Escherichia coli capable of producing 2'-fucosyllactose, and the mutant is introduced or integrated into the genome of the Escherichia coli; Preferably, the E. coli reduces the expression level of one or more genes among lacZ, wcaJ and nudD; Further preferably, the Escherichia coli further comprises: one or more of manB, manC, gmd and wcaG genes.

9. A method for improving the ability of Escherichia coli to produce 2'-fucosyllactose, characterized in that: include: The mutant according to any one of claims 1 to 5 is used to replace the α-1,2-fucosyltransferase in Escherichia coli, or the mutant according to any one of claims 1 to 5 is introduced or integrated into the genome of the Escherichia coli.

10. An Escherichia coli, characterized in that Prepared by the method described in claim 9.

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