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

Abstract

The present application relates to the technical field of fermentation engineering, and particularly relates to a mutant of alpha-1,2-fucosyltransferase and application thereof. The mutant is based on the amino acid sequence of alpha-1,2-fucosyltransferase and comprises any one or more of the following mutations: K125D, A242C, Y269A, P284L or V292L. The present application provides a plurality of mutants of alpha-1,2-fucosyltransferase, which 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 present application can solve the problem of by-product generation in the fermentation process while improving the yield of 2'-fucosyllactose, which has important significance in the field of 2'-fucosyllactose production.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a mutant of α-1,2-fucosyltransferase and its application. Background Technology

[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. HMOs play an important role in infant health. As prebiotics, HMOs have many benefits, including regulating gut microbiota, strengthening the intestinal barrier, promoting the development of the infant's immune system, and improving brain and cognitive development. Currently, more than 200 types of human milk oligosaccharides have been identified, which can be classified into sialylated lactose, neutralized lactose, and fucoidylated lactose based on their monomer composition.

[0003] 2'-Fucosyllactose (2'-FL) is a type of fucoidanized lactose, and it is the most abundant human milk oligosaccharide, accounting for approximately 30% of all human milk oligosaccharides. Furthermore, related studies have shown that 2'-FL has effects such as preventing intestinal diseases, enhancing immunity, and promoting brain development. The de novo synthesis of 2'-fucosyllactose involves multiple steps involving mannose-6-phosphate isomerase ManA, phosphogmannose mutase ManB, mannose-1-phosphate guanylate transferase ManC, GDP-D-mannose-4,6-dehydratase Gmd, and GDP-fucosyllase WcaG to synthesize GDP-L-fucose. The substrates GDP-L-fucose and lactose are then catalyzed by α-1,2-fucosyllase to synthesize 2'-fucosyllactose.

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

[0005] To address the problems existing in the prior art, this invention provides a mutant of α-1,2-fucosyltransferase and its application.

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

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

[0008] Furthermore, the mutant is based on the amino acid sequence of α-1,2-fucosyltransferase, and includes any one of the following:

[0009] i) Mutations in K125D and Y269A;

[0010] ii) In addition to i), it also includes the mutation A242C.

[0011] Furthermore, the α-1,2-fucosyltransferase is derived from... Escherichia coli O126.

[0012] Furthermore, the α-1,2-fucosyltransferase comprises any one of the following amino acid sequences:

[0013] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0014] (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids as shown in SEQ ID NO.1.

[0015] Furthermore, the gene encoding the α-1,2-fucosyltransferase includes any one of the following nucleotide sequences:

[0016] (1) The nucleotide sequence as shown in SEQ ID NO.2;

[0017] (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2;

[0018] (3) A nucleotide sequence that can hybridize with a nucleotide sequence as shown in SEQ ID NO.2 under strict conditions.

[0019] Secondly, the present invention provides the application of the aforementioned mutant in improving the ability of Escherichia coli to produce 2'-fucosylated lactose.

[0020] The present invention further provides the application of the aforementioned mutant in reducing the impurity level in the process of Escherichia coli producing 2'-fucosylated lactose;

[0021] The impurities preferably include lactose-fucotetraose and 3-fucosyllactose.

[0022] Furthermore, the *E. coli* is an *E. coli* species capable of producing 2'-fucosylated lactose, and the mutant is introduced into or integrated into the genome of the *E. coli*.

[0023] Preferably, the *E. coli* reduces the expression levels of the lacZ, wcaJ, and nudD genes.

[0024] More preferably, the *E. coli* further includes the manB, manC, gmd, and wcaG genes. These genes are integrated independently or in any combination into the genome of the genetically engineered bacterium, or they exist in a recombinant plasmid carried by the genetically engineered bacterium, and their genes may originate from exogenous or endogenous genes.

[0025] The lacZ gene described in this invention is β-galactosidase, wcaJ is UDP-glucose lipotransferase, nudD is GDP-mannosyl hydrolase, manB is phosphogannase mutase, manC is mannose-6-phosphate guanosine transferase, gmd is GDP-mannose-1,4-dehydratase, and wcaG is GDP-fucose synthase.

[0026] The reduction in expression levels of the lacZ, wcaJ, and nudD genes described in this invention can be achieved using techniques commonly used in the art to reduce gene expression levels, such as gene knockout resulting in the deletion of gene fragments or complete gene knockout, or RNA interference. Furthermore, the reduction in gene expression levels includes varying degrees of gene level reduction, including complete loss of function.

[0027] Thirdly, the present invention provides a method for improving the ability of Escherichia coli to produce 2'-fucosylated lactose, comprising: replacing the α-1,2-fucosylated transferase in Escherichia coli with the aforementioned mutant, or introducing the aforementioned mutant into Escherichia coli.

[0028] Furthermore, the mutant was introduced into E. coli through one or more of the following methods: plasmid transfection, CaCl2 transformation, electroporation, or phage transformation.

[0029] Fourthly, the present invention provides an Escherichia coli, which is prepared by the aforementioned method.

[0030] The present invention has the following beneficial effects:

[0031] This invention provides various mutants of α-1,2-fucosyltransferase, which have high catalytic activity and specificity. After being introduced into Escherichia coli, they can significantly increase the yield of 2'-fucosyllactose and reduce the yield of byproducts lactose-fucotetraose and 3-fucosyllactose, thereby solving the problems of improving the production performance of 2'-fucosyllactose and the generation of byproducts during fermentation, and have high economic value. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0034] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0035] Example 1

[0036] 1. Obtaining the chassis strain

[0037] The chassis strain used in this invention is *Escherichia coli* capable of producing 2'-fucosylated lactose (hereinafter referred to as 2'-FL), and any *E. coli* strain with the corresponding capability in the prior art can be used. This embodiment preferably uses a chassis strain obtained by the following method:

[0038] Starting with Escherichia coli BL21(DE3), the 2'-FL synthesis precursor-related metabolic genes, such as β-galactosidase (lacZ), UDP-glucose lipotransferase (wcaJ), and GDP-mannose mannosyl hydrolase (nudD), were knocked out in the genome to obtain the chassis strain FL001.

[0039] 2. Obtaining mutants

[0040] The α-1,2-fucosyltransferase (wbgL) involved in this embodiment is derived from... Escherichia coli O126, whose GenBank accession number is ADN43847.1 (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2).

[0041] A total of nine mutations were involved, including individual mutations such as L86K, K125D, V139P, A242C, Y269A, P284L, and V292L. There were also mutations involving K125D and Y269A, as well as mutations involving K125D, Y269A, and A242C simultaneously.

[0042] The corresponding nucleotide sequence mutations for these methods are as follows:

[0043] Table 1. Nucleotide sequence changes

[0044]

[0045] 3. Insert the nucleotide sequence of α-1,2-fucosyltransferase from step 2 into the restriction site (NcoⅠ / HindⅢ) of the expression vector pETDuet-1 to obtain plasmid pET-wbgL. Since the synthesis of 2'-FL requires the construction of the precursor substance GDP-L-fucose pathway, the genes manC (AAC75110.1), manB (AAC75109.1), gmd (AAC75114.1), and wcaG (AAC75113.1) from Escherichia coli K-12 are sequentially inserted into pRSFDuet-1 to obtain plasmid pRSF-CBGW. Plasmid pET-wbgL and pRSF-CBGW are co-transformed into the chassis strain FL001 to obtain the 2'-FL production strain named FL001-wbgL.

[0046] 4. Repeat step 3 for all mutants, and name the resulting strain as follows:

[0047] 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. These names correspond to different mutation patterns.

[0048] 5. Perform shake-flask fermentation on the production strains obtained in steps 3 and 4, as follows:

[0049] Shake-flask fermentation medium: glycerol 30 g / L, anhydrous glucose 10 g / L, disodium hydrogen phosphate dodecahydrate 17.9 g / L, potassium dihydrogen phosphate 3.1 g / L, ammonium chloride 2.0 g / L, ammonium phosphate 1 g / L, trisodium citrate dihydrate 2.2 g / L, yeast extract 2 g / L, tryptone 15 g / L, magnesium sulfate heptahydrate 10 g / L, anhydrous calcium chloride 0.015 g / L, vitamin B1 0.01 g / L, plus Triton X-100 0.3 mL / L and trace elements 10 mL / L.

[0050] Trace elements (g / L): Sodium hypotriacetate 13.74 g / L, ferric ammonium citrate 5.6 g / L, zinc sulfate heptahydrate 0.9 g / L, CoCl2·6H2O 0.2 g / L, manganese chloride tetrahydrate 1.0 g / L, CuCl2·2H2O 0.10 g / L, boric acid 0.2 g / L, Na2MoO4·2H2O 0.2 g / L.

[0051] Fermentation method: The seed culture was inoculated at a rate of 1% into the fermentation medium (containing 50 µg / mL ampicillin and 50 µg / mL kanamycin) and cultured at 37°C until OD reached. 600 The concentration of lactose and IPTG was 0.6-0.8, and the final concentrations were 12 g / L and 0.4 mmol / L, respectively, for induction. The mixture was cultured 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 then detected.

[0052] 6. The final fermentation results are as follows:

[0053] Table 2. Yields of 2'-FL, DFL, and 3-FL for each producing strain

[0054]

[0055] This shows that:

[0056] In this invention, the K residue at position 125 of the wbgL amino acid sequence was mutated to D, resulting in a strain with a 2'-FL yield of 8.06 g / L, an increase of 5.36%, a DFL yield of 0.02 g / L, and a 3-FL yield of 0.05 g / L, while significantly reducing byproducts.

[0057] Mutating residue A to C at position 242 of the wbgL amino acid sequence resulted in a strain with 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.

[0058] Mutating residue Y at position 269 of the wbgL amino acid sequence to A yielded a strain with a 2'-FL yield of 7.82 g / L and a DFL yield of 0.26 g / L, with no 3-FL byproduct generated.

[0059] The combination of K125D and Y269A yielded a 2'-FL production of 8.35 g / L, which was 9.15% higher than that of the control strain FL001-wbgL, and no byproducts DFL or 3-FL were generated.

[0060] The combination of K125D, Y269A and A242C yielded a strain with a 2'-FL yield of 10.43 g / L, which is 36.34% higher than the previous strain, demonstrating a good effect on increasing 2'-FL yield. At the same time, no byproducts DFL and 3-FL were detected in the fermentation broth.

[0061] The results above show that various mutation methods can increase the production of 2'-FL and decrease the production of DFL and 3-FL. The two combinations, K125D and Y269A and K125D, Y269A and A242C, can avoid the production of DFL and 3-FL, while significantly increasing the production of 2'-FL.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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 is formed by any of the following mutations in the amino acid sequence of α-1,2-fucosyltransferase: (1) K125D; (2) K125D and Y269A; (3) K125D, Y269A and A242C; The amino acid sequence of the α-1,2-fucosyltransferase is shown in SEQ ID NO.

1.

2. The mutant according to claim 1, characterized in that, The gene encoding the α-1,2-fucosyltransferase has the nucleotide sequence shown in SEQ ID NO.

2.

3. The application of the mutant according to any one of claims 1-2 in improving the ability of Escherichia coli to produce 2'-fucosylated lactose; wherein the Escherichia coli is an Escherichia coli capable of producing 2'-fucosylated lactose.

4. The application of the mutant according to any one of claims 1-2 in reducing the level of impurities in the production of 2'-fucosylated lactose by Escherichia coli; wherein the impurities are lactose difucotetraose and 3-fucosylated lactose.

5. The application according to any one of claims 3-4, characterized in that, The mutant is introduced into the E. coli or integrated into the genome of the E. coli; The *E. coli* strain reduced the expression levels of lacZ, wcaJ, and nudD. The *E. coli* species also include the manB, manC, gmd, and wcaG genes.

6. A method for improving the ability of *Escherichia coli* to produce 2'-fucosylated lactose, characterized in that, include: The mutant according to any one of claims 1-2 is used to replace the α-1,2-fucosyltransferase in Escherichia coli, or the mutant according to any one of claims 1-2 is introduced into the Escherichia coli; The *Escherichia coli* is an *Escherichia coli* species capable of producing 2'-fucosylated lactose.

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