CN119899815A - A mutant of α-1,2-fucosyltransferase and its application - Google Patents
A mutant of α-1,2-fucosyltransferase and its application Download PDFInfo
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Abstract
The invention relates to the technical field of fermentation engineering, in particular 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 K125D, A242C, Y269A, P L or V292L. The invention provides various mutants of alpha-1, 2-fucosyltransferase which are effective in improving the ability of E.coli to produce 2' -fucosyllactose and inhibiting the production of impurities. The mutant of the alpha-1, 2-fucosyltransferase provided by the invention can improve the yield of 2 '-fucosyllactose and solve the problem of byproduct generation in the fermentation process, and has important significance in the field of 2' -fucosyllactose production.
Description
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a mutant of alpha-1, 2-fucosyltransferase and application thereof.
Background
The human milk oligosaccharide (human milk oligosaccharides, HMOs) is the third largest solid component next to lactose and fat in breast milk, and the content of the human milk oligosaccharide in the human milk is 2-5 g/L. HMOs play an important role in infant health, and the HMOs can be used as prebiotics, and have the advantages of regulating intestinal flora, enhancing intestinal barrier, promoting infant immune system development, improving brain and cognitive development and the like. 200 human milk oligosaccharides have been identified, which can be classified into sialyllactose, neutralized lactose and fucosylated lactose, depending on their constituent monomers.
2' -Fucosyllactose (2 ' -Fucosyllactose,2' -FL) is one of the fucosylated lactose and is the highest in human milk oligosaccharides, accounting for about 30% of the human milk oligosaccharides. In addition, the related researches show that the 2' -FL has the effects of preventing intestinal diseases, improving immunity, promoting brain development and the like. De novo synthesis of 2'-fucosyllactose GDP-L-fucose was synthesized by multi-step transformations of mannose-6-phosphate isomerase ManA, phosphomannomutase ManB, mannose-1-guanosine transferase ManC, GDP-D-mannose-4, 6-dehydratase Gmd and GDP-fucose synthase WcaG, substrates GDP-L-fucose and lactose were synthesized by alpha-1, 2-fucosyltransferase catalysis to 2' -fucosyllactose.
In the synthesis process of 2' -FL, 3-fucosyllactose (3-FL) and lactodi-fucoidan (DFL) are easily generated as byproducts due to the non-specificity of alpha-1, 2-fucosyltransferase, which has alpha-1, 3-fucosyltransferase activity, thus limiting the production of 2' -FL and increasing the purification difficulty of 2' -FL product purification.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a mutant of alpha-1, 2-fucosyltransferase and application thereof.
In a first aspect, the invention provides a mutant of an alpha-1, 2-fucosyltransferase, which mutant comprises any one or more of the mutations K125D, A242C, Y269A, P L or V292L, based on the amino acid sequence of the alpha-1, 2-fucosyltransferase.
The expression mode of the mutation is the expression mode commonly used in the field, taking K125D as an example, the position of the mutation at 125 th position on uridine 5' -diphosphate-N-acetylglucosamine 2 epimerase, K is the amino acid type (lysine) before the mutation, D is the amino acid type (aspartic acid) after the mutation, namely the lysine at 125 th position is replaced by aspartic acid, A242C is the substitution of alanine at 242 th position by cysteine, Y269A is the substitution of tyrosine at 269 th position by alanine, P284L is the substitution of proline at 284 th position by leucine, and V292L is the substitution of valine at 292 nd position by leucine.
Further, the mutant is based on the amino acid sequence of alpha-1, 2-fucosyltransferase and comprises any one of the following:
i) Mutations K125D and Y269A;
ii) further comprises the mutation A242C on the basis of i).
Further, the alpha-1, 2-fucosyltransferase is derived from ESCHERICHIA COLIO126,126.
Further, the alpha-1, 2-fucosyltransferase comprises any one of the amino acid sequences:
(1) An amino acid sequence as shown in SEQ ID NO. 1;
(2) The amino acid sequence shown as SEQ ID NO.1 is obtained by replacing, inserting or deleting one or more amino acids to obtain the amino acid sequence of the protein with the same function.
Further, the coding gene of the alpha-1, 2-fucosyltransferase comprises any one of the following nucleotide sequences:
(1) A nucleotide sequence as shown in SEQ ID NO. 2;
(2) A nucleotide sequence which is obtained by replacing, deleting or inserting one or a plurality of nucleotides in the nucleotide sequence shown as SEQ ID NO.2 and can code proteins with the same functions;
(3) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence shown as SEQ ID NO. 2.
In a second aspect, the invention provides the use of a mutant as described above for increasing the ability of E.coli to produce 2' -fucosyllactose.
The invention further provides application of the mutant in reducing the impurity level in the process of producing 2' -fucosyllactose by escherichia coli;
the impurities preferably include lactose, di-fucoidan and 3-fucosyllactose.
Further, the E.coli is an E.coli having 2' -fucosyllactose-producing ability, and the mutant is introduced into or integrated into the genome of the E.coli;
preferably, the E.coli reduces the expression levels of the lacZ, wcaJ and nudD genes.
Further preferably, the E.coli further comprises manB, manC, gmd and wcaG genes. The genes are integrated independently or in any combination in the genome of the genetically engineered bacterium, or exist in a recombinant plasmid carried by the genetically engineered bacterium, and the genes can be derived from exogenous genes or endogenous genes.
The lacZ is beta-galactosidase gene, wcaJ is UDP-glucose lipid carrier transferase gene, nudD is GDP-mannosyl hydrolase gene, manB is mannose mutase gene, manC is mannose-6-guanosine phosphate transferase gene, gmd is GDP-mannose-1, 4-dehydratase gene, wcaG is GDP-fucose synthase gene.
The reduction of the expression levels of the lacZ, wcaJ and nudD genes can be achieved by adopting technical means commonly used in the field for reducing the expression levels of genes, such as deletion of gene fragments caused by gene knockout or complete knockout of genes, or RNA interference and the like. Meanwhile, decreasing the level of gene expression includes varying degrees of gene level decrease, including complete loss of function.
In a third aspect, the present invention provides a method for improving the ability of E.coli to produce 2' -fucosyllactose, comprising replacing alpha-1, 2-fucosyltransferase in E.coli with the aforementioned mutant or introducing the aforementioned mutant into E.coli.
Further, the mutants are introduced into E.coli by one or more of plasmid transfection, caCl 2 transformation, electroporation or phage transformation.
In a fourth aspect, the invention provides an E.coli prepared by the method described above.
The invention has the following beneficial effects:
The invention provides a plurality of mutants of alpha-1, 2-fucosyltransferase, which have higher catalytic activity and specificity, can obviously improve the yield of 2 '-fucosyllactose after being introduced into escherichia coli, and reduce the yields of byproducts lactose, disaccharide, fucose and 3-fucosyllactose, thereby solving the problems of improving the production performance of 2' -fucosyllactose and the production of byproducts in the fermentation process, and having higher economic value.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods referred to in the examples below, unless otherwise indicated, are conventional in the art, and may be, for example, referred to the laboratory manuals in the art or according to the conditions recommended by the manufacturer's instructions.
The experimental materials and reagents referred to in the examples below are commercially available unless otherwise indicated.
Example 1
1. Acquisition of Chassis strains
The chassis strain used in the invention is escherichia coli with the capability of producing 2 '-fucosyllactose (hereinafter referred to as 2' -FL), and any escherichia coli with the corresponding capability in the prior art can be used. The chassis strain obtained by the following method is preferably used in this example:
the E.coli BL21 (DE 3) was used as an initial strain, and metabolic genes related to 2' -FL synthesis precursor substances such as beta-galactosidase gene (beta-galactosidase, lacZ), UDP-glucose lipid carrier transferase gene (undecaprenyl-phosphate glucose-1-phosphate transferase, wcaJ) and GDP-mannosyl hydrolase gene (GDP-mannose mannosyl hydrolase, nudD) were knocked out on the genome to obtain chassis strain FL001.
2. Mutant acquisition
The alpha-1, 2-fucosyltransferase (wbgL for short) according to this example was derived from ESCHERICHIA COLIO126, and GenBank accession number was ADN43847.1 (the amino acid sequence was shown as SEQ ID NO.1, and the nucleotide sequence was shown as SEQ ID NO. 2).
A total of 9 mutation modes are involved, and the individual mutation modes comprise L86K, K125D, V139P, A242C, Y269A, P284L and V292L. In addition, there are mutations of K125D and Y269A, and mutations of both K125D, Y A and A242C.
The nucleotide sequence mutations corresponding to these modes are as follows:
TABLE 1 nucleotide sequence variation
3. The nucleotide sequence of α -1, 2-fucosyltransferase in step 2 was inserted into the cleavage site (NcoI/HindIII) of the expression vector pETDuet-1 to obtain plasmid pET-wbgL, because the synthesis of 2'-FL requires construction of the route of the precursor substance GDP-L-fucose, and genes manC (AAC 75110.1), manB (AAC 75109.1), gmd (AAC 75114.1) and wcaG (AAC 75113.1) derived from ESCHERICHIA COLI K-12 were sequentially inserted into pRSFDuet-1 to obtain plasmid pRSF-CBGW, and plasmids pET-wbgL and pRSF-CBGW were co-transformed into chassis strain FL001 to obtain 2' -FL producing strain designated FL001-wbgL.
4. Repeating step 3 for all mutants, the resulting strain was 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. Naming corresponds to each mutation pattern.
5. And (3) carrying out shake flask fermentation on each production strain obtained in the steps (3) and (4), wherein the method comprises the following steps:
The shake flask fermentation medium comprises 30g/L of glycerol, 10g/L of anhydrous glucose, 17.9g/L of disodium hydrogen phosphate dodecahydrate, 3.1g/L of potassium dihydrogen phosphate, 2.0g/L of ammonium chloride, 1g/L of ammonium phosphate, 2.2g/L of trisodium citrate dihydrate, 2g/L of yeast powder, 15g/L of tryptone, 10g/L of magnesium sulfate heptahydrate, 0.015g/L of anhydrous calcium chloride, 0.01g/L of vitamin B, and 0.3mL/L of additional triton X and 10mL/L of trace elements.
Microelements (g/L) are 13.74g/L of nitrilotriacetic acid sodium salt, 5.6g/L of ferric ammonium citrate, 0.9g/L of zinc sulfate heptahydrate, 0.2g/L of CoCl 2·6H2 O, 1.0g/L of manganese chloride tetrahydrate, 0.10g/L of CuCl 2·2H2 O, 0.2g/L of boric acid and 0.2g/L of Na 2MoO4·2H2 O.
The fermentation method comprises inoculating seed solution into fermentation medium (containing 50 μg/mL ampicillin and 50 μg/mL kanamycin) at 37deg.C until OD 600 is 0.6-0.8, adding lactose and IPTG to induce its final concentration to 12g/L and 0.4mmol/L, culturing at 28deg.C at 230r/min until fermentation is completed, and detecting 2' -FL, DFL and 3-FL content of fermentation broth.
6. The final fermentation results obtained were as follows:
TABLE 2 production of 2' -FL, DFL and 3-FL by the respective production strains
It can be seen from this:
The invention mutates the 125-bit residue K of wbgL amino acid sequence into D, the yield of the obtained strain 2' -FL is 8.06g/L, the yield of the DFL is 0.02g/L, the yield of the 3-FL is 0.05g/L, and the byproducts are greatly reduced.
Mutation of residue A at position 242 of wbgL amino acid sequence to C gave a strain with a 2' -FL yield of 9.24g/L, an improvement of 20.78%, a DFL yield of 0.75g/L and a 3-FL yield of 0.18g/L.
Mutation of residue Y at position 269 of the wbgL amino acid sequence to A gave a strain with a 2' -FL yield of 7.82g/L and a DFL yield of 0.26g/L, without by-product 3-FL production.
By combining K125D and Y269A, the strain obtained had a 2' -FL yield of 8.35g/L, 9.15% higher than that of the control strain FL001-wbgL, and did not produce by-product-free DFL and 3-FL.
When K125D, Y A and A242C were combined, the yield of 2'-FL of the obtained strain was 10.43g/L, which was 36.34% higher, and a better 2' -FL yield-improving effect was exhibited, while the byproducts DFL and 3-FL were not detected in the fermentation broth.
As can be seen from the above results, various mutation patterns can increase the 2'-FL yield of the strain, decrease the DFL yield and the 3-FL yield, and the two combination forms of the K125D and Y269A combination and the K125D, Y269A and A242C combination can avoid the production of the DFL and the 3-FL, and the 2' -FL yield is increased remarkably.
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.
Claims (10)
1. A mutant of alpha-1, 2-fucosyltransferase is characterized in that the mutant is based on the amino acid sequence of the alpha-1, 2-fucosyltransferase and comprises any one or more of K125D, A242C, Y269A, P L or V292L.
2. The mutant according to claim 1, wherein the mutant is based on the amino acid sequence of an α -1, 2-fucosyltransferase, and comprises any one of the following:
i) Mutations K125D and Y269A;
ii) further comprises the mutation A242C on the basis of i).
3. The mutant according to claim 1 or 2, wherein the α -1, 2-fucosyltransferase is derived from ESCHERICHIA COLI O126,126.
4. A mutant according to claim 3, wherein the α -1, 2-fucosyltransferase comprises any one of the amino acid sequences:
(1) An amino acid sequence as shown in SEQ ID NO. 1;
(2) The amino acid sequence shown as SEQ ID NO.1 is obtained by replacing, inserting or deleting one or more amino acids to obtain the amino acid sequence of the protein with the same function.
5. A mutant according to claim 4, wherein,
The coding gene of the alpha-1, 2-fucosyltransferase comprises any one of the following nucleotide sequences:
(1) A nucleotide sequence as shown in SEQ ID NO. 2;
(2) A nucleotide sequence which is obtained by replacing, deleting or inserting one or a plurality of nucleotides in the nucleotide sequence shown as SEQ ID NO.2 and can code proteins with the same functions;
(3) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence shown as SEQ ID NO. 2.
6. Use of a mutant according to any one of claims 1 to 5 for increasing the ability of escherichia coli to produce 2' -fucosyllactose.
7. Use of the mutant according to any one of claims 1-5 for reducing the impurity level during the production of 2' -fucosyllactose by e.
The impurities preferably include lactose, di-fucoidan and 3-fucosyllactose.
8. The use according to claim 6 or 7, wherein the escherichia coli is escherichia coli having 2' -fucosyllactose-producing ability, 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 of the genes lacZ, wcaJ and nudD;
Further preferably, the E.coli further comprises one or more of manB, manC, gmd and wcaG genes.
9. A method for improving the ability of E.coli to produce 2' -fucosyllactose, comprising replacing alpha-1, 2-fucosyltransferase in E.coli with the mutant according to any one of claims 1 to 5, or introducing or integrating the mutant according to any one of claims 1 to 5 into the genome of E.coli.
10. An escherichia coli prepared by the method of claim 9.
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| US20140031541A1 (en) * | 2012-07-25 | 2014-01-30 | Glycosyn LLC | Alpha (1,2) Fucosyltransferases Suitable for Use in the Production of Fucosylated Oligosaccharides |
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