CN118147103A - Alpha 1, 3/4-fucosyltransferase mutant and method for biosynthesis of difucosyl lactose by using same - Google Patents

Alpha 1, 3/4-fucosyltransferase mutant and method for biosynthesis of difucosyl lactose by using same Download PDF

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CN118147103A
CN118147103A CN202410283453.3A CN202410283453A CN118147103A CN 118147103 A CN118147103 A CN 118147103A CN 202410283453 A CN202410283453 A CN 202410283453A CN 118147103 A CN118147103 A CN 118147103A
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alpha
fucosyltransferase
lactose
gene
recombinant cell
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沐万孟
朱莺莺
杜智慧
张文立
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Jiangnan University
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Abstract

The invention discloses an alpha 1, 3/4-fucosyltransferase mutant and a method for biosynthesizing difucosyl lactose by using the same, belonging to the technical field of bioengineering. The invention obtains alpha 1, 3/4-fucosyltransferase mutant I46A, H, I, F, K, Y D and M134E through site-directed mutagenesis. Compared with the non-mutated wild-type strain, the recombinant strain containing the mutant has the advantages that the titer of the difucosyl lactose is obviously improved, and the accumulation of intermediate products is reduced to a certain extent. The highest titer of the difucosyl lactose is 6.35g/L, which is 1.2 times of that of the wild strain, exceeds the highest reported DFL shake flask yield at present, and has good industrial application prospect.

Description

Alpha 1, 3/4-fucosyltransferase mutant and method for biosynthesis of difucosyl lactose by using same
Technical Field
The invention relates to an alpha 1, 3/4-fucosyltransferase mutant and a method for biosynthesizing difucosyl lactose by using the same, belonging to the technical field of bioengineering.
Background
Breast milk oligosaccharides (HMOs) are the third largest solid component in breast milk, next to lactose and fat, in an amount of 5 to 15%. The prior study shows that the HMOs are beneficial to the health of infants: has the functions of regulating immunity, helping brain development and regulating intestinal flora, and is beneficial to the growth and development of infants. In addition, HMOs can also influence gastrointestinal motility and contraction, and treat related diseases such as intestinal pain. HMOs are generally classified into fucosylated HMOs, neutral HMOs and sialylated HMOs.2' -fucosyllactose (2 ' -FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL) and lactose-N-type-one fucose (LNFP I) are representative fucosyl-type HMOs, and tetrasaccharide DFL is of less interest than trisaccharides 2' -FL and 3-FL but is currently under active study.
At present, the production of 3-6 molecules of breast milk oligosaccharide by using a microbial cell factory becomes a mainstream synthesis mode. In contrast to 2' -FL and 3-FL, which have been widely studied to continue biosynthesis using microbial cells, difucosyl lactose has rarely been reported for biosynthesis. Typically, the first step in the synthesis of difucosylated lactose is to fucosylate the lactose by either alpha 1, 2-fucosyltransferase or alpha 1,3 fucosyltransferase, and then use 2' -FL and 3-FL as the receptors for the production of DFL. Enzymes having α1,2 activity and α1,3 activity are critical in the last step of DFL biosynthesis.
The alpha 1, 3/4-fucosyltransferases belong to the family of fucosyltransferases, catalyzing the transfer of GDP-fucosyl to a glucosyl-linked acceptor substrate, forming an alpha 1,3 glycosidic bond. For example, GDP-L-fucose may produce 3-fucosyllactose under the catalysis of alpha 1, 3/4-fucosyltransferase and 2' -fucosyllactose may produce Difucosyllactose (DFL) under the catalysis of alpha 1, 3/4-fucosyltransferase. Zhang et al produced 5.1g/L of DFL by the salvage pathway using 3g/L lactose and 2.6g/L fucose, but fucose was relatively expensive, so it was not very suitable for industrial production (Zhang,A.,Sun,L.,Bai,Y.,Yu,H.,McArthur,J.B.,Chen,X.,&Atsumi,S.(2021).Microbial production ofhuman milk oligosaccharide lactodifucotetraose.Metabolic Engineering,66,12-20.)., and Liang et al produced 6.19g/L of DFL by the de novo synthesis pathway using E.coli when 8g/L lactose was added, but the by-product 2' -FL was more, about 4.1g/L.(Liang,S.,He,Z.,Liu,D.,Yang,S.,Yan,Q.,&Jiang,Z.,(2024).Efficient biosynthesis of difucosyllactose via de novo GDP-L-fucose pathway in metabolically engineered Escherichia coli.Journal of Agricultural and Food Chemistry,72,4367-4375.)., and therefore further research was required to synthesize higher titres of difucosyl lactose using the helicobacter pylori-derived α1, 3/4-fucosyltransferase HP3/4FT catalytic substrate.
Disclosure of Invention
[ Technical problem ]
In microbial synthesis of difucosyl lactose, the production of difucosyl lactose by substrate synthesis is catalyzed by helicobacter pylori-derived alpha 1, 3/4-fucosyltransferase HP3/4FT, and higher accumulation of intermediates 2' -FL and 3-FL is present.
Technical scheme
In order to solve the technical problems, the mutant with enhanced titer of the difucosyl lactose is obtained through site-directed mutagenesis, so that the titer of the HP3/4FT catalytic substrate for synthesizing the difucosyl lactose is effectively enhanced, and the accumulation of intermediate products 2' -FL and 3-FL is reduced.
The first object of the present invention is to provide an alpha 1, 3/4-fucosyltransferase, wherein the alpha 1, 3/4-fucosyltransferase uses an amino acid sequence shown in SEQ ID NO.1 as a starting sequence, and the starting sequence is mutated as shown in any one of the following (a) to (e):
(a) Mutating isoleucine I at position 46 to alanine A;
(b) Mutating histidine H at position 48 to isoleucine I;
(c) Mutating phenylalanine F at position 49 to lysine K;
(d) Mutating tyrosine Y at position 131 to aspartic acid D;
(e) Methionine M at position 134 was mutated to glutamic acid E.
It is a second object of the present invention to provide a polynucleotide encoding the above alpha 1, 3/4-fucosyltransferase.
It is a third object of the present invention to provide a vector carrying the above polynucleotide.
It is a fourth object of the present invention to provide recombinant cells carrying the above polynucleotide or vector, or expressing the above alpha 1, 3/4-fucosyltransferase.
In one embodiment, the host of the recombinant cell comprises an animal cell, a plant cell, or a microbial cell.
In one embodiment, the recombinant cell uses escherichia coli as a starting strain, and the beta-galactosidase gene lacZ and the UDP-glucose lipid carrier transferase gene wcaJ on the genome of the starting strain are knocked out;
Promoter P J23119 is used for promoting the expression of manC-manB and gmd-wcaG gene clusters on genome;
The gene cluster manC-manB, GDP-D-mannose-4, 6-dehydratase-GDP-fucose synthase derived from phosphomannose-mannose-1-guanylate transferase, the gene cluster gmd-wcaG, the alpha 1, 2-fucosyltransferase FucT2 derived from helicobacter pylori, and the alpha 1, 3/4-fucosyltransferase are expressed.
In one embodiment, the GenBank accession numbers of the genes lacZ, wcaJ are: 945006, 946583; the nucleotide sequence of the gene cluster manC-manB is shown as SEQ ID NO. 3; the nucleotide sequence of the gene cluster gmd-wcaG is shown as SEQ ID NO. 4; the nucleotide sequence of the promoter P J23119 is shown in SEQ ID NO. 5; the NCBI accession number of the amino acid sequence of the alpha 1, 2-fucosyltransferase FucT2 is AAC99764.1.
In one embodiment, the gene cluster manC-manB and the gene cluster gmd-wcaG are expressed using plasmid pRSFDuet-1 as a vector; expressing the alpha 1, 2-fucosyltransferase FucT2 of helicobacter pylori by using a plasmid pCDDuet-1 as a vector; the alpha 1, 3/4-fucosyltransferase is expressed using plasmid pETDuet-1 as a vector.
It is a fifth object of the present invention to provide a method for producing a difucosyl lactose, which comprises the fermentation production of a difucosyl lactose using the above recombinant cells.
In one embodiment, the method comprises the steps of:
(1) Inoculating the recombinant cells into a seed culture medium, and culturing to obtain seed liquid;
(2) Transferring the seed liquid obtained in the step (1) to a fermentation medium, culturing until the OD 600 is 0.6-0.8, adding IPTG and lactose, and fermenting to produce the difucosyl lactose.
In one embodiment, the concentration of IPTG is between 0.05 and 0.5mM.
In one embodiment, the lactose concentration is 2-10g/L, or not less than 5g/L.
In one embodiment, the fermentation temperature is 20-30 ℃.
In one embodiment, the pH of the fermentation is from 6 to 8, alternatively from 6.5 to 7.
It is a sixth object of the present invention to provide the use of said alpha 1, 3/4-fucosyltransferase, or said polynucleotide, or said vector, or said recombinant cell, or said method for the preparation of difucosyllactose or a difucosyllactose containing product.
The beneficial effects are that:
The invention obtains alpha 1, 3/4-fucosyltransferase mutant I46A, H, I, F, K, Y D and M134E through site-directed mutagenesis. Compared with the non-mutated wild-type strain, the recombinant strain containing the mutant has the advantages that the titer of the difucosyl lactose is obviously improved, and the accumulation of intermediate products is reduced to a certain extent. The highest titer of the difucosyl lactose of the mutant Y131D expression strain is 6.35g/L, which is 1.2 times of that of the wild strain, exceeds the highest reported DFL shake flask yield at present, and has certain production and application values.
Drawings
FIG. 1 shows the production of difucosyl lactose titres by fermentation of recombinant bacteria containing mutants and the total content of the intermediate products 2' -fucosyl lactose and 3-fucosyl lactose.
Detailed Description
In order to make the technical scheme and the beneficial effects of the invention more obvious and understandable, the following detailed description is given by way of example.
In the present specification, the amino acids at the corresponding positions are indicated by the accepted single letter abbreviations of IUPAC, wherein each amino acid and its abbreviations are respectively: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V).
In the present specification, the mutation of an amino acid is expressed as "original amino acid, site, substituted amino acid". For example, the mutation of methionine to glutamic acid at position 134 is denoted by M134E.
The plasmids, endonucleases, PCR enzymes, column type DNA extraction kits, DNA gel recovery kits and the like used in the following examples are commercially available products, and specific operations are performed according to the kit instructions.
The following experimental procedures of competent cell preparation, colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electrotransformation, and extraction preservation of bacterial genome were carried out according to conventional methods. Sequencing of the following plasmids and DNA products was performed by the Biotechnology Co., inc. of Jin Weizhi, suzhou.
The initial seed solution is cultured by using LB liquid culture medium, and the composition of the initial seed solution is 10g/L peptone, 5g/L yeast extract and 10g/L sodium chloride. 15g/L agar powder is additionally added into the LB solid medium.
Fermentation medium: 20g/L glycerin, 13.5g/L potassium dihydrogen phosphate, 4.0g/L diammonium hydrogen phosphate, 1.7g/L citric acid, 1.4g/L magnesium sulfate heptahydrate, 10ml/L trace metal elements (10 g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 0.35g/L manganese sulfate monohydrate, 0.23g/L sodium borate decahydrate, 0.11g/L ammonium molybdate, 2.0g/L calcium chloride dihydrate).
The DFL and FL were detected by HPLC with the following configuration and detection parameters:
Chromatographic column: carbohydrate Analysis (Rezex ROA organic acid H + (8%)); a detector: a differential detector; mobile phase: 5mmol/L, H 2SO4; flow rate: 0.6mL/min; column temperature: 60 ℃; sample injection amount: 10 mu L.
Example 1: construction of recombinant plasmids
The primers used for the plasmid construction described below are listed in Table 1.
(1) The nucleotide sequence of the HP3/4FT gene fragment is shown as SEQ ID NO.2, and the HP3/4FT gene fragment is amplified by using primers HP3/4FT-F and HP3/4 FT-R. And (3) using pET-V-F/pET-V-R as a primer, amplifying a corresponding carrier fragment by using a pETDuet-1 template, and recovering the DNA fragment by glue.
The HP3/4FT gene fragment obtained by amplification is inserted between the cleavage sites NcoI and KpnI of the vector pETDuet-1, the gene fragment and the vector fragment are connected through a Jeep kit (NEB reagent company in the United states), and the obtained plasmid is named pET-HP3/4FT after sequencing verification.
(2) Acquisition of plasmid pRSF-CBGW
The nucleotide sequence of the gene cluster manC-manB is shown as SEQ ID NO.3, and the nucleotide sequence of the gene cluster gmd-wcaG is shown as SEQ ID NO. 4. The gene cluster manC-manB is amplified from the genome of Escherichia coli by a primer manCB-F/R, a plasmid vector pRSFDuet-1 is amplified by a primer RSF-V-F/R, a PCR product is obtained, agarose gel electrophoresis is performed, and the obtained two DNA fragments are purified, and are connected by a Jeep kit (NEB reagent company in the United states), so that an intermediate plasmid pRSF-CB (pRSFDuet-1-manC-manB) is constructed. Similarly, the intermediate plasmid is amplified by using a primer RSFCB-V-F/R to obtain a vector DNA fragment, the E.coli genome is amplified by using a primer F-GW-F/R to obtain a gmd-wcaG gene cluster DNA fragment, and the two fragments are assembled to obtain a plasmid pRSF-CBGW (pRSFDuet-1-manC-manB-gmd-wcaG).
(3) FucT2 acquisition of Gene fragment and acquisition of plasmid pCD-FucT2
The nucleotide sequence of FucT gene fragment is shown as SEQ ID NO.6, fucT fragment is amplified by using primer FucT2-F/FucT2-R, corresponding carrier fragment is amplified by using pCD-V-F/pCD-V-R as primer and pCDDuet-1 template, and DNA fragment is recovered. The amplified fucT2 gene fragment was inserted between the cleavage sites NcoI and KpnI of the vector pCDDuet-1, and the gene fragment and the vector fragment were ligated by using a Jeep kit (NEB reagent Co. In the U.S.) to give a plasmid designated pCD-fucT2.
TABLE 1 primers for plasmid construction
Example 2: construction of Single Point mutation plasmid vector
Primers used for single point mutant plasmid construction are listed in table 2.
PCR linear amplification was performed using the recombinant plasmid pET-HP3/4FT constructed in example 1 as a template, and 12 pairs of primers shown in Table 2, respectively, were digested with restriction enzyme DpnI at 37℃for 1.5 hours to completely remove the template. After completion of the reaction, 5. Mu.l of the mixture was transferred to competent E.coli JM109 cells, and cultured overnight on a plate containing ampicillin resistance (100. Mu.g/mL). 2-3 single clones are selected for each plate, sequenced and identified, and finally 12 single-point mutation plasmids and different single-point mutation plasmids are obtained, and mutation sites are respectively designated as pET-HP3/4FT(R43K)、pET-HP3/4FT(D44K)、pET-HP3/4FT(I46A)、pET-HP3/4FT(H48I)、pET-HP3/4FT(F49K)、pET-HP3/4FT(K52R)、pET-HP3/4FT(Q53E)、pET-HP3/4FT(Y128R)、pET-HP3/4FT(H130D)、pET-HP3/4FT(Y131D)、pET-HP3/4FT(A133D)、pET-HP3/4FT(M134E), brackets.
TABLE 2 primers for construction of Single Point mutant plasmids
Example 3: construction of escherichia coli mutant engineering strain and fermentation production of difucosyl lactose
(1) Strain construction
Coli BL21 (DE 3) is taken as an original strain, the beta-galactosidase gene lacZ and the UDP-glucose lipid carrier transferase gene wcaJ of the original strain are knocked out, the gene knockdown method is specifically disclosed in the patent with publication number CN110804577A, and a strong constitutive promoter P J23119 is utilized to start the expression of manC-manB and gmd-wcaG gene clusters on the genome of the original strain, and the constitutive promoter strengthening method is specifically disclosed in the patent with application number CN 202210666616.7. GenBank accession numbers of the gene lacZ and the gene wcaJ are respectively as follows: 945006, 946583. The nucleotide sequence of the promoter P J23119 is shown in SEQ ID NO. 5. The resulting strain was designated E.coli BL21 (DE 3) ΔwcaJ ΔlacZ ΔP manC-manB::PJ23119ΔPgmd-wcaG::PJ23119.
Plasmids pRSF-CBGW, pCD-FucT2 and pET-HP3/4FT obtained in example 1 were simultaneously transformed into strain E.coli BL21 (DE 3) ΔwcaJ ΔlacZΔP manC-manB::PJ23119ΔPgmd-wcaG::PJ23119, giving the recombinant strain designated BLC09. The alpha 1, 3/4-fucosyltransferase in recombinant strain BLC09 was wild-type and was not mutated.
Plasmids pRSF-CBGW and pCD-FucT2 obtained in example 1 were transformed into strain E.coli BL21 (DE 3) ΔwcaJ ΔlacZΔP manC-manB::PJ23119ΔPgmd-wcaG::PJ23119, while plasmid pET-HP3/4FT(R43K)、pET-HP3/4FT(D44K)、pET-HP3/4FT(I46A)、pET-HP3/4FT(H48I)、pET-HP3/4FT(F49K)、pET-HP3/4FT(K52R)、pET-HP3/4FT(Q53E)、pET-HP3/4FT(Y128R)、pET-HP3/4FT(H130D)、pET-HP3/4FT(Y131D)、pET-HP3/4FT(A133D)、pET-HP3/4FT(M134E), obtained in example 2 was transformed to obtain recombinant strains BLC09-1 to BLC09-12, respectively. The constructed strains and plasmids and genotypes contained therein are shown in Table 3.
(2) Fermentation production of difucosyl lactose
And (3) respectively inoculating the BLC09, BLC09-1, BLC09-2, BLC09-3, BLC09-4, BLC09-5, BLC09-6, BLC09-7, BLC09-8, BLC09-9, BLC09-10, BLC09-11 and BLC09-12 engineering strains constructed in the step (1) to an LB liquid culture medium, culturing at 37 ℃ at 200rpm overnight for 12 hours to obtain seed liquid, taking 500 mu L of the seed liquid, inoculating to 25mL of fermentation culture medium, culturing at 37 ℃ at 200rpm until OD 600 is 0.8, adding 0.5mM IPTG at the final concentration, and simultaneously adding 5g/L lactose, and continuously performing induction culture at 200rpm for 72 hours. 1mL of the fermentation broth was centrifuged at 10,000rpm for 10min, and the supernatant was used for HPLC to determine the yield of the disaccharide-based lactose (DFL).
As a result, as shown in FIG. 1, 5 engineering strains (BLC 09-3, BLC09-4, BLC09-5, BLC09-10, BLC 09-12) produced more difucosyl lactose in the 12 groups of mutants than the wild type engineering strain, and the yields of 2' -FL and 3-FL were lower than those of the wild type. Mutants expressed by engineering strains BLC09-5 and BLC09-10 were HP3/4FT-F49K and HP3/4FT-Y131D, respectively, yielding DFL of 6.05 and 6.35g/L, and total residues of 2' -FL and 3-FL were 1.04g/L and 1.26g/L, respectively. Wherein, the yield of the DFL of the engineering strain BLC09-10 is 1.2 times that of BLC09, which proves that the HP3/4FT-Y131D is more beneficial to the catalytic synthesis of the DFL.
TABLE 3 genotype of engineering strains and fermentation information
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. An alpha 1, 3/4-fucosyltransferase, wherein the alpha 1, 3/4-fucosyltransferase has an amino acid sequence as set forth in SEQ ID No.1, and wherein the set forth sequence is mutated as set forth in any one of (a) to (e):
(a) Mutating isoleucine I at position 46 to alanine A;
(b) Mutating histidine H at position 48 to isoleucine I;
(c) Mutating phenylalanine F at position 49 to lysine K;
(d) Mutating tyrosine Y at position 131 to aspartic acid D;
(e) Methionine M at position 134 was mutated to glutamic acid E.
2. A polynucleotide encoding the alpha 1, 3/4-fucosyltransferase of claim 1.
3. A vector carrying the polynucleotide of claim 2.
4. A recombinant cell expressing the alpha 1, 3/4-fucosyltransferase of claim 1, or carrying the polynucleotide of claim 2 or the vector of claim 3.
5. The recombinant cell of claim 4, wherein the host of the recombinant cell comprises an animal cell or a microbial cell.
6. The recombinant cell according to claim 5, wherein the recombinant cell uses escherichia coli as a starting strain, and the beta-galactosidase coding gene lacZ and the UDP-glucose lipid carrier transferase coding gene wcaJ on the genome of the starting strain are knocked out;
Promoter P J23119 is used for promoting the expression of manC-manB and gmd-wcaG gene clusters on genome;
Expressing the phosphomannose-mannose-1-guanylate transferase gene cluster manC-manB, GDP-D-mannose-4, 6-dehydratase-GDP-fucose synthase gene cluster gmd-wcaG, alpha 1, 2-fucosyltransferase FucT2 and the alpha 1, 3/4-fucosyltransferase;
GenBank accession numbers of the gene lacZ and the gene wcaJ are respectively as follows: 945006, 946583; the nucleotide sequence of the gene cluster manC-manB is shown as SEQ ID NO. 3; the nucleotide sequence of the gene cluster gmd-wcaG is shown as SEQ ID NO. 4; the nucleotide sequence of the promoter P J23119 is shown in SEQ ID NO. 5; the NCBI accession number of the amino acid sequence of the alpha 1, 2-fucosyltransferase FucT2 is AAC99764.1.
7. A method for producing a difucosyl lactose, characterized in that the method comprises the fermentation production of a difucosyl lactose using the recombinant cell according to any one of claims 4-6.
8. The method according to claim 7, characterized in that it comprises the steps of:
(1) Inoculating the recombinant cells into a seed culture medium, and culturing to obtain seed liquid;
(2) Transferring the seed liquid obtained in the step (1) to a fermentation culture medium, culturing until the OD 600 is 0.6-0.8, adding IPTG and lactose, and fermenting to produce the difucosyl lactose; the concentration of IPTG is 0.05-0.5mM; the lactose concentration is not less than 5g/L.
9. Use of an alpha 1, 3/4-fucosyltransferase according to claim 1, or a polynucleotide according to claim 2, or a vector according to claim 3, or a recombinant cell according to any one of claims 4 to 6, or a method according to claim 7 or 8 in the food, chemical or pharmaceutical field.
10. The application according to claim 9, characterized in that it comprises: the alpha 1, 3/4-fucosyltransferase, or the polynucleotide, or the vector, or the recombinant cell, or the method is used to prepare a difucosyl lactose or a difucosyl lactose containing product.
CN202410283453.3A 2024-03-13 2024-03-13 Alpha 1, 3/4-fucosyltransferase mutant and method for biosynthesis of difucosyl lactose by using same Pending CN118147103A (en)

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