Construction method and application of high-yield lactyl-N-difucotetraose engineering bacteria

Abstract

The invention relates to a construction method and application of high-yield lactyl-N-difucotetraose engineering bacteria, and belongs to the technical field of bioengineering and food biology. According to the method, genes in a GDP-1-fucose synthesis path are combined and regulated in host bacteria by utilizing a synthetic biological means, alpha-1, 2-fucosyltransferase and alpha-1, 3-fucosyltransferase are heterologously expressed on the basis, and then construction of a DFL high-yield strain is realized through modification strategies such as site-specific mutagenesis and random saturated mutation of rate-limiting enzyme. The invention also provides a method for synthesizing a high-added-value product DFL by taking glycerol as a substrate. The method has reference significance for solving the technical bottleneck of the DFL, provides an effective way for industrial production of the DFL, and has relatively high theoretical research value and social and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a high-yield Difucosyllactose (DFL) engineering strain and its application, belonging to the field of bioengineering and food biotechnology. BACKGROUND

[0002] Difucosyllactose (DFL) is an important functional oligosaccharide in breast milk, accounting for about 4% of total human milk oligosaccharides (HMOs). Its biosynthesis is usually based on the modification of 2'-FL or 3-FL as the backbone. Studies have shown that DFL has obvious antibacterial activity against Streptococcus agalactiae, which causes neonatal sepsis, but 2'-FL and 3-FL have no similar effect on this pathogen. In addition, DFL has been found to promote the colonization of probiotics and reduce the secretion of interleukin-8 in immature epithelial cells. So far, DFL has been approved by the European Food Safety Authority and the US Food and Drug Administration as a new food additive. In recent years, microbial fermentation and enzymatic reaction have been successfully established and used for the production of individual HMOs. In particular, enzymatic synthesis requires high synthesis costs and harsh reaction conditions, while microbial fermentation has low cost and mild reaction conditions. The currently reported DFL biosynthesis pathways include the salvage pathway and the de novo synthesis pathway. However, due to the high cost of substrates and poor economy of the salvage pathway, the de novo synthesis pathway has gradually become the mainstream method.

[0003] Recent studies have successfully improved the yield of human milk oligosaccharides by non-rational or semi-rational design of key enzymes. Li et al. used a semi-rational site-directed mutagenesis strategy to optimize the catalytic activity and thermal stability of HpFutC by combining multiple site mutations through mutant library screening. However, the biosynthesis ability of the mutants obtained by random mutation still needs to be verified by large-scale fermentation and analysis. On the other hand, the catalytic activity of α-1,3-fucosyltransferase towards 2'-FL acceptors and GDP-l-fucose donors plays a crucial role in the biosynthesis of DFL. Therefore, it is crucial to improve the enzyme activity of α-1,3-fucosyltransferase, and thus, it is necessary to screen new α-1,2-fucosyltransferases. At the same time, the overall yield of DFL biosynthesis needs to be improved. Compared with mature HMOs such as 2'-FL, the efficient synthesis of DFL is less studied and the yield is slightly lower, which restricts its industrial production and commercial application. SUMMARY

[0004] The present application provides α-1,3-fucosyltransferases with improved catalytic activity and genetically engineered strains producing Difucosyllactose (DFL) to solve the problem of low yield of Difucosyllactose (DFL) synthesized by existing biological methods.

[0005] The present application provides an alpha-1, 3-fucosyltransferase mutant with improved catalytic activity, which is based on the amino acid sequence shown in SEQ ID NO. 2 and has one or more mutations at positions N25K, N216K, E99Q, N276D.

[0006] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO. 2 and has any one of mutations (a)~(g): (a) the 25th asparagine is mutated to lysine; (b) the 216th asparagine is mutated to lysine; (c) the 25th asparagine is mutated to lysine and the 99th glutamic acid is mutated to glutamine; (d) the 25th asparagine is mutated to lysine and the 276th asparagine is mutated to aspartic acid; (e) the 25th asparagine is mutated to lysine, the 99th glutamic acid is mutated to glutamine, and the 276th asparagine is mutated to aspartic acid; (f) the 25th asparagine is mutated to lysine, the 98th threonine is mutated to lysine, the 99th glutamic acid is mutated to glutamine, and the 276th asparagine is mutated to aspartic acid; (g) the 22nd phenylalanine is mutated to tyrosine, the 25th asparagine is mutated to lysine, the 98th threonine is mutated to lysine, the 99th glutamic acid is mutated to glutamine, and the 276th asparagine is mutated to aspartic acid.

[0007] The present application also provides a gene encoding the mutant.

[0008] The present application also provides a genetically engineered bacterium expressing the mutant.

[0009] In one embodiment, the genetically engineered bacterium expresses phosphomannomutase manB, mannose-1-phosphate guanylyltransferase manC, GDP-l-mannose-4, 6-dehydratase gmd, GDP-l-fucose synthase wcaG, alpha-1, 2-fucosyltransferase FutC, and the alpha-1, 3-fucosyltransferase mutant.

[0010] In one embodiment, the genetically engineered bacterium has Escherichia coli as the host.

[0011] In one embodiment, the Escherichia coli is E. coli BL21(DE3) ΔnudDΔlacZΔnudKΔwcaJΔclpYQΔLonΔlacAΔiclRΔpoxB.

[0012] In an embodiment, FutC and the α-1, 3-fucosyltransferase mutant are expressed separately using plasmid pRSFDuet-1. manB manC gmd wcaG

[0013] In an embodiment, FutC and the α-1, 3-fucosyltransferase mutant are expressed separately using plasmid pETDuet-1.

[0014] In an embodiment, expression of manB manC gmd wcaG is initiated using a strong promoter.

[0015] In an embodiment, expression of FutC and BfFucT is initiated using a strong promoter.

[0016] In an embodiment, the strong promoter is T7 promoter.

[0017] In an embodiment, the nucleotide sequence of the phosphomannomutase gene manB and the nucleotide sequence of the mannose-l-phosphate guanylyltransferase gene manC are as shown in SEQ ID NO. 10, and the nucleotide sequence of the GDP-l-mannose-4, 6-dehydratase gene gmd and the nucleotide sequence of the GDP-fucose synthase gene wcaG are as shown in SEQ ID NO. 11.

[0018] In an embodiment, the genetically engineered bacteria also express a Gsk gene, and the nucleotide sequence of the Gsk gene is as shown in SEQ ID NO. 12.

[0019] The present application also provides a method for preparing lacto-N-difucohexaose, comprising: culturing the engineered bacteria in a culture medium with glycerol as a carbon source.

[0020] In an embodiment, IPTG is used for induction during the culturing process, and lactose is added.

[0021] In an embodiment, IPTG is used for induction during the culturing process, and the final concentration of IPTG is 0.2 mM.

[0022] In an embodiment, the induction is performed at 24-26°C.

[0023] ​​​​​​​In one embodiment, the fermentation medium comprises 30 g / L glycerol, 6.725 g / L potassium dihydrogen phosphate, 1.4 g / L magnesium sulfate heptahydrate, 2.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid monohydrate, 5 g / L industrial yeast extract, 2.5 g / L bone peptone, and 10 mL / L trace metal ion solution; wherein the trace metal ion solution comprises 10.0 g / L magnesium sulfate heptahydrate, 0.5 g / L manganese sulfate tetrahydrate, 0.1 g / L ammonium molybdate, 3.0 g / L copper sulfate pentahydrate, 2.25 g / L zinc sulfate heptahydrate, and 0.23 g / L sodium borate.

[0024] The present invention also provides the use of the α-1,3-fucosyltransferase mutant, the genetically engineered bacteria, or the method in the preparation of lactoyl-N-difucotetrasaccharide or products containing lactoyl-N-difucotetrasaccharide.

[0025] Beneficial effects: (1) This invention, through the application of... Bacillus fengqiuensis By rationally and semi-rationally designing α-1,3-fucosyltransferases derived from this source, and combining them with a high-throughput screening platform for reducing sugars using the DNS colorimetric method, a series of mutants with enhanced catalytic activity were constructed.

[0026] (2) This invention combines the regulation of GDP-1-fucose pathway genes in Escherichia coli ( manB Phosphomannanotropic enzyme gene manC mannose-1-phosphate guanosine transferase gene, gmd GDP-l-mannose-4,6-dehydrogenase gene and wcaG The GDP-l-fucosylate synthase gene was used, and based on this, α-1,2-fucosylate transferase FutC and the constructed mutant MM5 (F22Y / N25K / T98K / E99Q / N276D) were heterologously expressed, resulting in a DFL yield of 9.9 g / L from shake-flask fermentation.

[0027] (3) The genetically engineered bacteria constructed in this application were fermented in batches in a 3-L fermenter. The yield of lactoyl-N-difucotetrasaccharide reached 65.12 g / L, with a yield of 0.83 g / L / h, which is the highest yield reported so far. This invention lays the foundation for the industrial production of lactoyl-N-difucotetrasaccharide. Attached Figure Description

[0028] Figure 1 This is a metabolic pathway diagram of lactyl-N-difucotetrasaccharide.

[0029] Figure 2 Comparison of DFL yields between BfFucT single-point and multi-point mutants based on rational and semi-rational designs.

[0030] Figure 3 This document presents a flowchart and results for screening high-performance BfFucT mutants based on a high-throughput screening strategy.

[0031] Figure 4 The feed-by-batch fermentation curves of strain BZ26 in a 3-L fermenter are shown. Detailed Implementation

[0032] The specific implementation of the present invention will be further described below with reference to examples and accompanying drawings.

[0033] The plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products, and the specific operations are performed according to the kit instructions; the embodiments of the present invention are not limited thereto, and other experimental operations and process parameters not specified are performed according to conventional techniques.

[0034] (1) Strains and vectors Vectors pRSFDuet-1 and pETDuet-1 were purchased from Addgene.

[0035] The sequencing of DNA products and plasmids was completed by Suzhou Genewise Biotechnology Co., Ltd. and Beijing Qingke Biotechnology (Suzhou) Co., Ltd.

[0036] (2) Culture medium LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0037] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar powder.

[0038] Fermentation medium: glycerol 30 g / L, potassium dihydrogen phosphate 6.725 g / L, magnesium sulfate heptahydrate 1.4 g / L, diammonium hydrogen phosphate 2.0 g / L, citric acid monohydrate 1.7 g / L, industrial yeast extract 5 g / L, bone peptone 2.5 g / L, trace metal ion solution 10 mL / L (magnesium sulfate heptahydrate 10.0 g / L, manganese sulfate tetrahydrate 0.5 g / L, ammonium molybdate 0.1 g / L, copper sulfate pentahydrate 3.0 g / L, zinc sulfate heptahydrate 2.25 g / L, sodium borate 0.23 g / L), pH 7.2.

[0039] (3) Shake flask fermentation conditions: A single colony of the engineered bacteria was inoculated into LB liquid medium and cultured in a shake flask at 37℃ and 200 rpm for 12 h to obtain a seed culture; the seed culture was inoculated into 50 mL of fermentation medium at an inoculation rate of 1% (v / v) and cultured in a shake flask at 37℃ and 200 rpm until the OD600 was 0.6-0.8; isopropyl-β-D-thiopyranogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and lactose was added to a final concentration of 5 g / L. The culture was induced at 25℃ and 200 rpm for 72 h to obtain the fermentation broth. The content of lactyl-N-difucotetraose was determined by HPLC.

[0040] (4) Determination of lactyl-N-difucotetraose: 1 mL of fermentation broth was boiled at 100℃ for 15 min, centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm membrane. The amount of lactyl-N-difucotetraose and 2'-fucosylated lactose, as well as the amount of lactose and glycerol consumed, were determined by HPLC. HPLC detection conditions: differential refractive index detector; chromatographic column: Rezex ROA-organic acid (Phenomenex, USA); column temperature: 50℃; mobile phase: 5 mmol / L H2SO4 aqueous solution; flow rate: 0.5 mL / min; injection volume: 10 μL.

[0041] Example 1: De novo synthesis of lactyl-N-difucotetraose and module-optimized gene expression Using Escherichia coli BWLAI (genotype BL21 (DE3) Δ nudD Δ lacZ Δ nudK Δ wcaJ Δ clpYQ Δ Lon Δ lacA Δ iclR It has been published in the paper "High-Yield Synthesis of 2′-Fucosyllactose from Glycerol and Glucose in Engineered..." Escherichia coli The BL21 (DE3) Δ chassis cells were constructed by knocking out the poxB gene, as described in the paper (e.g., Genbank: NZ_CP053602.1, pp. 915333-917051). nudD Δ lacZ Δ nudK Δ wcaJ Δ clpYQ Δ Lon Δ lacA Δ iclR (abbreviated as BZ), according to the metabolic pathway diagram (Figure 1 The pathway genes were expressed using a dual-plasmid system. The specific steps for constructing the recombinant plasmid and strain are as follows (the primer sequences involved are shown in Table 1): Obtaining manB, manC, gmd, and wcaG fragments: Using the genome of Escherichia coli K12 as a template, PCR amplification was performed using primers manCB-F / R and gmd-wcaG-F / R. The DNA was recovered by gel extraction to obtain the manC-manB (nucleotide sequence as shown in SEQ ID NO. 10) and gmd-wcaG gene fragments (nucleotide sequence as shown in SEQ ID NO. 11).

[0042] Using pRSFDuet-1 as a template, the vector backbone sequence was amplified using primers CB-F / R and GW-F / R. The manC-manB and gmd-wcaG gene fragments were ligated into the MCS1 and MCS2 regions of the vector pRSFDuet-1, respectively, using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.). Positive clones were screened and sequenced, ultimately yielding the recombinant plasmid pRSF-CBGW.

[0043] Obtaining the FutC, BfFucT genes and the gsk fragment: The FutC gene sequence (SEQ ID NO.1) from Helicobacter pylori UA 802 and the BfFucT gene sequence (SEQ ID NO.2) from Bacillus cereus were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. FutC and BfFucT were amplified by PCR using primers pET-FutC-F / R and BfFucT-FutC-F / R, respectively. DNA was recovered from the gel to obtain the FutC and BfFucT gene fragments. Using the genome of Escherichia coli K12 as a template, PCR amplification was performed using primer pET-Gsk-F / R. DNA was recovered from the gel to obtain the Gsk gene fragment (nucleotide sequence shown in SEQ ID NO.12).

[0044] Gsk-BfFucT fragment acquisition: Gsk and BfFucT were amplified by PCR using primers pET-Gsk-F and BfFucT-FutC-R, and the DNA was recovered by gel extraction to obtain the Gsk-BfFucT gene fragment.

[0045] Using pETDuet-1 as a template, the vector backbone sequence was amplified using pET-F / R. The Gsk-BfFucT and FutC gene fragments were ligated into the MCS1 and MCS2 regions of the pETDuet-1 vector, respectively, using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.). Positive clones were screened and sequenced, ultimately yielding the recombinant plasmid pET-Gsk-BfFucT-FutC.

[0046] Table 1 Primers for plasmid construction

[0047] The recombinant plasmids pRSF-CBGW and pET-Gsk-BfFucT-FutC were transformed into the chassis strain BZ to construct the recombinant strain BZ01. The recombinant strain BZ01 was inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 12 h in a shake flask to obtain the seed culture. The seed culture was then inoculated into 50 mL of fermentation medium at a 1% (v / v) inoculation rate and cultured at 37°C and 200 rpm until OD (dose retardation). 600 The concentration was 0.6-0.8; isopropyl-β-D-thiopyranogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and lactose was added to a final concentration of 10 g / L. The mixture was induced and cultured at 25℃ and 200 rpm for 72 h to obtain the fermentation broth. The content of lactyl-N-difucotetraose in the fermentation broth was determined to be 8.03 g / L.

[0048] Example 2: BfFucT mutants based on rational and semi-rational design To explore more information to support rational design and modification, the protein structures of published α-1,3-fucosyltransferases were studied. Starting with the sequence shown in SEQ ID NO.2, several single sites (N25K, K32D, V38D, D65N, N86D, L96N, Y97E, E99Q, E117R, Y139N, S159G, N181S, S210A, N216K, N239K, I247M, and N276D) and combinatorial mutants (N25K / N86D, N25K / E99Q, N25K / N216K, N25K / I247M, N25K / N276D, N25K / E99Q / N276D) were selected and applied to the fermentation production of lactyl-N-difucotetraose. Based on the recombinant plasmid pET-Gsk-BfFucT-FutC constructed in Example 1, mutants were constructed. The specific process and primers used are shown in Table 2.

[0049] Taking the N25K mutant as an example, the construction method is as follows: Using the recombinant plasmid pET-Gsk-BfFucT-FutC constructed in Example 1 as a template, and using N25K-F / R as upstream and downstream primers, the expression plasmid was linearized and amplified by reverse PCR. Then, the template plasmid was digested with DpnI enzyme to remove excess circular template. It was then transformed into JM109 and cultured overnight. Single clones were picked from the plate and transferred to 10 mL of LB medium for expansion culture. The plasmid was then extracted and sequenced. The successfully constructed plasmid was named pET-Gsk-N25K-FutC. The plasmid templates for the other single-point mutants are all N25K. The combined mutants V1, V2, V3, V4, V5, and V6 are based on the single-point mutants with superposition mutations, and the template is N25K. The final single-point mutant plasmids obtained are pET-Gsk-N25K-FutC, pET-Gsk-K32D-FutC, pET-Gsk-V38D-FutC, pET-Gsk-D65N-FutC, pET-Gsk-N86D-FutC, pET-Gsk-L96N-FutC, pET-Gsk-Y97E-FutC, pET-Gsk-E99Q-FutC, pET-Gsk-E117R-FutC, pET-Gsk-Y139N-FutC, pET-Gsk-S159G-FutC, pET-Gsk-N181S-FutC, and pET-Gsk-S2. The plasmids obtained from pET-Gsk-N216K-FutC, pET-Gsk-N239K-FutC, pET-Gsk-I247M-FutC, and pET-Gsk-N276D-FutC were pET-Gsk-N25K / N86D-FutC, pET-Gsk-N25K / E99Q-FutC, pET-Gsk-N25K / N216K-FutC, pET-Gsk-N25K / I247M-FutC, pET-Gsk-N25K / N276D-FutC, and pET-Gsk-N25K / E99Q / N276D-FutC, respectively.

[0050] Table 2 Primer sequences for the α-1,3-fucosyltransferase BfFucT mutant The mutant-containing plasmid and the recombinant plasmid pRSF-CBGW, successfully constructed in the above steps, were simultaneously transformed into the chassis strain BZ. The resulting engineered strains are shown in Table 3. Fermentation was carried out using the same method as in Example 1, and the DFL yield in the fermentation broth was detected. The results are shown in Table 3. Figure 2 As shown, strain BZ24 produced 9.48 g / L of DFL after 72 h of fermentation, which is 18.05% higher than that of strain BZ01.

[0051] Table 3. Detailed information on engineered bacteria with different α-1,3-fucosyltransferase BfFucT mutants.

[0052] Example 3: A high-throughput screening method for reducing sugar determination based on DNS colorimetry The fact that the fucosyltransferase-catalyzed reaction in the de novo DFL synthesis pathway does not directly produce fluorescence or light absorption signals makes high-throughput screening of this enzyme mutant library difficult. Therefore, to solve this problem, the principle of the DNS colorimetric method for measuring reducing sugars was learned through research, and its application was attempted to screen high-yielding DFL strains to establish a high-throughput screening method. The specific steps are as follows (the primer sequences involved are shown in Table 4): Based on the sequence shown in SEQ ID NO.2, its structure was simulated using AlphaFold 3 software. Combined with predictions from the HotSpotWizard website, six residue sites (F22, L26, F91, F95, Y97, and T98) near the catalytic active site of BfFucT were selected.

[0053] Using the recombinant plasmid pET-Gsk-N25K / E99Q / N276D containing the mutant N25K / E99Q / N276D constructed in Example 2 as a template, reverse PCR amplification was performed using degenerate primers containing the NNK codon. The amplified product was digested with the restriction endonuclease Dpn I to remove excess circular plasmids. The plasmid was then transformed into JM109 cells and cultured overnight. The cells on the plate were washed with LB liquid and transferred to a 1.5 mL centrifuge tube for plasmid extraction, yielding a mixed plasmid library, which was the mutant plasmid library. This library was transformed into competent cells of strain BZ and cultured overnight.

[0054] Table 4 Primer sequences for the α-1,3-fucosyltransferase BfFucT mutant library

[0055] Single clones from the plate were transferred to a 96-well plate containing four control strains BZ24 and cultured at 37°C and 750 rpm in a 96-well plate vortex incubator for 12 h. Subsequently, 60 μL of seed culture was transferred to a 96-well deep-well plate containing 600 μL of fermentation medium, sealed with sterile gauze, and cultured at 25°C and 750 rpm in a 96-well plate vortex incubator for 72 h.

[0056] After culturing, the 96-well plate was boiled to inactivate the enzyme and centrifuged at 4000 r / min for 20 min. First, the lactase and buffer were mixed in the correct proportion, and 380 μL was transferred to another sterile deep-well plate. Then, 20 μL of fermentation supernatant was added, and the mixture was rinsed and incubated at room temperature. 600 μL of DNS was added, and the mixture was boiled for 7 min to develop color. 1 mL of deionized water was added and mixed well. 200 μL of the reaction solution was then transferred to an ELISA plate, and the absorbance of the reaction solution at 540 nm was measured using a multi-mode microplate reader.

[0057] Strains with absorbance values ​​lower than the control strain were selected and subjected to shake-flask fermentation verification according to the method in Example 1. This yielded strain BZ25, which expressed the dominant mutant (BfFucT-N25K / T98K / E99Q / N276D), and its fermentation produced a DFL yield of 9.67 g / L. Figure 3 ).

[0058] Using the constructed recombinant plasmid pET-Gsk-N25K / T98K / E99Q / N276D-FutC as a template, a mutant library was prepared again. The mutant library was fermented according to the method in Example 1, with strain BZ25 as a control. The above steps were repeated to finally obtain the dominant mutant MM5 (F22Y / N25K / T98K / E99Q / N276D). The DFL production yield of strain BZ26 expressing this mutant was 9.9 g / L, showing a 1.23-fold improvement in DFL biosynthesis compared to the initial wild-type strain.

[0059] Example 4: Production of lactyl-N-difucotetrasaccharide by fed-batch fermentation in a 3-L fermenter The recombinant strain BZ26 constructed in Example 3 was subjected to high-density fed-batch fermentation in a 3-L fermenter.

[0060] Fermentation conditions: The glycerol bacteria stored at -80℃ were thawed on ice. A small amount of bacterial culture was streaked onto a solid LB agar plate (ampicillin 100 μg / mL, kanamycin 50 μg / mL) using an inoculation loop and incubated overnight at 37℃. A single colony from the plate was inoculated into 10 mL of LB liquid medium and incubated at 37℃ and 200 rpm for 12 h. 200 μL of the bacterial culture was inoculated into 100 mL of LB liquid medium and incubated at 37℃ and 200 rpm for 10 h to obtain the seed culture. A 3-L fermenter containing 1-L of fermentation medium (initial glycerol concentration 30 g / L) was used for bacterial culture at an inoculation rate of 10% (v / v). The pre-induction culture temperature was set at 37℃ until OD was reached. 600Once the pH reaches 10-15, IPTG at a final concentration of 0.2 mmol / L is added to induce protein expression, along with lactose at a final concentration of 10 g / L. The fermentation temperature is 25°C. Ammonia is used to control the pH of the tank throughout the fermentation process. To maintain cell growth and the synthesis of fucoidan-lactose, 600 g / L of glycerol (containing 22 g / L MgSO4·7H2O, 5.76 g / L industrial yeast extract, and 2.88 g / L pork bone peptone) is added after the initial glycerol is consumed to supplement the carbon source. The concentration of glycerol in the fermentation system is maintained at a low level (glycerol is used for cell growth and metabolism, and the concentration is approximately 0 g / L, i.e., just consumed by the strain) through pH feedback adjustment until the end of fermentation. After the initial lactose is consumed, 200 g / L of lactose is added to maintain its final concentration in the fermentation system at around 10 g / L. If the lactose concentration drops to a low level during fermentation, lactose is added again until the end of fermentation. The system is cascaded and controlled during fermentation. By adjusting the rotation speed, aeration rate and oxygen, the dissolved oxygen in the tank is kept at 30% ± 5%.

[0061] Sampling was performed regularly throughout the fermentation process, and cell OD was measured. 600 1 mL of fermentation broth was centrifuged at 12000 r / min for 5 min, and the supernatant was boiled for 15 min to completely lyse the cells. The supernatant was then centrifuged at 12000 r / min for 5 min and filtered through a 0.22 μm membrane. During fermentation, the production of DFL and the consumption of lactose and glycerol were detected by HPLC. The results showed that the concentration of DFL in the product reached 65.12 g / L after 78 hours of fermentation. Figure 4 ).

[0062] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A mutant of α-1,3-fucosyltransferase with improved catalytic activity, characterized in that, Based on the amino acid sequence shown in SEQ ID NO. 2, one or more mutations in the following positions: N25K, N216K, E99Q, N276D.

2. The mutant according to claim 1, wherein Based on the amino acid sequence shown in SEQ ID NO. 2, one or more mutations in the following positions: N25K, N216K, E99Q, N276D. (a) mutating the asparagine at position 25 to lysine; (b) mutating the asparagine at position 216 to lysine; (c) mutating the asparagine at position 25 to lysine and mutating the glutamic acid at position 99 to glutamine; (d) mutating the asparagine at position 25 to lysine and mutating the asparagine at position 276 to aspartic acid; (e) mutating the asparagine at position 25 to lysine, mutating the glutamic acid at position 99 to glutamine, and mutating the asparagine at position 276 to aspartic acid; (f) mutating the asparagine at position 25 to lysine, mutating the threonine at position 98 to lysine, mutating the glutamic acid at position 99 to glutamine, and mutating the asparagine at position 276 to aspartic acid; (g) mutating the phenylalanine at position 22 to tyrosine, mutating the asparagine at position 25 to lysine, mutating the threonine at position 98 to lysine, mutating the glutamic acid at position 99 to glutamine, and mutating the asparagine at position 276 to aspartic acid.

3. A gene encoding the mutant of claim 1 or 2.

4. A genetically engineered bacterium expressing the mutant of claim 1 or 2.

5. The genetically engineered bacteria according to claim 4, characterized in that, expressing phosphomannomutase manB, mannose-1-phosphate guanosyltransferase manC, GDP-l-mannose-4,6-dehydratase gmd, GDP-l-fucose synthase wcaG, α-1,2-fucosyltransferase FutC, and the mutant of α-1,3-fucosyltransferase of claim 1. 6.The genetically engineered bacterium of claim 5, characterized in that, FutC and the a-1,3-fucosyltransferase mutant were expressed free using the plasmid pRSFDuet-1 manB , manC , gmd and wcaG ; FutC and the a-1,3-fucosyltransferase mutant were expressed free using the plasmid pETDuet-1.

7. The genetically engineered bacteria according to any one of claims 4 to 6, characterized in that, Expression initiated using a strong promoter manB , manC , gmd and wcaG Expression initiated using a strong promoter; the strong promoter includes but is not limited to T7 promoter.

8. Process for the preparation of lacto-N-difucohexaose, characterized in that, The genetically engineered bacterium of any one of claims 4-7 is cultured in a medium with glycerol as the carbon source.

9. The method of claim 8, wherein, IPTG is added during the culturing process, and lactose is added.

10. Use of the mutant of α-1, 3-fucosyltransferase of claim 1 or 2, or the genetically engineered bacterium of any one of claims 4-7, or the method of any one of claims 8-9 in the preparation of lacto-N-difuco-tetraose or a product containing lacto-N-difuco-tetraose.

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