CN114990037B

CN114990037B - Construction method and application of recombinant escherichia coli for high-yield lactoyl-N-tetraose

Info

Publication number: CN114990037B
Application number: CN202210573394.4A
Authority: CN
Inventors: 沐万孟; 张文立; 李泽宇; 朱莺莺
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2024-03-26
Anticipated expiration: 2042-05-24
Also published as: CN114990037A

Abstract

The invention provides a construction method and application of recombinant escherichia coli for high-yield lactoyl-N-tetraose, and belongs to the technical field of microbial metabolic engineering. The invention takes the recombinant escherichia coli with high yield of lactoyl-N-trisaccharide II obtained in the previous study as an original strain, integrates single copy galE on the escherichia coli genome, optimizes expression of lgtA, pF and galE through plasmid combination, knocks out ugD on the escherichia coli host genome through a CRISPR-Cas9 gene editing system to relieve metabolic diversion of intermediate products, simultaneously strengthens regeneration and circulation of cofactors in the product synthesis process, further improves the yield of lactoyl-N-tetrasaccharide, has the capacity of producing the lactoyl-N-tetrasaccharide of 6.16g/L in the shake flask fermentation experiment process, has the yield of 57.5g/L of the lactoyl-N-tetrasaccharide in a 5L fermentation tank, and lays a foundation for further industrialized production of the lactoyl-N-tetrasaccharide.

Description

Construction method and application of recombinant escherichia coli for high-yield lactoyl-N-tetraose

Technical Field

The invention relates to a construction method and application of recombinant escherichia coli for high-yield lactoyl-N-tetraose, and belongs to the technical field of microbial metabolic engineering.

Background

Breast milk is considered to be the best source of nutrients to promote infant development, and breast milk oligosaccharides (Human milk oligosaccharides, HMOs) are the third largest solid nutritional component in breast milk other than lactose and lipids, which provide unique health benefits to infants that conventional formula cannot provide. Studies show that the breast milk oligosaccharide plays an important physiological function in the aspects of intestinal probiotics growth, intestinal immunoregulation, antiviral protection and infant brain development. In breast milk, more than 200 different structures of HMOs were found, with lacto-N-tetraose being about 6% (w/w) of all HMOs, and with lacto-N-tetraose as the core unit, a number of different breast milk oligosaccharides could be further sialylated or fucosylated. Thus, efficient and economical production of lacto-N-tetraose is the focus of current research.

At present, lacto-N-tetraose can be synthesized by chemical methods, enzymatic methods and cell factory methods. The chemical synthesis of lactoyl-N-tetraose involves complex chemical reaction processes, stringent reaction conditions and toxic and harmful chemical reagents, which are disadvantageous for the industrial synthesis of lactoyl-N-tetraose. The enzymatic synthesis of lacto-N-tetraose requires the participation of expensive substrates and cofactors, and the conversion rate of the enzyme and the industrial scale-up limit its development. In contrast, the cell factory method has low cost of substrate, less byproducts, higher yield and easy industrialized production. Production of lacto-N-tetraose by cell factory method involves two key genes encoding beta-1, 3-acetylglucosamine transferase from Neisseria meningitidis (Neisseria meningitidis) and beta-1, 3-galactosyltransferase from E.coli O55:7 or Pseudomonas, and has been used for microbial fermentation production of lacto-N-tetraose. The German team integrates the gene (lgT A) encoding beta-1, 3-acetylglucosamine transferase and the gene (wbgO) encoding beta-1, 3-galactosyltransferase through the escherichia coli genome, optimizes the culture medium condition, strengthens the supply of UDP-galactose, and finally realizes the yield of lactoyl-N-tetraose of 12.72g/L through a fed-batch culture mode. In addition, chinese team screened novel gene encoding beta-1, 3-galactosyltransferase (encoding glycosyltransferase from Pseudomonas solafluorooxidans) to realize efficient synthesis of lactoyl-N-tetraose, and the yield reaches 25.49g/L. Compared with a chemical method and an enzyme method, the efficient synthesis of the lactoyl-N-tetraose by a cell factory method is safer and more efficient, but currently, aiming at the synthesis of the lactoyl-N-tetraose, the adopted strategy technology is single, the regulation and control balance of metabolic flow in the synthesis process of a target product are not concerned, and different strategies are adopted to further improve the yield of the lactoyl-N-tetraose, so that the reasonable transformation of strains is realized, and the purpose of industrialized mass production is realized.

Disclosure of Invention

In order to solve the related problems existing at present, the invention integrates a single copy of uridine diphosphate-glucose-4-epimerase gene (galE) on the escherichia coli host genome, thereby strengthening the supply of UDP-galactose, optimizes the coding of beta-1, 3-acetylglucosamine transferase gene (lgtA) through plasmid combination, is derived from the expression level of pseudobulbenianiferoxadans coding beta-1, 3-galactosyltransferase gene (Pf beta 3 GalT) and coding of uridine diphosphate-glucose-4-epimerase gene (galE), so as to achieve the balance of metabolic flows, knocks out coding of uridine diphosphate-glucose dehydrogenase gene (ugD) on the escherichia coli host genome through a CRISPR-Cas9 gene editing system, relieves the metabolic split of auxiliary products, and simultaneously strengthens the regeneration and circulation of factors in the product synthesis process, and further improves the yield of lactoyl-N-tetraose.

A first object of the present invention is to provide a recombinant E.coli producing lactoyl-N-tetraose, knocking out genes related to catabolism of substrate lactose and key intermediates in the E.coli genome, integrating gene galE encoding uridine diphosphate-glucose-4-epimerase at recA gene of E.coli genome, heterologously expressing gene lgtA encoding beta-1, 3-acetylglucosamine transferase and gene Pf beta 3GalT encoding beta-1, 3-galactosyltransferase.

In one embodiment, the E.coli includes, but is not limited to E.coli BL21 (DE 3).

In one embodiment, the galE gene, which is initiated using tac, is integrated at the recA gene, genBank number ACT44368.1 of which recA gene.

In one embodiment, the gene wecB encoding UDP N acetylglucosamine 2 epimerase, the gene nagB encoding glucosamine 6 phosphate deaminase, and the gene lacZ encoding β -galactosidase, the gene ugD encoding uridine diphosphate-glucose dehydrogenase are knocked out.

In one embodiment, the UDPN acetylglucosamine 2 epimerase WecB has NCBI sequence No. yp_026253.1, glucosamine 6 phosphate deaminase NagB has NCBI sequence No. np_415204.1, and beta galactosidase LacZ has NCBI sequence No. np_414878.1.

In one embodiment, lgtA is expressed using the pETDuet-1 plasmid and pF is expressed using the pACYCDuet-1 plasmid.

Preferably, galE is expressed simultaneously with pF expression using plasmid pACYCDuet-1, on the basis of lgtA expression using pET plasmid.

Preferably, the gene lgtA encoding a β -1, 3-acetylglucosamine transferase is derived from neisseria meningitidis (Neisseria meningitidis) and the gene pF encoding a β -1, 3-galactosyltransferase is derived from pseudobulbenk niaaforoxydans.

Preferably, the nucleotide sequence of the gene lgtA for encoding the beta-1, 3-acetylglucosamine transferase is shown in SEQ ID NO. 1.

Preferably, the nucleotide sequence of the gene pF encoding the beta-1, 3-galactosyltransferase is shown in SEQ ID NO. 2.

Preferably, the nucleotide sequence of the gene galE encoding uridine diphosphate-glucose-4-epimerase is shown in SEQ ID NO. 3.

In one embodiment, the expression of udK, ndK, pyrF or pyrH is enhanced, or both udK and ndK are enhanced, or both udK-pyrF are enhanced, or both udK and pyrH are enhanced, or both pF and galE are enhanced, or both udK and pyrF are enhanced.

In one embodiment, the enhanced expression of the above gene udK, ndK, pyrF, pyrH is achieved using the pCOLADuet-1 plasmid.

In one embodiment, genBanK No. udK: ACT43823.1; genBank number of ndK: ACT44230.1; genBank accession number of pyrF: ACT43146.1; genBank number of pyrH: ACT42070.1.

Preferably, the expression of gene udK encoding guanosine kinase and gene pyrF of guanosine 5' -phosphate decarboxylase is enhanced.

More preferably, gene udK encoding guanosine kinase and gene pyrF encoding guanosine 5' -phosphate decarboxylase are integrated on the pCOLADuet-1 plasmid to achieve expression of udK and pyrF.

Preferably, the nucleotide sequence of the gene udK encoding guanosine kinase is shown as SEQ ID NO. 4.

Preferably, the nucleotide sequence of the gene pyrF encoding guanosine 5' -phosphate decarboxylase is shown in SEQ ID NO. 5.

Preferably, the nucleotide sequence of pyrH is shown in SEQ ID NO. 6.

Preferably, the nucleotide sequence of ndK is shown in SEQ ID NO. 7.

In one embodiment, the glgC, rffH, yihX, nrdD or umpH is knocked out on the basis of the knockdown ugD.

In one embodiment, genBank No. ugD: ACT43781.1; genBank No. of the glgC: ACT45084.2; genBank number of the rffH: ACT45460.1; genBank number of the yihX: ACT45563.1; genBank number of nrdD: ACT42508.1; genBank number of the umpH: ACT45894.1.

In one embodiment, the expression vectors pETDuet-1 and pACYCDuet-1 are utilized; the expression vector pETDuet-1 contains a gene (lgtA) for encoding beta-1, 3-acetylglucosamine transferase; the expression vector pACYCDuet-1 contains a gene (pF) encoding a beta-1, 3-galactosyltransferase and a gene (galE) encoding a uridine diphosphate-glucose-4-epimerase.

The invention provides a method for improving the production capacity of recombinant escherichia coli lactoyl-N-tetraose, which comprises the following steps of containing expression vectors pETDuet-1, pACYCDuet-1 and pCOLADuet-1 in escherichia coli; the expression vector pETDuet-1 contains a gene lgtA for encoding beta-1, 3-acetylglucosamine transferase; the expression vector pACYCDuet-1 contains a gene pF for encoding beta-1, 3-galactosyltransferase and a gene galE for encoding uridine diphosphate-glucose-4-epimerase; the expression vector pCOLADuet-1 contains a gene udK encoding guanosine kinase and a gene pyrF encoding guanosine 5' -phosphate decarboxylase.

The invention provides a method for producing lactoyl-N-tetraose, which utilizes recombinant escherichia coli as a microbial fermentation strain, takes glycerol as a carbon source and takes lactose as a substrate to synthesize the lactoyl-N-tetraose.

Culturing the recombinant escherichia coli in a shake flask system containing a DM culture medium until the recombinant escherichia coli reaches a thallus OD ₆₀₀ Reaching 0.6-0.8, adding inducer IPTG to a final concentration of 0.5mM and lactose to a final concentration of 5g/L, and culturing at 25 ℃ and 200rpm for not less than 96 hours;

alternatively, the recombinant E.coli is cultured in a fermenter system containing DM medium to a cell OD ₆₀₀ When the glycerol concentration in the tank is lower than 8g/L, glycerol is added to maintain the glycerol concentration in the fermentation system at 8-15 g/L, ammonia water is added to maintain the pH value in the fermentation tank at 6.6-6.8, and dissolved oxygen is maintained at 20% -30%.

In one embodiment, the DM medium contains 15-20 g/L glycerol, 10-13.5 g/L potassium dihydrogen phosphate, 1.0-2.0 g/L citric acid, 3.0-5.0 g/L diammonium hydrogen phosphate, 1.0-2.0 g/L magnesium sulfate heptahydrate and 5-10 ml/L trace metal elements.

In one embodiment, the trace metal elements comprise 2.25g/L of zinc sulfate hydrate, 10g/L of ferrous sulfate, 0.35g/L of manganese sulfate monohydrate, 1.0g/L of anhydrous copper sulfate, 0.23g/L of sodium borate decahydrate, 2.0g/L of calcium chloride dihydrate and 0.11g/L of ammonium molybdate.

The invention provides application of the recombinant escherichia coli in the fields of medicine, food and chemical industry.

The invention provides application of the recombinant escherichia coli strain in preparation of lactoyl-N-tetraose and derivative products thereof.

The invention has the beneficial effects that:

the invention utilizes genetic engineering technology to modify an escherichia coli host, integrates single copy of uridine diphosphate-glucose-4-epimerase gene (galE) on the escherichia coli host genome, thereby strengthening the supply of UDP-galactose, optimizing the expression levels of beta-1, 3-acetylglucosamine transferase gene (lgtA), beta-1, 3-galactosyltransferase gene (pF) and nucleoside diphosphate-glucose-4-epimerase gene (galE) by plasmid combination, achieving the balance of metabolic flows in bacteria, and knocking out the uridine diphosphate-glucose dehydrogenase gene (ugD) on the escherichia coli host genome by a CRISPR-Cas9 gene editing system, relieving metabolic split of intermediate products, strengthening the regeneration and circulation of cofactors in the synthesis process of the products, and further improving the yield of lactoyl-N-tetraose. In shake flask fermentation experiments, the yield of lactoyl-N-tetraose produced by recombinant escherichia coli can reach 6.16g/L, and in 5L fermentation tank experiments, after fed-batch of the recombinant escherichia coli for 65h, the yield of lactoyl-N-tetraose can reach 57.5g/L, so that the method has good industrial application prospect.

Drawings

FIG. 1 is a diagram of the metabolic pathway of recombinant E.coli synthesized lacto-N-tetraose;

FIG. 2 is a graph showing the yield of lacto-N-tetraose after the E.coli host genome integration gene galE;

FIG. 3 is a graph of the yield of lacto-N-tetraose from metabolic pathways regulated by plasmids of different copy numbers;

FIG. 4 is a graph showing comparison of the yield of lacto-N-tetraose produced by fermentation of different knockout strains;

FIG. 5 is a graph comparing the yields of lacto-N-tetraose after strengthening the cofactor regeneration pathway;

FIG. 6 is a graph of the yield of lacto-N-tetraose fed-batch to a 5L fermenter.

Detailed Description

The invention is further illustrated by the following specific examples of implementation.

All commercial products such as plasmids, endonucleases, PCR enzymes, column type plasmid extraction cassettes and DNA gel recovery kits are used, and specific operations are carried out according to the instruction of the kit.

Routine Molecular biology experimental procedures such as colony PCR, heat shock transformation, nucleic acid agarose gel electrophoresis, competent cell preparation, and bacterial genome extraction were performed according to Molecular cloning: ALaboratory Manua (fourier Edition).

Sequencing of the plasmid and DNA products was done by the company Anshengda, suzhou.

E.coli competent preparation: TAKARA kit

LB solid medium: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 15g/L of agar powder.

LB liquid medium: 5g/L of yeast extract, 10g/L of peptone and 10g/L of sodium chloride.

Fermentation medium (DM): 20g/L of glycerin, 13.5g/L of monopotassium phosphate, 1.7g/L of citric acid, 4.0g/L of diammonium phosphate, 1.4g/L of magnesium sulfate heptahydrate and 10ml/L of trace metal elements, and adjusting the pH to 6.8 by using sodium hydroxide.

Trace metal elements: zinc sulfate heptahydrate 2.25g/L, ferrous sulfate 10g/L, manganese sulfate monohydrate 0.35g/L, anhydrous copper sulfate 1.0g/L, sodium borate decahydrate 0.23g/L, calcium chloride dihydrate 2.0g/L, ammonium molybdate 0.11g/L, and dissolved in 5M hydrochloric acid.

Fed-batch fermentation feed liquid: (1) feeding a carbon source: 600g/L glycerin, 0.2g/L thiamine and 20g/L magnesium sulfate heptahydrate; (2) pH adjustment: 14% aqueous ammonia (v/v).

Antibiotic concentration: 50mg/L kanamycin, 200mg/L ampicillin (solid medium), 100mg/L ampicillin (liquid medium), 50mg/L chloramphenicol, 50mg/L spectinomycin, and 50mg/L streptomycin.

Concentration of inducer: isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the shake flask at a final concentration of 0.5mM, the fermentation tank is fed in batches at a final concentration of 0.2mM, and the final concentration is 1mM when the relevant gene is knocked out by the CRISPR-Cas9 gene editing system, namely pTarget-T plasmid is removed. The final concentration of L-arabinose addition was 30mM.

Strain culture and fermentation:

single colonies with normal colony morphology are selected from the corresponding flat plates and added into 4mL test tube LB culture medium added with corresponding antibiotics, after overnight culture for 10-12 hours, 0.4mL of culture seed liquid is inoculated into 20mL shaking flask DM culture medium added with the corresponding antibiotics, and the culture conditions are 37 ℃,200rpm and 250mL triangular flask. When the thallus OD ₆₀₀ When reaching 0.6 to 0.8, adding the inducer IPTG to the final concentration of 0.5mM and simultaneously adding the bottomAfter the addition, the induction temperature is changed to 25 ℃ and the rotation speed of a shaking table is unchanged, and after 96 hours of induction, the fermentation broth is collected and a sample is prepared for the yield of lactoyl-N-tetraose.

The upper tank fermentation is carried out in a 5L fermentation tank, the initial DM culture medium is 2L, the seed liquid inoculation amount is 10% (v/v), the initial temperature is 37 ℃, and the culture medium is used as the bacterial OD in the tank ₆₀₀ When the glycerol grows to 12-15, the fermentation tank is cooled to the induction temperature of 25 ℃ in a gradient way, an inducer IPTG is added to the final concentration of 0.2mM and the lactose as a reaction substrate is added to the final concentration of 10g/L, and when the glycerol concentration in the tank is consumed to be lower than about 8g/L, the glycerol is added by a constant flow pump to carry out feeding, so that the glycerol concentration in a reaction system is maintained at 8-15 g/L. The pH in the tank is maintained within the range of 6.6 to 6.8 by supplementing 14% ammonia water (v/v) through a constant flow pump, and an antifoaming agent is added to control the amount of foam in the tank, and the stirring speed (300-900 rpm) and the aeration rate (2-6 vvm) are related to the dissolved oxygen value, so that the dissolved oxygen is maintained at a level of 20% -30%.

Yield detection of lacto-N-tetraose:

to examine the yield of lactoyl-N-tetraose in the fermentation broth, the fermentation broth was centrifuged at 10000g for 15min, and the supernatant was collected and filtered with a 0.22 μm aqueous needle filter. The diluted collected supernatant was subjected to High Performance Anion Exchange Chromatography (HPAEC) under conditions of CarboPac PA10 column (4 mm×250 mm) and pulse amperometric detector, and was analyzed by linear gradient elution with 3 mobile phases as eluent a: ultrapure water; eluent B:1M sodium acetate; eluent C:250mm NaOH; the flow rate of elution is 1.0mL/min; sample injection amount: 20. Mu.L.

TABLE 1 primer sequences

TABLE 2 PCR System

PCR reaction system conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 2kb/min; and (3) 32 cycles, preserving at 4 ℃, and performing gel recovery on the PCR product.

TABLE 3 Gibbsen packaging reaction System

Gibbsen assembly reaction conditions: 50℃for 30min.

X/Y is calculated according to the formula to obtain the linearization vector amount and the insert amount:

x= (0.02 Xcloning vector base pair number/gel recovery corresponding DNA fragment concentration) ng,

y= (0.02×insert base pair number/gel recovery corresponding DNA fragment concentration) ng.

TABLE 4 overlap extension PCR System

Overlap extension PCR reaction system conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 2kb/min; and (3) performing 32 cycles, preserving at 4 ℃, taking part of PCR products for performing nucleic acid electrophoresis verification, then performing enzyme digestion by using DPN1 to remove template plasmids, and performing transformation verification.

TABLE 5 knockout related primer sequence listing

TABLE 6 Gene targeting plasmid information

Example 1: construction of recombinant E.coli integrating gene galE

Carrying out PCR amplification on the pTarget-F plasmid by using a primer GalE-N20-F/R to obtain a pTarget plasmid targeting a target site;

the galE gene, i.e., its upstream and downstream homology arms and pTarger vector fragment, was amplified by PCR using primers GalE-T-F/R, galE-UP-F/R, galE-Down-F/R and GalE-T-V-F/R, respectively;

assembling the amplified four fragments by using a Gibbsen assembly kit, thereby constructing a plasmid pTarget-galE for integrating genes;

the pCas plasmid was transferred into a starting host e.coli BL21 (DE 3) Δwecb Δnagb Δlacz by means of electric shock transformation (knockout method of gene wecB, nagB, lacZ is disclosed in CN111979168 a) and 30mM arabinose was added to induce expression of the lambda-Red recombinase system;

electrotransferring pTarget-galE plasmid into the escherichia coli containing pCas, coating the plasmid on an LB plate containing spectinomycin and kanamycin, culturing for 15-24 hours at 30 ℃, and performing colony PCR verification on single colonies on the plate;

after colony PCR verification is successful, sample sending and sequencing are carried out to verify whether the integrated genes of related genomes are complete, and related sequencing work is responsible for the related genes by the Suzhou Anshengda company;

transferring the single colony which is verified to be successful into a 4mL LB liquid test tube, adding 0.1mM IPTG to induce removal of pTarget plasmid, culturing overnight by streaking part of bacterial liquid after shaking overnight, and then verifying whether the pTarget plasmid is removed;

selecting a single colony for successfully removing the pTarget plasmid, inoculating the single colony into a non-resistant LB liquid test tube, culturing at 42 ℃ at 200rpm overnight, streaking part of bacterial liquid onto a non-resistant LB solid plate, and then selecting the single colony to verify whether pCas plasmid is removed, wherein the success is E.coli BL21 (DE 3) delta wecB delta nagB delta lacZ delta recA, namely Ptac-galE, namely EN0.

Example 2: construction of recombinant E.coli with knocked out Gene ugD, glgC, rffH, yihX, umpH and nrdD

The operational procedure for knockout gene is the same as that for integration gene, but when the pTarget plasmid is constructed, knockout plasmid pTargrt-F consists of three amplified DNA fragments: linearizing the vector, knocking out the upstream homology arm of the gene and knocking out the downstream homology arm of the gene, and referring to the integrated gene operation flow by other experimental operations, the related primers are shown in tables 5 and 6, namely:

the ugD gene of the strain EN0 was knocked out in the same manner as in example 1 to construct a strain EN1;

the glgC gene of the strain EN1 was knocked out in the same manner as in example 1 to construct a strain EN2;

the rffH gene of the strain EN1 was knocked out in the same manner as in example 1, and a strain EN3 was constructed;

knocking out yihX gene of the strain EN1 according to the method of the example 1, and constructing a strain EN4;

the umpH gene of strain EN1 was knocked out as in example 1 to construct strain EN5;

the nrdD gene of the strain EN1 was knocked out in the same manner as in example 1 to construct a strain EN6.

Example 3: construction of recombinant plasmid vector

The specific steps are as follows, the related primers are shown in Table 1, the related PCR system is shown in Table 2, the Gibbs assembly system is shown in Table 3, and the overlap extension PCR system is shown in Table 4:

the primers Pf-GalE-F/R, pf-F/R, pf-GalE-V1-F/R and Pf-GalE-V2-F/R are used for amplifying galE genes (source E.coliMG1655 genome), pF genes (source plasmid synthesized by codon optimization in Suzhou's lifting), a vector fragment 1 and a vector fragment 2 respectively through PCR, four fragments are assembled through a Gibbsen assembly kit, then are transformed into DH5 alpha, are coated on a corresponding resistant LB plate for overnight culture, then a single colony extraction plasmid is picked for sequencing, and the construction of the recombinant plasmid vector pAC-Pf-galE is completed after sequencing successfully. Amplifying related plasmid vectors by using primers Pf-HP1-F/R and Pf-HP2-FR through overlap extension PCR, then converting the amplified related plasmid vectors into DH5 alpha, coating the DH5 alpha on a LB plate with corresponding resistance for overnight culture, then picking single colony and extracting plasmids for sequencing, and obtaining recombinant plasmid vectors pCD-Pf and pAC-Pf after successful sequencing. Construction of plasmids pRSF (29) M (29) US and pET-lgT A is described in the patent publication No. CN111979168A, and construction of pCD-pf-galE is described in the patent publication No. CN 113652385A.

Example 4: construction of recombinant E.coli to enhance regeneration of UTP

The expression of genes udK, pyrF, pyrH and ndK was enhanced in E.coli. The relevant primers are shown in Table 1.

Gene udK (nucleotide sequence shown as SEQ ID NO. 4), pyrF (nucleotide sequence shown as SEQ ID NO. 5), pyrH (nucleotide sequence shown as SEQ ID NO. 6) and ndK (nucleotide sequence shown as SEQ ID NO. 7) in E.coli MG1655 were amplified by primers UdK-F/R, pyrF-F/R, pyrH-F/R and NdK-F/R and by primers UdK-V-F/R, pyrF-V-F/R, pyrH-V-F/R and NdK-V-F/R to amplify the vector fragments, followed by gel recovery and Gibbsen assembly to give recombinant plasmids pCO-ud, pCO-pyrF, pCO-pyrH and pCO-ndK. Recombinant plasmids pCO-udK-ndK, pCO-udK-pyrF and pCO-udK-pyrH were obtained by similar methods.

Transferring the obtained recombinant plasmids into corresponding hosts in different combination modes to obtain shake flask fermentation information in Table 7, namely, combination 1 (recombinant strain EN11 in Table 7): recombinant E.coli EN1 contains plasmids pET-lgtA, pAC-pf-galE and pCO-udK; combination 2 (recombinant strain EN12 in Table 7), recombinant E.coli EN1 contained plasmids pET-lgT A, pAC-pf-galE and pCO-ndK; combination 3 (recombinant strain EN13 in Table 7), recombinant E.coli EN1 contained plasmids pET-lgT A, pAC-pf-galE and pCO-pyrF; combination 4 (recombinant strain EN14 in Table 7), recombinant E.coli EN1 contained plasmids pET-lgT A, pAC-pf-galE and pCO-pyrH; combination 5 (recombinant strain EN15 in Table 7), recombinant E.coli EN1 contained plasmids pET-lgT A, pAC-pf-galE and pCO-udK-ndK; combination 6 (recombinant strain EN16 in Table 7), recombinant E.coli EN1 contained plasmids pET-lgT A, pAC-pf-galE and pCO-udK-pyrF; combination 7 (recombinant strain EN17 in Table 7), recombinant E.coli EN1 contained plasmids pET-lgT A, pAC-pf-galE and pCO-udK-pyrH; combination 8 (recombinant strain EN18 in Table 7), recombinant E.coli EN5 contained plasmids pET-lgT A and pAC-pf-galE; combination 9 (recombinant strain EN19 in Table 7), recombinant E.coli EN5 contained plasmids pET-lgT A, pAC-pf-galE and pCO-udK-pyrF; combination 10 (recombinant strain EN20 in Table 7), recombinant E.coli EN6 contained plasmids pET-lgT A and pAC-pf-galE; recombinant E.coli EN6 containing plasmids pET-lgT A, pAC-pf-galE and pCO-udK-pyrF was obtained from combination 11 (recombinant strain EN21 in Table 7). The shake flask fermentation experiment result shows that the yield of the recombinant escherichia coli EN16 lactoyl-N-tetraose of the overexpression genes udK and pyrF can reach 6.16g/L.

TABLE 7 shake flask fermentation details of recombinant E.coli

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Example 5: batch feeding production of lactoyl-N-tetraose in 5L fermenter

The optimal strain EN16 is selected for fermentation tank expansion fermentation experiments, the related experimental operation is as described in the specific embodiment, the final fermentation time is 65h, the yield of lactoyl-N-tetraose reaches 57.5g/L, and the maximum OD ₆₀₀ 94.4, the relevant fermentation process variation is shown in FIG. 6.

TABLE 8 dynamic changes in the number of cells and the synthesis of lacto-N-tetraose during fermentation

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.

SEQUENCE LISTING

<110> university of Jiangnan

<120> construction method and application of recombinant escherichia coli for high yield of lactoyl-N-tetraose

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<160> 7

<170> PatentIn version 3.3

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gaagattttg aactggcgcg tcgtttcctg taccagtgct tcaaacgtac cgataccctg 900

ccggcgggcg cttggctgga tttcgcggcg gatggccgta tgcgtcgtct gttcaccctg 960

cgtcagtact tcggtatcct gcaccgtctg ctgaaaaacc gttaa 1005

<210> 2

<211> 828

<212> DNA

<213> artificial sequence

<400> 2

atggataaaa tcaaacaggg tagcgctagc ctggttgttg gtgatcagca ggaaaaacac 60

ccggttgtta gcgttctgct gccggttaac cgtgttgatc gtttcttcat cccggcggtt 120

gaatctatcc tgacccagac cctgcaggat ttcgaactga tcattatcgc taacggttgc 180

agcaccgaac acctgaacaa aatccgtctg acctacggtg atcacaaccg cgttcgtatc 240

ctgaacaccg aaatcaaagg cctgccgttc gcgctgaacc tgggtgttca caacgcgcgt 300

ggcctgtaca tcgcgcgtat ggatgcggat gatatctcta tcccggaacg tctggaaaaa 360

cagctgaaca ccctggaaca gaacaagaaa atcggcgttg tttctagcgg tgttgatttc 420

atcgatgaaa acgatcaggc gatccgtgaa ggcaaattcc cggaactgac cgataaagat 480

caccgtcgtc tgctgccgct gatctgctgc atcgcgcacc cgaccgttat ggttcgtaaa 540

gaaatcatca acaaactggg tggttacagc ttcggcagct tcagcgaaga ttacgatctg 600

tggctgcgta tcatgcgtga actgccggaa gttgaattct accgtatccc ggaatccctg 660

ctgaaatacc gtcgtcacgg taaccaggct accagcagca aaaacatcaa aaagatccgt 720

gcgtacaact ctgcgctgaa aatccgtgaa ctgttcctga gccgtaaact gaaattcatc 780

atcggtatca tcctgccggc gcgtatggtg accctgtggc gtaaataa 828

<210> 3

<211> 1017

<212> DNA

<213> artificial sequence

<400> 3

atgagagttc tggttaccgg tggtagcggt tacattggaa gtcatacctg tgtgcaatta 60

ctgcaaaacg gtcatgatgt catcattctt gataacctct gtaacagtaa gcgcagcgta 120

ctgcctgtta tcgagcgttt aggcggcaaa catccaacgt ttgttgaagg cgatattcgt 180

aacgaagcgt tgatgaccga gatcctgcac gatcacgcta tcgacaccgt gatccacttc 240

gccgggctga aagccgtggg cgaatcggta caaaaaccgc tggaatatta cgacaacaat 300

gtcaacggca ctctgcgcct gattagcgcc atgcgcgccg ctaacgtcaa aaactttatt 360

tttagctcct ccgccaccgt ttatggcgat cagcccaaaa ttccatacgt tgaaagcttc 420

ccgaccggca caccgcaaag cccttacggc aaaagcaagc tgatggtgga acagatcctc 480

accgatctgc aaaaagccca gccggactgg agcattgccc tgctgcgcta cttcaacccg 540

gttggcgcgc atccgtcggg cgatatgggc gaagatccgc aaggcattcc gaataacctg 600

atgccataca tcgcccaggt tgctgtaggc cgtcgcgact cgctggcgat ttttggtaac 660

gattatccga ccgaagatgg tactggcgta cgcgattaca tccacgtaat ggatctggcg 720

gacggtcacg tcgtggcgat ggaaaaactg gcgaacaagc caggcgtaca catctacaac 780

ctcggcgctg gcgtaggcaa cagcgtgctg gacgtggtta atgccttcag caaagcctgc 840

ggcaaaccgg ttaattatca ttttgcaccg cgtcgcgagg gcgaccttcc ggcctactgg 900

gcggacgcca gcaaagccga ccgtgaactg aactggcgcg taacgcgcac actcgatgaa 960

atggcgcagg acacctggca ctggcagtca cgccatccac agggatatcc cgattaa 1017

<210> 4

<211> 642

<212> DNA

<213> artificial sequence

<400> 4

atgactgatc agtctcatca gtgcgtcatt atcggtatcg ctggcgcatc ggcttccggc 60

aagagtctta ttgccagtac cctttatcgt gaattacgtg agcaagtcgg tgatgaacac 120

atcggcgtaa ttcccgaaga ctgctattac aaagatcaaa gccatctgtc gatggaagaa 180

cgcgttaaga ccaactacga ccatcccagc gcgatggatc acagcctgct gcttgagcat 240

ttacaagcgt tgaaacgcgg ctcggcaatt gacctgccgg tttacagcta tgttgaacat 300

acgcgtatga aagaaacggt gacggttgag ccgaagaagg tcatcattct cgaaggcatt 360

ttgttgctga cggatgcgcg tttgcgtgac gaacttaact tctccatttt cgttgatacc 420

ccgctggata tctgcctgat gcgccgcatc aagcgtgacg ttaacgagcg tggacgttca 480

atggattcag tgatggcgca atatcaaaaa accgtgcgcc cgatgttcct gcaattcatt 540

gagccttcta aacaatatgc ggacattatc gtgccgcgcg gcgggaaaaa ccgcatcgcg 600

atcgatatat tgaaagcgaa aataagtcag ttctttgaat aa 642

<210> 5

<211> 738

<212> DNA

<213> artificial sequence

<400> 5

atgacgttaa ctgcttcatc ttcttcccgc gctgttacga attctcctgt ggttgttgcc 60

cttgattatc ataatcgtga tgacgcgctg tcctttgtcg acaagatcga cccacgcgat 120

tgtcgtctga aggtcggcaa agagatgttt acattgtttg ggccacagtt tgtgcgcgaa 180

cttcaacagc gtggttttga tatctttctt gacctgaaat tccacgatat tcctaacact 240

gcagcgcacg ctgtcgctgc tgcagctgac ttaggcgtgt ggatggtgaa tgttcatgcc 300

tctggtgggg cgcgtatgat gaccgcagcg cgtgaggcac tggttccgtt tggcaaagat 360

gcaccgcttt tgattgctgt gacagtgttg accagcatgg aagccagcga cctggtcgat 420

cttggcatga cactgtcacc tgcagattat gcagaacgtc tggcggcact gacgcaaaaa 480

tgtggccttg atggtgtggt gtgttctgct caggaagctg tgcgctttaa acaggtattc 540

ggtcaggagt tcaaactggt tacgccgggc attcgtccgc aggggagtga agctggtgac 600

cagcgccgca ttatgacgcc agaacaggcg ttgtcggctg gtgttgatta tatggtgatt 660

ggtcgcccgg taacgcaatc ggtagatcca gcgcagacgc tgaaagcgat caacgcctct 720

ttacagcgga gtgcatga 738

<210> 6

<211> 726

<212> DNA

<213> artificial sequence

<400> 6

atggctacca atgcaaaacc cgtctataaa cgcattctgc ttaagttgag tggcgaagct 60

ctgcagggca ctgaaggctt cggtattgat gcaagcatac tggatcgtat ggctcaggaa 120

atcaaagaac tggttgaact gggtattcag gttggtgtgg tgattggtgg gggtaacctg 180

ttccgtggcg ctggtctggc gaaagcgggt atgaaccgcg ttgtgggcga ccacatgggg 240

atgctggcga ccgtaatgaa cggcctggca atgcgtgatg cactgcaccg cgcctatgtg 300

aacgctcgtc tgatgtccgc tattccattg aatggcgtgt gcgacagcta cagctgggca 360

gaagctatca gcctgttgcg caacaaccgt gtggtgatcc tctccgccgg tacaggtaac 420

ccgttcttta ccaccgactc agcagcttgc ctgcgtggta tcgaaattga agccgatgtg 480

gtgctgaaag caaccaaagt tgacggcgtg tttaccgctg atccggcgaa agatccaacc 540

gcaaccatgt acgagcaact gacttacagc gaagtgctgg aaaaagagct gaaagtcatg 600

gacctggcgg ccttcacgct ggctcgtgac cataaattac cgattcgtgt tttcaatatg 660

aacaaaccgg gtgcgctgcg ccgtgtggta atgggtgaaa aagaagggac tttaatcacg 720

gaataa 726

<210> 7

<211> 432

<212> DNA

<213> artificial sequence

<400> 7

atggctattg aacgtacttt ttccatcatc aaaccgaacg cggtagcaaa aaacgtcatt 60

ggtaatatct ttgcgcgctt tgaagctgca gggttcaaaa ttgttggcac caaaatgctg 120

cacctgaccg ttgaacaggc acgtggcttt tatgctgaac acgatggaaa accgttcttt 180

gatggtctgg ttgaattcat gacctctggc ccgatcgtgg tttccgtgct ggaaggtgaa 240

aacgccgttc agcgtcaccg cgatctgctg ggcgcgacca atccggcaaa cgcactggct 300

ggtactctgc gcgctgatta cgctgacagc ctgaccgaaa acggtaccca cggttctgat 360

tccgtcgaat ctgccgctcg cgaaatcgct tatttctttg gcgaaggcga agtgtgcccg 420

cgcacccgtt aa 432

Claims

1. High-yield lactoylNRecombinant E.coli strain containing tetraose, characterized in that the gene related to catabolism of substrate lactose and key intermediate is knocked out from E.coli genomerecAIntegration of the Gene encoding uridine diphosphate-glucose-4-epimerase at the Gene, heterologous expression encodingβGenes encoding-1, 3-acetylglucosamine transferaseβ-genes for 1, 3-galactosyltransferase and uridine diphosphate-glucose-4-epimerase;

the knockout is specifically as follows: knocking out gene encoding UDP-N-acetylglucosamine-2-epimerasewecBGene encoding glucosamine-6 phosphate deaminasenagBAnd a gene encoding beta-galactosidaselacZCoded uridine diphosphate-glucose desquamationHydrogenase geneugDThe method comprises the steps of carrying out a first treatment on the surface of the The UDP-N-acetylglucosamine-2-epimerase WecB has NCBI sequence number of YP_026253.1, glucosamine-6 phosphate deaminase NagB has NCBI sequence number of NP_415204.1, beta-galactosidase LacZ has NCBI sequence number of NP_414878.1, and uridine diphosphate-glucose dehydrogenase geneugDIs ACT43781.1;

the heterologous expression is specifically as follows: expression using pETDuet-1 plasmidlgtAExpression using pACYCDuet-1 plasmidPfβ 3GalTAnd uridine diphosphate-glucose-4-epimerase; the codeβThe nucleotide sequence of the gene of the-1, 3-acetylglucosamine transferase is shown as SEQ ID NO. 1; the codeβThe nucleotide sequence of the gene of the-1, 3-galactosyltransferase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for encoding the uridine diphosphate-glucose-4-epimerase is shown as SEQ ID NO. 3;

further enhancing expression of a gene encoding guanosine kinase and a gene encoding guanosine 5' -phosphate decarboxylase in the recombinant E.coli; the nucleotide sequence of the gene for encoding guanosine kinase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene encoding guanosine 5' -phosphate decarboxylase is shown as SEQ ID NO. 5.

2. Production of lactoylNA method for synthesizing lactoyl-tetraose, characterized in that the recombinant E.coli as defined in claim 1 is used as a microorganism fermentation strain, glycerol is used as a carbon source, and lactose is used as a substrateN-tetraose.

3. The method according to claim 2, wherein the recombinant E.coli is cultured to a bacterial OD in a shake flask system containing DM medium ₆₀₀ Reaching 0.6-0.8, adding an inducer IPTG to a final concentration of 0.5mM and simultaneously adding lactose to a final concentration of 5g/L, and then culturing at 25 ℃ and 200rpm for not less than 96 hours;

alternatively, the recombinant E.coli is cultured in a fermenter system containing DM medium to a cell OD ₆₀₀ When the concentration reaches 12-15, IPTG is added to a final concentration of 0.2mM and lactose is added to a final concentration of 10g/LThe concentration of glycerol in the tank is lower than 8g/L, glycerol is added to maintain the concentration of glycerol in the fermentation system at 8-15 g/L, ammonia water is added to maintain the pH value in the fermentation tank at 6.6-6.8, dissolved oxygen is maintained at 20% -30%, and the fermentation time is not less than 34 hours.

4. The method of claim 3, wherein the DM medium contains 15-20 g/L glycerol, 10-13.5 g/L potassium dihydrogen phosphate, 1.0-2.0 g/L citric acid, 3.0-5.0 g/L diammonium hydrogen phosphate, 1.0-2.0 g/L magnesium sulfate heptahydrate, and 5-10 ml/L trace metal elements.

5. The method of claim 4, wherein the trace metal elements comprise zinc sulfate hydrate 2.25g/L, ferrous sulfate 10g/L, manganese sulfate monohydrate 0.35g/L, copper sulfate anhydrate 1.0g/L, sodium borate decahydrate 0.23g/L, calcium chloride dihydrate 2.0g/L, and ammonium molybdate 0.11g/L.

6. The recombinant E.coli of claim 1 for the production of lactoyl in the pharmaceutical, food and chemical fieldsN-the use of tetraose.