The invention relates to a method for efficiently synthesizing 2apos; genetically engineered bacterium of fucosyllactose as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for efficiently synthesizing 2 '-fucosyllactose as well as a construction method and application of the genetically engineered bacterium. According to the genetically engineered bacterium, a CRISPR-transposition technology is combined on the basis of a modular metabolic engineering modification strategy; a fucose module gene and a glycosyltransferase module gene in a 2 '-fucosyllactose synthesis route are subjected to genome integration, a multi-copy strain library for synthesizing 2'-fucosyllactose is constructed, and finally, a plurality of integrated expression strains for efficiently synthesizing 2 '-fucosyllactose are obtained. Compared with a plasmid type expression strain, the integrative expression strain is higher in yield and better in stability. By knockout or interruption of a glucokinase coding gene glk or a pyruvate oxidase coding gene poxB, the yield can be further increased, the unit cell yield of a glk interruption strain DH1W6G6glk:: VerA is 5.14 g / g DCW, and the yield of 2 '-fucosyllactose relative to lactose reaches 0.991 mol / mol which is the highest value of an integrated strain taking escherichia coli as a chassis at the present stage, so that the 2'-fucosyllactose has a good industrial prospect.

Description

Technical Field

[0001] The invention relates to a genetic engineering bacterium for efficiently synthesizing 2'-fucosyllactose and a construction method and application thereof, belonging to the field of biotechnology. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest component in breast milk after lactose and lipids, and are one of the main components that distinguish breast milk from cow's milk powder. HMOs are composed of five structural units: glucose, D-galactose, N-acetylglucosamine (GlcNAC), L-fucose, and N-acetylneuraminic acid (Neu5Ac). According to the different spatial configurations, glycosyl sequences, and chain lengths of the monosaccharide structural units, HMOs can be divided into three categories: neutral fucosyllactose, acidic sialyllactose, and neutral non-fucosylated lactose, of which fucosylated HMOs account for about 50% of the total. 2'-fucosyllactose (2'-FL) is the most widely studied type of fucosyllactose. In October 2023, the National Health Commission issued an announcement, officially approving 2'-fucosyllactose as a food nutrient fortifier, which can be used in infant formula, modified milk powder (for children), and special medical baby food, and has broad market prospects.

[0003] In recent years, microbial synthesis has become the mainstream method for the synthesis of 2'-fucosyllactose. Escherichia coli is widely used in the production of 2'-fucosyllactose because of its efficient introduction of DNA molecules, high-level expression and rapid growth. The synthesis pathway of 2'-fucosyllactose involves a variety of enzymes, and its metabolic modification usually involves regulating the co-expression of multiple genes. According to the function of the gene or the characteristics of the product, the 2'-fucosyllactose synthesis pathway can be divided into five modules: carbon source module, lactose module, fucose module, glycosyltransferase module and efflux pump module. Different modules are optimized or combined to regulate the coordinated expression of each module, which can effectively achieve a balance between cell growth and product synthesis. The literature reported so far mainly focuses on the fucose module and the glycosyltransferase module, and uses plasmids with different copy numbers to regulate the metabolic flux of each module to achieve the purpose of balancing the 2'-fucosyllactose synthesis pathway (Mol Biotechnol. 2023, 65, 1485-1497). Plasmid-based gene expression systems are commonly used tools for modifying metabolic pathways in microbial systems. However, in the process of industrial microbial production, recombinant plasmids often mutate, lose, or vary in copy number, resulting in reduced production levels. Even in the presence of antibiotic selection, 33% of cells still lost plasmids after 24 hours of induction (J Ind Microbiol Biot. 2023, 50, 1, kuad033).

[0004] Genome integration is one of the effective means to solve the instability of plasmids. In recent years, there have been reports of integrated strains producing 2'-fucosyllactose. Some of the cases with higher yields in current public publications are as follows:

[0005] (1) A genetically engineered bacterium for producing 2'-fucosyllactose and its application (Publication No.: CN116622602A), wherein one copy of the fucose module gene and one copy of the glycosyltransferase module gene were respectively integrated into the genome of Escherichia coli. The recombinant strain constructed had a production of 126.37 g / L in a 5L fermentation tank, which is the highest titer of 2'-fucosyllactose synthesized by microorganisms reported so far.

[0006] (2) Parschat et al. integrated and expressed the fucose module gene, genes related to sucrose transport, hydrolysis, and metabolism, key genes for the metabolic pathway and transport of intracellular lactose synthesis, and 2'-fucosyllactose synthesis and transport genes in the genome, creating a precedent for the synthesis of 2'-fucosyllactose using sucrose as a single carbon source. The fermentation tank yield was 60 g / L, and the yield of 2'-fucosyllactose relative to sucrose was 0.18 mol / mol (ACS Synth. Biol. 2020, 9, 10, 2784–2796).

[0007] (3) Chen et al. used a strong constitutive promoter to in situ overexpress the fucose module gene and simultaneously integrated two copies of the glycosyltransferase module gene into the genome. The shake flask yield was 7.22 g / L, the 5L fermenter yield was 50.1 g / L, and the yield of 2'-fucosyllactose relative to lactose was 0.93 mol / mol (J. Agric. Food Chem. 2023, 71, 40, 14678–14686).

[0008] However, none of these cases examined the effects of different gene dosages between modules on the synthesis of 2'-fucosyllactose. Optimizing gene dosages between modules and combining and regulating the coordinated expression of each module may further improve the production indicators of microbial synthesis of 2'-fucosyllactose. Summary of the invention

[0009] Purpose of the invention: The technical problem to be solved by the present invention is to provide a genetically engineered bacterium for efficiently synthesizing 2'-fucosyllactose and a construction method and application thereof in view of the low yield and lactose utilization rate when producing 2'-fucosyllactose in the prior art.

[0010] In order to solve the above technical problems, the present invention discloses a method for constructing a genetically engineered bacterium that efficiently synthesizes 2'-fucosyllactose, focusing on the fucose module and glycosyltransferase module in the 2'-fucosyllactose synthesis pathway, and using CRISPR transposition technology to quickly achieve multi-copy integration of the two genomes on the Escherichia coli genome in order to stably express them on the basis of optimizing the flux of the two. The method comprises the following steps:

[0011] (1) Knock out the UDP-glucose lipid carrier transferase encoding gene wcaJ, β-galactosidase encoding gene lacZ, α-galactosidase encoding gene melA, β-thiogalactoside transacetylase encoding gene lacA, N-acetylneuraminic acid decomposition-related gene cluster nanKETA and glucan glucosyltransferase encoding gene mdoH in the genome of the starting strain Escherichia coli to obtain DH1Δ6;

[0012] (2) integrating the fucose module gene and the glycosyltransferase module gene into the genome of DH1Δ6 obtained in step (1) to obtain DH1WxGy;

[0013] (3) knocking out or interrupting the glucose kinase encoding gene glk, or knocking out or interrupting the pyruvate oxidase encoding gene poxB in the genome of DH1WxGy obtained in step (1), thereby obtaining.

[0014] Wherein, the Escherichia coli includes Escherichia coli K12 DH1.

[0015] Among them, the Gene ID of the UDP-glucose lipid carrier transferase encoding gene wcaJ is 946583, the Gene ID of the β-galactosidase encoding gene lacZ is 945006, the Gene ID of the α-galactosidase encoding gene melA is 948636, the Gene ID of the β-thiogalactoside transacetylase encoding gene lacA is 945674, the Gene IDs of the nanK, nanE, nanT and nanA genes of the N-acetylneuraminic acid decomposition related gene cluster nanKETA are 947757, 947745, 947740 and 947742 respectively, the Gene ID of the glucan glucosyltransferase encoding gene mdoH is 945624, the Gene ID of the glucose kinase encoding gene glk is 946858, and the Gene ID of the pyruvate oxidase encoding gene poxB is 946132.

[0016] Among them, the fucose module gene includes a phosphomannose mutase encoding gene manB, a mannose-1-phosphate guanyltransferase encoding gene manC, a GDP-mannose-6-dehydrogenase encoding gene gmd and a GDP-fucose synthase encoding gene fcl; the Gene IDs of the phosphomannose mutase encoding gene manB, the mannose-1-phosphate guanyltransferase encoding gene manC, the GDP-mannose-6-dehydrogenase encoding gene gmd and the GDP-fucose synthase encoding gene fcl are 946574, 946580, 946562 and 946563 respectively.

[0017] The glycosyltransferase module gene includes an α1,2-fucosyltransferase encoding gene wbgL; the α1,2-fucosyltransferase encoding gene wbgL is derived from Escherichia coli O126; the Genebank accession number of the α1,2-fucosyltransferase is ADN43847.1.

[0018] Among them, the integrated fucose module gene and glycosyltransferase module gene are respectively connected in series with the inducible promoter Ptac to form an expression cassette Ptac-gmd-fcl-manC-manB and an expression cassette Ptac-wbgL, and the expression cassette Ptac-gmd-fcl-manC-manB and the expression cassette Ptac-wbgL are integrated into the Escherichia coli genome for expression using CRISPR transposition technology; the nucleotide sequences of the expression cassette Ptac-gmd-fcl-manC-manB and the expression cassette Ptac-wbgL are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0019] Wherein, a single copy or multiple copies of the expression cassette Ptac-gmd-fcl-manC-manB and the expression cassette Ptac-wbgL are integrated into the Escherichia coli genome.

[0020] Preferably, the CRISPR-transposition system is a VchCAST double plasmid system or a PtrCAST double plasmid system.

[0021] Further preferably, the fucose module genome uses the VchCAST double-plasmid system for genome integration of 1 to 8 copies, the integration site is a meaningless site between genes, and the nucleotide sequence of its spacer is shown in SEQ ID NO.3 to 10; the spacer is the recognition sequence of the genome integration site.

[0022] Further preferably, the phycosyltransferase module gene is integrated into the genome in 1 to 7 copies using the PtrCAST double plasmid system, and the nucleotide sequence of its spacer is shown in SEQ ID NO.11; the spacer is the recognition sequence of the genomic integration site.

[0023] Wherein, the knockout or interruption of the glucose kinase encoding gene glk is to use CRISPR / Cas9 technology to knock out or use CRISPR transposition technology to interrupt the glucose kinase encoding gene glk in the Escherichia coli genome; the knockout or interruption of the pyruvate oxidase encoding gene poxB is to use CRISPR / Cas9 technology to knock out or use CRISPR transposition technology to interrupt the pyruvate oxidase encoding gene poxB in the Escherichia coli genome. The knockout or interruption of the glucose kinase encoding gene glk, or the knockout or interruption of the pyruvate oxidase encoding gene poxB further increases the yield of 2'-fucosyllactose produced by the genetically engineered bacteria.

[0024] Wherein, when the CRISPR / Cas9 technology is used to knock out the glucose kinase encoding gene glk or the pyruvate oxidase encoding gene poxB in the Escherichia coli genome, the N20 sequences targeting the glucose kinase encoding gene glk and the pyruvate oxidase encoding gene poxB are shown as SEQ ID NO.12 and SEQ ID NO.13, respectively; when the CRISPR transposition technology is used to interrupt the glucose kinase encoding gene glk or the pyruvate oxidase encoding gene poxB in the Escherichia coli genome, the spacer sequences targeting the glucose kinase encoding gene glk and the pyruvate oxidase encoding gene poxB are shown as SEQ ID NO.14 and SEQ ID NO.15, respectively; the spacer is the recognition sequence of the targeted gene.

[0025] Specifically, the construction method of the genetically engineered bacteria for efficiently synthesizing 2'-fucosyllactose is as follows:

[0026] 1. Obtaining the chassis strain

[0027] Knock out genes wcaJ, lacZ, melA, lacA, nanKETA and mdoH in the Escherichia coli DH1 genome. For detailed steps, refer to patent CN109609422A.

[0028] (1) Construct sgRNA plasmids pTargetF-N20(wcaJ), pTargetF-N20(lacZ), pTargetF-N20(melA), pTargetF-N20(lacA), pTargetF-N20(nanKETA) and pTargetF-N20(mdoH) targeting genes wcaJ, lacZ, melA, lacA, nanKETA and mdoH in the Escherichia coli DH1 genome, respectively.

[0029] (2) Taking the knockout gene wcaJ as an example, the E. coli DH1 genome was used as a template, and the upstream and downstream homologous arms of the gene wcaJ were obtained by PCR amplification, and the repair fragment up-wcaJ-down of the knockout gene wcaJ was connected by overlapping PCR. Prepare E. coli DH1 competent cells containing pEcCas9, and co-transform the sgRNA plasmid pTargetF-N20 (wcaJ) targeting wcaJ in step (1) and the above-mentioned repair fragment up-wcaJ-down into the above-mentioned E. coli DH1 competent cells containing pEcCas9, and screen the transformants on the corresponding LB resistance plate.

[0030] Preferably, the LB resistance plate is an LB plate containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin.

[0031] (3) The clones with successful knockout of the wcaJ gene were verified by colony PCR combined with sequencing screening. After inserting the plasmid pTargetF-N20 (wcaJ) and pEcCas9, the strain DH1ΔwcaJ with genomic knockout of the wcaJ gene was obtained.

[0032] (4) On this basis, the above steps are repeated until the genes lacZ, melA, lacA, nanKETA and mdoH in the E. coli genome are all knocked out. The resulting strain is the desired chassis strain, recorded as DH1Δ6.

[0033] 2. Genomic transposition integration of glycosyltransferase module gene expression cassette Ptac-wbgL

[0034] (1) The codon-optimized expression cassette Ptac-wbgL encoding the α1,2-fucosyltransferase gene wbgL was synthesized and ligated to the vector pDonor(Ptr) to obtain the recombinant plasmid pDonor(Ptr)-Ptac-wbgL.

[0035] (2) The recombinant plasmid pDonor(Ptr)-Ptac-wbgL obtained in step (1) and the plasmid pQCasTns(Ptr) targeting multiple genomic sites were electroporated into the DH1Δ6 competent cells obtained in step 1, and transformants were screened on the corresponding LB resistance plates.

[0036] Preferably, the plasmid pQCasTns(Ptr) targeting multiple genomic sites is pQCasTns(Ptr)-IS1.

[0037] Preferably, the LB resistance plate is an LB plate containing 50 μg / mL kanamycin and 50 μg / mL chloramphenicol.

[0038] (3) The transformants obtained in step (2) were streaked sequentially on LB resistance plates containing different concentrations of anhydrotetracycline, and transformants were selected for colony PCR to verify the gene transposition status.

[0039] Preferably, the concentration of anhydrotetracycline is 100 ng / mL and 1000 ng / mL, respectively.

[0040] (4) The clones that successfully underwent transposition verification were subjected to plasmid loss. The clones that successfully lost the plasmid were strains DH1Wx (x represents the number of copies of Ptac-wbgL, x = 1 to 7) with different numbers of Ptac-wbgL copies integrated into their genome.

[0041] 3. Genomic transposition integration of the fucose module gene expression cassette Ptac-gmd-fcl-manC-manB

[0042] (1) Using the Escherichia coli DH1 genome as a template, the gmd-fcl and manC-manB gene fragments were obtained by PCR amplification, respectively. Using Ptac-wbgL as a template, the promoter Ptac fragment was obtained by PCR amplification. Ptac, gmd-fcl and manC-manB were connected by overlapping PCR to construct the expression cassette Ptac-gmd-fcl-manC-manB.

[0043] (2) The expression cassette Ptac-gmd-fcl-manC-manB obtained in step (1) is cloned into the vector pDonor(Vch) to obtain the recombinant plasmid pDonor(Vch)-GFCB.

[0044] (3) The recombinant plasmid pDonor(Vch)-GFCB obtained in step (2) and the plasmid pQCasTns(Vch) targeting multiple genomic sites were electroporated into the strain DH1Wx (x=1-7) obtained in step 2, which was integrated with 1 to 7 copies of Ptac-wbgL, and transformants were selected on the corresponding LB resistance plates.

[0045] Preferably, the plasmid pQCasTns(Vch) targeting multiple genomic sites is pQCasTns(Vch)-array8.

[0046] Preferably, the LB resistance plate is an LB plate containing 50 μg / mL streptomycin and 100 μg / mL ampicillin.

[0047] (4) The transformants obtained in step (3) were streaked on LB resistance plates containing different concentrations of anhydrotetracycline, and transformants were selected for colony PCR to verify the gene transposition status.

[0048] Preferably, the concentration of anhydrotetracycline is 100 ng / mL and 1000 ng / mL, respectively.

[0049] (5) The clones with successful transposition verification were subjected to the loss of the tool plasmid. The one with successful loss was the multi-copy strain DH1WxGy (x represents the number of copies of Ptac-wbgL and y represents the number of copies of Ptac-gmd-fcl-manC-manB; x=1-7, y=1-8) with multiple copies of Ptac-wbgL and Ptac-gmd-fcl-manC-manB integrated into the genome.

[0050] 4. The strain DH1WxGy obtained in step 3 is screened, representative strains are selected, the glucose kinase encoding gene glk in its genome is knocked out using CRISPR / Cas9 technology or the CRISPR transposition technology is interrupted, or the pyruvate oxidase encoding gene poxB in its genome is knocked out using CRISPR / Cas9 technology or the CRISPR transposition technology is interrupted, and finally the genetically engineered strains DH1WxGyΔglk (DH1WxGy with glk knocked out), DH1WxGyglk::VerA (DH1WxGy with glk interrupted), DH1WxGyΔpoxB (DH1WxGy with poxB knocked out) and DH1WxGypoxB::VerA (DH1WxGy with poxB interrupted) are obtained.

[0051] The invention also discloses the genetic engineering bacteria constructed by the construction method of the genetic engineering bacteria for efficiently synthesizing 2'-fucosyllactose.

[0052] The invention also discloses the application of the genetic engineering bacteria in the fermentation production of 2'-fucosyllactose.

[0053] Specifically, the seed liquid of the genetically engineered bacteria is inoculated into a fermentation medium for cultivation, and then an IPTG inducer and lactose are added to the fermentation medium to ferment and synthesize 2'-fucosyllactose.

[0054] The seed solution of the genetically engineered bacteria was inoculated into the fermentation medium and cultured until the OD 600 The value is 0.6~0.8.

[0055] Wherein, the fermentation medium is a glycerol-containing medium. Preferably, the components of the fermentation medium include: 25 g / L glycerol and 2 g / L yeast extract. More preferably, the concentration of the glycerol is 25 g / L and the concentration of the yeast extract is 2 g / L. The addition amounts of the lactose and the IPTG (isopropylthiogalactoside) inducer are 5-10 g / L and 0.1-0.3 mM, respectively. The fermentation synthesis conditions are: fermentation at 28-30°C and 200-220 rpm for 48-72 hours.

[0056] The invention also discloses the application of the genetic engineering bacteria in preparing products containing 2'-fucosyllactose.

[0057] Wherein, the product is a food additive or infant food.

[0058] The 2'-fucosyllactose-producing Escherichia coli genetically engineered bacteria DH1W6G6 glk::VerA of the present invention has a unit cell yield of 5.14 g / g DCW for producing 2'-fucosyllactose, which is the highest value of integrated strains based on Escherichia coli at this stage. The yield of 2'-fucosyllactose relative to lactose is 0.991 mol / mol, reaching a historical high, and has good prospects for industrial production.

[0059] Beneficial effects:

[0060] (1) The genetically engineered bacteria constructed by the present invention can efficiently synthesize 2'-fucosyllactose. The unit cell yield of 2'-fucosyllactose in a 24-deep-well plate is 5.14 g / g DCW, and the yield of 2'-fucosyllactose relative to lactose is 0.991 mol / mol, which is the highest value of the integrated strain with Escherichia coli as the chassis at this stage. The fermentation yield in a 5L fermenter can reach 120-130 g / L, which has good prospects for industrial application.

[0061] (2) The genetically engineered bacteria described in the present invention are mainly constructed by modular metabolic engineering transformation based on CRISPR-transposition technology. Compared with the currently reported 2'-fucosyllactose production strains that use plasmids with different copy numbers to regulate the metabolic flux of each module, the constructed genetically engineered bacteria have higher stability, more precise control of gene expression cassette dosage, avoid the use of antibiotics, reduce production costs, and can promote the industrial application of 2'-fucosyllactose. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0063] Figure 1 This is a diagram of the 2'-fucosyllactose metabolic pathway.

[0064] Figure 2 This is a nucleic acid gel verification image of Ptac-wbgL at the multi-copy integration of the genomic IS1 site of some clones in Example 2.

[0065] Figure 3 This is a nucleic acid gel verification image of the multi-copy integration of Ptac-gmd-fcl-manC-manB at 8 genomic sites partially cloned in Example 2.

[0066] Figure 4 The yield of 2'-fucosyllactose produced by fermentation of the multi-copy integrated strain library in Example 3 is shown.

[0067] Figure 5 This is the yield of 2'-fucosyllactose produced by fermentation after the multi-copy strain in Example 4 has its glucokinase encoding gene glk knocked out or interrupted.

[0068] Figure 6 This is the yield of 2'-fucosyllactose produced by fermentation after the pyruvate oxidase encoding gene poxB is knocked out or interrupted in the multi-copy strain in Example 5.

[0069] Figure 7 These are the loss rate results of plasmids pBBR1-GFCB and pMD-wbgL in Comparative Example 7. DETAILED DESCRIPTION

[0070] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0071] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0072] The plasmids, endonucleases, high-fidelity enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are carried out according to the instructions of the kits. The following competent state preparation, colony PCR, nucleic acid agarose gel electrophoresis, electrotransformation and bacterial genome extraction and storage and other experimental operations are carried out according to conventional methods.

[0073] The gene knockout system described below is the CRISPR-Cas9 dual-plasmid system of pTargetF and pEcCas9, and the gene editing system in the multi-copy strain construction stage described below is the PtrCAST dual-plasmid system of pQCasTns(Ptr) and pDonor(Ptr) and the VchCAST dual-plasmid system of pQCasTns(Vch) and pDonor(Vch), all of which were donated by Researcher Yang Sheng from the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0074] The sequencing of the following plasmids and DNA products was completed by Anhui General Biotechnology Co., Ltd.

[0075] The fermentation medium of the strain in the following examples is a glycerol quantitative medium, including 25 g / L glycerol, 2 g / L yeast extract, 5 g / L dipotassium hydrogen phosphate, 12 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 1.2 g / L calcium chloride, 0.25 g / L manganese sulfate, 0.02 g / L sodium molybdate dihydrate, 2.0 g / L zinc sulfate heptahydrate, 0.5 g / L cupric chloride dihydrate, 0.5 g / L boric acid, 0.5 g / L ferrous sulfate heptahydrate, and 1.5 g / L sodium citrate.

[0076] The specific method of fermenting the genetically engineered strain in the well plate in the following embodiment is: a single colony of the genetically engineered bacteria is inoculated into LB liquid medium, and cultured overnight at 37°C and 220rpm to obtain a seed solution; the seed solution is inoculated into a 24-deep well plate containing 2mL of glycerol quantitative medium at an inoculum volume of 2.5% (v / v), and cultured at 37°C and 220rpm until OD 600 The induction temperature was 0.6, and IPTG inducer and lactose were added to the glycerol quantitative culture medium to make the initial concentrations of the two in the glycerol quantitative culture medium 0.1-0.3 mM and 5-10 g / L, respectively. The fermentation was induced at 28-30°C and 220 rpm. The 2'-fucosyllactose fermentation liquid was obtained after fermentation for 72 hours.

[0077] High Performance Liquid Chromatography (HPLC) was used to determine the lactose concentration and 2'-fucosyllactose concentration in the fermentation broth. The determination method was as follows: 1 mL of fermentation broth was boiled at 100°C for 10 min, centrifuged at 12000 rpm for 5 min, the supernatant was diluted and filtered through a 0.22 μm filter membrane, and the lactose concentration and 2'-fucosyllactose concentration in the fermentation broth were analyzed by HPLC. HPLC detection method: differential refractive index detector; chromatographic column was BioRad HPX-87H (9 μm, 300 mm × 7.8 mm), column temperature was 60°C; mobile phase was 0.5 mmol / L H2SO4 aqueous solution, flow rate was 0.5 mL / min, isocratic elution; injection volume was 5 μL.

[0078] Example 1 Knockout of wcaJ, lacZ, lacA, melA, mdoH, and nanKETA in the Escherichia coli genome Figure 1 The diagram shows the metabolic pathway of 2'-fucosyllactose. The genes wcaJ, lacZ, lacA, melA, mdoH and nanKETA are all synthetic branch genes. Knocking them out is beneficial to the carbon flux to the target product 2'-fucosyllactose.

[0079] Using Escherichia coli DH1 as the starting strain, the CRISPR / Cas9 gene editing system was used to knock out the wcaJ, lacZ, lacA, melA, mdoH and nanKETA genes in the Escherichia coli DH1 genome. For specific experimental steps, please refer to patent CN109609422A to obtain the recombinant Escherichia coli DH1Δ6.

[0080] Example 2 Construction of a multi-copy strain library

[0081] The CRISPR-transposition system was used to integrate multiple copies of the fucose module genes (manC, manB, gmd and fcl) and the glycosyltransferase module gene (wbgL) into the Escherichia coli DH1Δ6 genome constructed in Example 1. The primer sequences involved are shown in Table 1. The specific construction steps are as follows:

[0082] 1. Construction of recombinant plasmid

[0083] (1) Using the original pDonorGFP (Ptr) plasmid (Addgene: #175578) as a template, primers ptr-F / ptr-R were used to amplify the linearized vector fragment pDonor (Ptr); using the original pDonor-GDH plasmid (Addgene: #140629) as a template, primers vch-F / vch-R were used to amplify the linearized vector fragment pDonor (Vch).

[0084] (2) Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the expression cassette Ptac-wbgL, wherein the α1,2-fucosyltransferase gene wbgL was codon-optimized, and the nucleotide sequence of the expression cassette Ptac-wbgL was shown in SEQ ID NO.2.

[0085] (3) Using the Escherichia coli DH1 genome as a template, primers gmd-fcl-F / gmd-fcl-R and manCB-F / manCB-R were used to amplify the target gene fragments gmd-fcl and manC-manB, respectively. Using the expression cassette Ptac-wbgL as a template, primers Ptac-F / Ptac-R were used to amplify the promoter Ptac fragment. Using the Ptac, gmd-fcl and manC-manB fragments as templates, primers Ptac-F / manCB-R were used to perform overlapping PCR on the fragments to construct the expression cassette Ptac-gmd-fcl-manC-manB. The PCR reaction system and reaction conditions are shown in Table 2.

[0086] (4)Use ClonExpress TM II recombination reaction system (ClonExpress II One Step Cloning Kit; Cat: C112-01 / 02) connects the target gene fragment Ptac-wbgL with the linearized vector fragment pDonor (Ptr) obtained in step (1). After the connection reaction is completed, the reaction tube is immediately placed in an ice water bath to cool for 5 minutes, and then the connection product is transformed into Escherichia coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.), coated on LB agar plates with chloramphenicol resistance, and cultured at 37°C overnight. Pick a single clone to extract the plasmid and verify the transformant plasmid. The one that is successfully verified is the constructed recombinant plasmid pDonor (Ptr) -Ptac-wbgL. The method for constructing the recombinant plasmid pDonor (Vch) -GFCB with the target gene fragment Ptac-gmd-fcl-manC-manB and the linearized vector fragment pDonor (Vch) refers to the construction method of pDonor (Ptr) -Ptac-wbgL.

[0087] 2. Construction of recombinant strains

[0088] (1) 300 ng of each of the recombinant plasmid pDonor(Ptr)-Ptac-wbgL constructed in step 1 and the plasmid pQCasTns(Ptr)-IS1 targeting multiple genomic sites were electroporated into the electroporation competent cells of the recombinant bacterium Escherichia coli DH1Δ6 constructed in Example 1, and spread them on LB resistance plates containing 50 μg / mL kanamycin and 50 μg / mL chloramphenicol. Transformants were picked and plated on LB resistance plates containing 100 ng / mL and 1000 ng / mL anhydrotetracycline, respectively. Induce, pick a single clone and perform colony PCR with primers IS1-150R / IS1-1, IS1-150R / IS1-2, IS1-150R / IS1-3, IS1-150R / IS1-4, IS1-150R / IS1-5, IS1-150R / IS1-6 and IS1-7 / IS1-7R to verify the transposition of the expression cassette Ptac-wbgL, pick a positive clone for sequencing, and if the sequencing is correct, the expression cassette Ptac-wbgL is successfully integrated into the Escherichia coli genome in multiple copies.

[0089] Figure 2 This is a nucleic acid gel verification diagram of the multi-copy integration of Ptac-wbgL in the genomic IS1 site of some clones in this example. If the expression cassette Ptac-wbgL is not integrated, the nucleic acid band sizes of the IS1-1 to IS1-7 sites should be 453bp, 461bp, 515bp, 522bp, 484bp, 456bp, and 509bp, respectively; if the integration is successful, the nucleic acid band sizes of the IS1-1 to IS1-7 sites should increase by 1980bp on the original basis.

[0090] (2) Pick the positive clones with 1 to 7 copies of Ptac-wbgL in step (1), prepare electroporation competent cells respectively, take 100 ng of plasmid pFREE (Addgene: #92050) and electroporate into the competent cells of the recombinant strain with 1 to 7 copies of Ptac-wbgL respectively, after 2 h of recovery in LB medium, transfer 25 μL of the recovered bacterial liquid into 5 mL LB liquid medium (containing 2 g / L rhamnose, 200 ng / mL anhydrotetracycline and 50 μg / mL bleomycin), and culture at 30°C, 220 rpm for 24 h. A small amount of bacterial liquid was streaked onto an LB solid plate, and single clones were randomly picked and spotted onto non-resistant LB plates, kanamycin-resistant LB plates (containing 50 μg / mL kanamycin), chloramphenicol-resistant LB plates (containing 25 μg / mL chloramphenicol), and bleomycin-resistant LB plates (50 μg / mL bleomycin), respectively. The clones that grew on the non-resistant LB plates and did not grow on the above three LB-resistant plates were strains in which the tool plasmid was successfully lost. Finally, the recombinant strains DH1Wx (DH1W1, DH1W2, DH1W3, DH1W4, DH1W5, DH1W6, and DH1W7) with integrated expression cassette Ptac-wbgL were obtained.

[0091] (3) Prepare electroporation competent cells from the recombinant strains DH1W1, DH1W2, DH1W3, DH1W4, DH1W5, DH1W6 and DH1W7 in step (2), respectively, and electroporate 300 ng of the recombinant plasmid pDonor(Vch)-GFCB constructed in step 1 and the plasmid pQCasTns(Vch)-array8 targeting multiple genomic sites into the above competent cells for induced transposition of the expression cassette Ptac-gmd-fcl-manC-manB, pick a single clone and clone it with primers cr1UF / cr1DR, cr2UF / cr2DR, cr3UF / cr3DR, cr4UF / cr4DR, cr5UF / cr5DR, cr6UF / cr6DR, cr7UF / cr7DR and cr8UF / cr8DR were used for colony PCR to verify the transposition of the expression cassette Ptac-gmd-fcl-manC-manB, and the positive clones were picked for sequencing. If the sequencing was correct, the expression cassette Ptac-gmd-fcl-manC-manB was successfully integrated into the genome of the recombinant strain DH1Wx in multiple copies.

[0092] Figure 3This is a nucleic acid gel verification diagram of the multi-copy integration of Ptac-gmd-fcl-manC-manB at 8 loci of the genome partially cloned in this example. If the expression cassette Ptac-gmd-fcl-manC-manB is not integrated, the nucleic acid band sizes at 8array-1 to 8array-8 should be 844bp, 652bp, 735bp, 664bp, 666bp, 724bp, 741bp, and 787bp, respectively; if the integration is successful, the nucleic acid band sizes at 8array-1 to 8array-8 should increase by 5287bp on the original basis.

[0093] (5) Refer to step (2) to lose the tool plasmid, pick the positive clones with 1 to 8 copies of Ptac-gmd-fcl-manC-manB, prepare electroporation competent cells respectively, take 100ng of plasmid pCutamp (Addgene: #140632) and electroporate into the competent cells of the recombinant strain with 1 to 8 copies of Ptac-gmd-fcl-manC-manB to lose the tool plasmid. After the plasmid is successfully lost, the recombinant Escherichia coli genetic engineering strain DH1W with multiple copies of Ptac-wbgL and Ptac-gmd-fcl-manC-manB is obtained. x G y (x=1~7; y=1~8).

[0094] Table 1 Primer sequences

[0095]

[0096] Table 2 PCR reaction system and reaction conditions

[0097]

[0098] Example 3 Screening of multi-copy strain library

[0099] The multi-copy strain DH1W constructed in Example 2 x G y The cells were inoculated into 96-deep-well plates containing 0.8 mL LB medium and cultured overnight at 37°C and 220 rpm to obtain seed solution. The seed solution was inoculated into 24-deep-well plates containing 2 mL glycerol quantitative medium at an inoculum of 2.5% (v / v) and cultured at 37°C and 220 rpm until OD 600 The pH value was 0.6, and IPTG and lactose were further added to the 24-deep-well plate to make their initial concentrations in the fermentation reaction system 0.1 mM and 5 g / L, respectively; the fermentation was induced at 30°C and 220 rpm, and the fermentation broth was collected after 72 hours of fermentation. The lactose concentration and 2'-fucosyllactose concentration in the fermentation broth were analyzed by HPLC.

[0100] Figure 4 The yield of 2'-fucosyllactose fermented by the multi-copy integrated strain library in this example. Figure 4 As shown in the figure, with the increase of the copy number of the expression cassettes Ptac-wbgL and Ptac-gmd-fcl-manC-manB, the 2'-fucosyllactose production generally showed an upward trend. After reaching a certain level, the continued stacking of the genome copy number could not further increase the production. A series of strains with higher production were obtained through screening. Figure 4 From the heat map, it can be seen that the 2'-fucosyllactose production of the strains obtained when 4 or more copies of Ptac-wbgL were integrated into the genome was mostly high. Among them, DH1W4G5, DH1W5G4 and DH1W6G6 were representative strains (with the highest 2'-fucosyllactose production) when the copy numbers of Ptac-wbgL were 4, 5 and 6, respectively. The titers of 2'-fucosyllactose synthesized by them were 4.87 g / L, 5.47 g / L and 6.05 g / L, respectively, and the yields of 2'-fucosyllactose relative to lactose were 0.910 mol / mol, 0.951 mol / mol and 0.972 mol / mol, respectively.

[0101] Example 4 Knockout or interruption of Escherichia coli chromosomal gene glk

[0102] Using the strains obtained in Example 3 (DH1W6G2, DH1W4G5, DH1W5G4 and DH1W6G6, wherein DH1W4G5, DH1W5G4 and DH1W6G6 are the dominant strains obtained in Example 3) as the chassis, the gene glk was knocked out or interrupted, and the required primers are shown in Table 3. The specific steps are as follows:

[0103] 1. Knockout of the glucokinase encoding gene glk

[0104] (1) Using the Escherichia coli DH1 genome as a template, the upstream homology arm primers up-F (glk) / up-R (glk) and the downstream homology arm primers down-F (glk) / down-R (glk) were used to amplify the upstream and downstream fragments of the glk gene respectively and recovered by gel. Then, the upstream and downstream fragments were connected by overlapping PCR and recovered by gel to obtain the complete repair fragment of the glk gene knockout.

[0105] (2) Using the original pTargetF plasmid (Addgene: #62226) as a template, PCR amplification was performed with primers sg-glk-F / pTargetF-R to replace the N20 sequence on the original plasmid with an N20 sequence complementary to the glk sequence. The amplified product was digested with restriction endonuclease Dpn I to eliminate the excess circular pTargetF plasmid, and the amplified product was transformed into Escherichia coli JM109 competent cells, which were spread on LB resistance plates (containing 50 μg / mL spectinomycin), and a single clone was picked to extract the plasmid and sequenced. The one with the correct sequencing was the pTargetF plasmid with targeting glk, which was named pTargetF-N20 (glk).

[0106] (3) Take 100 ng of pEcCas plasmid (Addgene: #60847) and add it to 100 μL of competent cells of the strains (DH1W6G2, DH1W4G5, DH1W5G4 and DH1W6G6) screened in Example 3, mix gently, and then transfer to a pre-cooled electrode cup. After 2.5 kV electric shock for 5 ms, quickly add 900 μL of LB liquid culture medium, mix gently and transfer to a sterilized 1.5 mL centrifuge tube; place the centrifuge tube at 37°C and shake gently at 200 rpm for 60 min for recovery, then take 100 μL of bacterial solution and spread it on the LB solid plate containing the corresponding resistance; incubate inverted in a 37°C constant temperature incubator overnight to culture a single clone.

[0107] (4) Pick a single colony from step (3) and place it in LB liquid medium, and culture it at 37°C, 200 rpm overnight; transfer the bacterial solution to 50 mL LB liquid medium at a 1% (v / v) inoculation volume, and culture it at 37°C, 200 rpm for 1 h. Add L-arabinose (to a concentration of 30 mM) to induce the expression of the λ-red system, and then culture it until the OD 600 When the pH reaches 0.6-0.8, prepare E. coli competent cells containing pEcCas9.

[0108] (5) 200 ng of the plasmid pTargetF-N20 (glk) constructed in step (2) and 1000 ng of the glk homologous repair template fragment constructed in step (1) were electroporated into the competent E. coli cells containing pEcCas9 prepared in step (4), spread on LB resistance plates (containing kanamycin and spectinomycin), and cultured at 37°C until single colonies appeared. The single colonies grown on the plates were verified by colony PCR using primers glkYZ-F / glkYZ-R, and positive transformants were screened and gene sequencing was performed. The strain with correct sequencing was the strain in which glk was knocked out.

[0109] (6) The positive clone colonies that were successfully knocked out by sequencing in step (5) were picked into 5 mL LB liquid culture medium (containing kanamycin), rhamnose was added to an initial concentration of 10 mM, and cultured at 37 ° C for 6 h to induce pEcCas9 to cut the pTargetF-N20 (glk) plasmid. The bacterial liquid was streaked on a kanamycin-resistant LB solid plate, cultured at 37 ° C until a single colony appeared, and the single colony was spotted on a kanamycin-resistant LB plate and a spectinomycin-resistant LB plate, respectively. If the strain grows on a kanamycin-resistant LB plate and does not grow on a spectinomycin-resistant LB plate, it indicates that the plasmid pTargetF-N20 (glk) in the strain has been successfully eliminated.

[0110] (7) Plasmid pEcCas9 contains a sucrose lethal gene. The single colony successfully eliminated from pTargetF-N20 (glk) in (6) was transferred to an LB liquid medium without antibiotics, and the bacterial liquid was streaked on an LB solid plate containing 10 g / L sucrose, and cultured at 37°C until a single colony appeared. The single colony was spotted on an LB plate without antibiotics and a kanamycin-resistant LB plate, respectively. If the strain did not grow on the kanamycin-resistant LB plate, it indicated that the plasmid pEcCas9 was successfully eliminated. Finally, a series of recombinant strains DH1W6G2Δglk, DH1W4G5Δglk, DH1W5G4Δglk, and DH1W6G6Δglk with the glucose kinase encoding gene glk knocked out were obtained.

[0111] 2. Disruption of the Glucokinase Encoding Gene glk

[0112] (1) A spacer sequence targeting the genomic glucose kinase encoding gene glk was synthesized by Jinweizhi Biotechnology Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.14, and it is used to replace the spacer sequence targeting the IS1 site on the genome in the plasmid pQCasTns(Ptr)-IS1 to construct a recombinant plasmid pQCasTns(Ptr)-glk.

[0113] (2) 300 ng of the recombinant plasmid pQCasTns(Ptr)-glk obtained in step (1) and 300 ng of pDonor(Ptr)-VerA were introduced into the competent cells of the strains (DH1W6G2, DH1W4G5, DH1W5G4 and DH1W6G6) screened in Example 3, and gently mixed. Then, the cells were transferred to a pre-cooled electrode cup, and 900 μL of LB liquid culture medium was quickly added after 2.5 kV electric shock for 5 ms. After gentle mixing, the cells were transferred to a sterilized 1.5 mL centrifuge tube; the centrifuge tube was placed at 37°C and shaken at 200 rpm for 60 min for recovery, and then 100 μL of the bacterial solution was spread on an LB solid plate containing the corresponding resistance; the cells were inverted and cultured in a 37°C constant temperature incubator overnight until a single clone appeared.

[0114] (3) Referring to Example 2, the clones obtained in (2) were induced on plates containing 100 ng / mL and 1000 ng / mL of dehydrotetracycline, respectively. Single clones were selected for colony PCR verification of terminator transposition using primers glk-CAST-F / glk-CAST-R. Positive clones were selected for sequencing. If the sequencing was correct, the glk gene was successfully interrupted.

[0115] (4) Plasmid loss step: Referring to Example 2, the recombinant strains obtained after successful loss are the strains DH1W6G2 glk::VerA, DH1W4G5 glk::VerA, DH1W5G4 glk::VerA, and DH1W6G6 glk::VerA in which the glucose kinase encoding gene glk is interrupted.

[0116] The strains with knockout or interrupted glucokinase encoding gene glk were inoculated into fermentation medium, respectively, and plate fermentation was performed and the yield of 2'-fucosyllactose was detected. The specific fermentation method and 2'-fucosyllactose detection method were referred to Example 3.

[0117] Figure 6 The yield of 2'-fucosyllactose produced by fermentation after the multi-copy strain in this example has knocked out or interrupted the gene glk. Figure 6 As shown in the results, knocking out or interrupting the glk gene can increase the yield of 2'-fucosyllactose, among which interrupting glk has a better effect on yield than knocking out, and the improvement effect is particularly obvious when the initial strain yield is low. For example, after the glk gene was interrupted in strain DH1W6G2, the yield increased by 151% compared with DH1W6G2 and 7.36% compared with the glk knockout strain. After the glk gene was interrupted in strain DH1W6G6, the unit cell yield of 2'-fucosyllactose was 5.14g / g DCW, which is the highest value of the integrated strain with Escherichia coli as the chassis at this stage. The yield of 2'-fucosyllactose relative to lactose is 0.991mol / mol, and the fermentation yield in a 5L fermenter can reach 120-130g / L, which has good prospects for industrial application.

[0118] Table 3 Primer sequences

[0119]

[0120] Example 5 Knockout or interruption of Escherichia coli chromosomal gene poxB

[0121] The strains with higher yields (DH1W6G2, DH1W4G5, DH1W5G4 and DH1W6G6) screened in Example 3 were used as the chassis to knock out or interrupt the gene poxB. The required primers are shown in Table 4. The specific experimental steps refer to Example 4.

[0122] 1. Knockout of the pyruvate oxidase encoding gene poxB

[0123] (1) Using the Escherichia coli DH1 genome as a template, upstream homology arm primers up-F (poxB) / up-R (poxB) and downstream homology arm primers down-F (poxB) / down-R (poxB) were used to amplify the upstream and downstream fragments of the poxB gene respectively and recovered by gel. The upstream and downstream fragments were then connected and recovered by gel by overlapping PCR to obtain a complete repair fragment of the poxB gene knockout.

[0124] (2) Using the original pTargetF plasmid (Addgene: #62226) as a template, PCR amplification was performed with primers sg-poxB-F / pTargetF-R to replace the N20 sequence on the original plasmid with the N20 sequence complementary to the poxB sequence to construct a pTargetF plasmid targeting poxB, which was named pTargetF-N20(poxB).

[0125] (3) 200 ng of the plasmid pTargetF-N20 (poxB) constructed in step (2) and 1000 ng of the poxB homologous repair template fragment constructed in step (1) were electroporated into the competent E. coli cells containing pEcCas9 prepared in Example 4, and the single colonies grown on the plate were verified by colony PCR using primers poxBYZ-F / poxBYZ-R. Positive transformants were screened and gene sequencing was performed. The strain with correct sequencing was the poxB knockout strain.

[0126] (4) Sequencing to verify the positive clones with successful poxB knockout The tool plasmid was lost according to Example 4 to obtain a series of knockout strains of the pyruvate oxidase encoding gene poxB: DH1W6G2ΔpoxB, DH1W4G5ΔpoxB, DH1W5G4ΔpoxB and DH1W6G6ΔpoxB.

[0127] 2. Disruption of the pyruvate oxidase encoding gene poxB

[0128] (1) A spacer sequence targeting the genomic pyruvate oxidase encoding gene poxB was synthesized by Jinweizhi Biotechnology Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.15, and it was used to replace the spacer sequence targeting the IS1 site on the genome in the plasmid pQCasTns(Ptr)-IS1 to construct the recombinant plasmid pQCasTns(Ptr)-poxB.

[0129] (2) Take 300 ng of the recombinant plasmid pQCasTns(Ptr)-poxB obtained in step (1) and 300 ng of pDonor(Ptr)-VerA and electroporate them into competent cells of the dominant strains (DH1W6G2, DH1W4G5, DH1W5G4 and DH1W6G6) screened in Example 3, and culture them in an inverted incubator at 37°C overnight until a single clone appears.

[0130] (3) Referring to Example 2, the clones obtained in (2) were induced on plates containing 100 ng / mL and 1000 ng / mL of dehydrotetracycline, respectively. Single clones were selected for colony PCR verification of terminator transposition using primers poxB-CAST-F / poxB-CAST-R. Positive clones were selected for sequencing. If the sequencing was correct, the poxB gene was successfully interrupted.

[0131] (4) The plasmid loss step was carried out as in Example 2. The recombinant strains obtained after successful loss were the strains DH1W6G2 poxB::VerA, DH1W4G5 poxB::VerA, DH1W5G4poxB::VerA, and DH1W6G6 poxB::VerA in which the pyruvate oxidase encoding gene poxB was interrupted.

[0132] The strains with knockout or interrupted pyruvate oxidase encoding gene poxB were inoculated into fermentation medium, respectively, and plate fermentation was performed and the yield of 2'-fucosyllactose was detected. The specific fermentation method and 2'-fucosyllactose detection method were referred to Example 3.

[0133] Figure 7 The yield of 2'-fucosyllactose produced by fermentation after knocking out or interrupting the gene poxB in the multi-copy strain in this example. Figure 7 As shown in the figure, knockout or interruption of the gene poxB can increase the production of 2'-fucosyllactose, among which interruption of poxB has a better effect on improving the yield than knockout, and the improvement effect is particularly obvious when the initial strain yield is low. For example, after the poxB gene was interrupted in the strain DH1W6G2, the yield increased by 98% compared with DH1W6G2 and increased by 12.19% compared with the poxB knockout strain.

[0134] Table 4 Required primers

[0135]

[0136] Comparative Example 6 Comparison of 2'-fucosyllactose fermentation effects between plasmid strains and genome multi-copy integrated strains

[0137] The plasmid strain was constructed as a control for the genome multi-copy integrated recombinant strain (DH1WxGy) producing 2'-fucosyllactose of the present invention. The fucose module genes (manC, manB, gmd and fcl) and the glycosyltransferase module gene (wbgL) were cloned into the vectors pBBR1-MCS2 and pMD-18T, respectively, to construct the recombinant plasmids pBBR1-GFCB and pMD-wbgL, respectively. The recombinant plasmids pBBR1-GFCB and pMD-wbgL were introduced together into the Escherichia coli DH1Δ6 competent cells constructed in Example 1 to construct the plasmid strain. For the specific construction method, refer to the patent with publication number CN109609422A.

[0138] The above-mentioned plasmid strain was used to synthesize 2'-fucosyllactose by the fermentation method of Example 3. The titer of 2'-fucosyllactose produced by the plasmid strain was 3.26 g / L, and the yield of 2'-fucosyllactose relative to lactose was 0.595 mol / mol. Compared with the fermentation effect of the genome multi-copy integrated strain of the present invention in Example 3, it can be seen that the genome multi-copy integrated fucose module gene and glycosyltransferase module gene can significantly increase the titer of 2'-fucosyllactose and greatly improve the utilization rate of lactose, which is of great significance for industrial production.

[0139] Comparative Example 7 Plasmid stability of plasmid strains

[0140] The plasmid strain constructed in Comparative Example 6 was subcultured and its plasmid stability was analyzed, and the specific steps were as follows:

[0141] (1) The plasmid strain constructed in Comparative Example 1 was inoculated into 5 mL of LB liquid culture medium (containing 50 μg / mL kanamycin and 100 μg / mL ampicillin) and cultured overnight at 37° C. and 220 rpm.

[0142] (2) inoculating the bacterial solution obtained in step (1) into 50 mL of LB liquid medium without corresponding resistance at a transfer volume of 1%, and culturing at 37°C, 200 rpm for 12 h; transferring the bacterial solution into a new 50 mL of LB medium without corresponding resistance at a transfer volume of 1%, and subculturing for 3 times, once every 12 h;

[0143] (3) The bacterial suspensions from passage 1, 2, and 3 were streaked onto LB plates without corresponding resistance and incubated at 37°C until single colonies appeared. Fifty single colonies were randomly picked from each plate and spotted onto LB plates without resistance, kanamycin-resistant LB plates (containing 50 μg / mL kanamycin), and ampicillin-resistant LB plates (containing 100 μg / mL ampicillin). The number of colonies on the three plates was counted and the plasmid loss rate was calculated.

[0144] Figure 5 The loss rate results of plasmid pBBR1-GFCB and pMD-wbgL in this comparative example are shown in Table 2. Figure 5 As shown in the figure, plasmids pBBR1-GFCB and pMD-wbgL were lost to varying degrees during the passage process, and the plasmid loss rate of plasmid pMD-wbgL reached more than 50% after the first passage. α1,2-fucosyltransferase wbgL is a key rate-limiting enzyme in the 2'-fucosyllactose synthesis pathway. The extreme instability of pMD-wbgL hinders the industrial-scale production of 2'-fucosyllactose. Genome integration expression is one of the feasible methods to solve the instability of plasmid expression.

[0145] Comparative Example 8 Summary of cases of synthesizing 2'-fucosyllactose using Escherichia coli as a chassis in current public publications

[0146] The examples of synthesizing 2'-fucosyllactose using E. coli as the chassis in the current public publications are summarized in Table 5 below:

[0147] Table 5 Summary of the cases of synthesizing 2'-fucosyllactose using E. coli as the chassis in the current publications

[0148]

[0149]

[0150] Note: The values ​​of unit cell yield in references 1 to 6 are given in the articles; the values ​​of unit cell yield in references 7 to 13 are calculated based on the values ​​of OD600 and yield. The specific formula is: Unit cell yield (g / g DCW) = yield (g / L) / (OD 600 (g / L)*0.36).

[0151] As can be seen from the above table, the unit cell yield of the strain reported in document 13 is the highest value at present, but the strain adopts a salvage synthesis pathway to produce 2'-fucosyllactose. In addition to the commonly used carbon sources glycerol and lactose, fucose needs to be added in the production process. Fucose is expensive, and the fermentation cost is high compared with the de novo synthesis pathway, and it does not have industrial advantages. And the strain is a plasmid strain, which is prone to genetic instability in industrial applications. The integrated strain constructed by the present invention adopts a de novo synthesis pathway to produce 2'-fucosyllactose, avoiding the above problems, and its unit cell yield is 5.14g / g DCW, which is the highest value of the integrated strain with Escherichia coli as the chassis at this stage, and has good prospects for industrial application.

[0152] The references in Table 5 are as follows:

[0153] [1]F.Baumgaertner,L.Seitz,G.A.Sprenger,C.Albermann,Construction ofEscherichia coli strains with chromosomally integrated expression cassettesfor the synthesis of 2’-fucosyllactose,Microbial Cell Factories.12(2013).

[0154] [2]J.W.Lee,S.Kwak,J.-J.Liu,E.J.Yun,Y.-S.Jin,2’-Fucosyllactoseproduction in engineered Escherichia coli with deletion of waaF and wcaJ andoverexpression of FucT2,Journal of Biotechnology.340(2021)30-38.

[0155] [3]M.Li,C.Li,M.Hu,T.Zhang,Metabolic engineering strategies of de novopathway for enhancing 2’-fucosyllactose synthesis in Escherichia coli,Microbial Biotechnology.15(5)(2022)1561-1573.

[0156] [4]B.S.Park,Y.H.Choi,M.W.Kim,B.G.Park,E.-J.Kim,J.Y.Kim,J.H.Kim,B.-G.Kim,Enhancing biosynthesis of 2’-Fucosyllactose in Escherichia coli throughengineering lactose operon for lactose transport and alpha-1,2-Fucosyltransferase for solubility,Biotechnology and Bioengineering.119(5)(2022)1264-1277.

[0157] [5]R.Chen,L.Wan,Y.Zhu,Y.Liu,W.Zhang,W.Mu,Spatial organization ofpathway enzymes via self-assembly to improve 2’-fucosyllactose biosynthesisin engineered Escherichia coli,Biotechnology and Bioengineering.(2022).

[0158] [6]C.Li,M.Li,M.Hu,T.Zhang,Metabolic Engineering of De Novo Pathwayfor the Production of 2’-fucosyllactose in Escherichia coli,MolecularBiotechnology.65(9)(2023)1485-1497.

[0159] [7]Y.L.Liu,Y.Y.Zhu,L.Wan,R.L.Chen,W.L.Zhang,W.M.Mu,High-Level De NovoBiosynthesis of 2’-Fucosyllactose by Metabolically Engineered Escherichiacoli,Journal of Agricultural and Food Chemistry.70(29)(2022)9017-9025.

[0160] [8]Y.-W.Chin,N.Seo,J.-H.Kim,J.-H.Seo,Metabolic Engineering ofEscherichia coli to Produce 2’-Fucosyllactose via Salvage Pathway ofGuanosine 5’-Diphosphate(GDP)-L-Fucose,Biotechnology and Bioengineering.113(11)(2016)2443-2452.

[0161] [9]M.Li,Y.Luo,M.Hu,C.Li,Z.Liu,T.Zhang,Module-Guided MetabolicRewiring for Fucosyllactose Biosynthesis in Engineered Escherichia coli withLactose De Novo Pathway,Journal of Agricultural and Food Chemistry.70(46)(2022)14761-14770.

[0162]

[10] Y.Chen,Y.Zhu,H.Wang,R.Chen,Y.Liu,W.Zhang,W.Mu,De novobiosynthesis of 2-fucosyllactose in a metabolically engineered Escherichiacoli using a novel 1,2-fucosyltransferase from Azospirillum lipoferum,Bioresource Technology.374(2023).

[0163]

[11] Y.Zhu,R.Chen,H.Wang,Y.Chen,Y.Liu,J.Zhou,W.Mu,Elimination ofByproduct Generation and Enhancement of 2’-Fucosyllactose Synthesis byExpressing aNovelα1,2-Fucosyltransferase in Engineered Escherichia coli,Journal of Agricultural and Food Chemistry.71(12)(2023)4915-4923.

[0164]

[12] Y.-W.Chin, J.-Y.Kim, W.-H.Lee, J.-H.Seo, Enhanced production of 2'-Fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation oflactose metabolism and fucosyltransferase, Journal of Biotechnology.210(2015)107-115.

[0165]

[13] S. Liang, Z. He, D. Liu, S. Yang, Q. Yan, Z. Jiang, Construction of anengineered Escherichia coli for effective synthesis of 2'-Fucosyllactose via the salvage pathway, Synthetic and Systems Biotechnology. 9(1)(2024)108-114.

[0166] The present invention provides a genetically engineered bacterium for efficiently synthesizing 2'-fucosyllactose and a construction method and application thereof. There are many methods and approaches to specifically implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing a genetically engineered bacterium for efficiently synthesizing 2′-fucosyllactose, characterized in that: The steps include: (1) Knock out the UDP-glucose lipid carrier transferase encoding gene wcaJ, β-galactosidase encoding gene lacZ, α-galactosidase encoding gene melA, β-thiogalactoside transacetylase encoding gene lacA, N-acetylneuraminic acid decomposition-related gene cluster nanKETA and glucan glucosyltransferase encoding gene mdoH in the genome of the starting strain Escherichia coli to obtain DH1Δ6; (2) integrating the fucose module gene and the glycosyltransferase module gene into the genome of DH1Δ6 obtained in step (1) to obtain DH1WxGy; (3) knocking out or interrupting the glucose kinase encoding gene glk, or knocking out or interrupting the pyruvate oxidase encoding gene poxB in the genome of DH1WxGy obtained in step (1), thereby obtaining.

2. The construction method according to claim 1, characterized in that: The Escherichia coli is Escherichia coli K12DH1.

3. The construction method according to claim 1, characterized in that: The Gene ID of the UDP-glucose lipid carrier transferase encoding gene wcaJ is 946583, the Gene ID of the β-galactosidase encoding gene lacZ is 945006, the Gene ID of the α-galactosidase encoding gene melA is 948636, the Gene ID of the β-thiogalactoside transacetylase encoding gene lacA is 945674, the Gene IDs of the nanK, nanE, nanT and nanA genes of the N-acetylneuraminic acid decomposition related gene cluster nanKETA are 947757, 947745, 947740 and 947742 respectively, the Gene ID of the glucan glucosyltransferase encoding gene mdoH is 945624, the Gene ID of the glucokinase encoding gene glk is 946858, and the Gene ID of the pyruvate oxidase encoding gene poxB is 946132.

4. The construction method according to claim 1, characterized in that: The fucose module gene includes a phosphomannose mutase encoding gene manB, a mannose-1-phosphate guanyl transferase encoding gene manC, a GDP-mannose-6-dehydrogenase encoding gene gmd and a GDP-fucose synthase encoding gene fcl; the Gene IDs of the phosphomannose mutase encoding gene manB, the mannose-1-phosphate guanyl transferase encoding gene manC, the GDP-mannose-6-dehydrogenase encoding gene gmd and the GDP-fucose synthase encoding gene fcl are 946574, 946580, 946562 and 946563 respectively.

5. The construction method according to claim 1, characterized in that: The glycosyltransferase module gene includes an α1,2-fucosyltransferase encoding gene wbgL; the α1,2-fucosyltransferase encoding gene wbgL is derived from Escherichia coli O126; the Genebank accession number of the α1,2-fucosyltransferase is ADN43847.

1.

6. The construction method according to any one of claims 1 or 4 to 5, characterized in that: The integrated fucose module gene and glycosyltransferase module gene are respectively connected in series with the inducible promoter Ptac to form an expression cassette Ptac-gmd-fcl-manC-manB and an expression cassette Ptac-wbgL, and the expression cassette Ptac-gmd-fcl-manC-manB and the expression cassette Ptac-wbgL are integrated into the Escherichia coli genome by using CRISPR transposition technology for expression; the nucleotide sequences of the expression cassette Ptac-gmd-fcl-manC-manB and the expression cassette Ptac-wbgL are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the expression cassette Ptac-gmd-fcl-manC-manB and the expression cassette Ptac-wbgL are integrated into the Escherichia coli genome in single or multiple copies.

7. The construction method according to claim 1, characterized in that: The knockout or interruption of the glucose kinase encoding gene glk is to use CRISPR / Cas9 technology to knock out or use CRISPR transposition technology to interrupt the glucose kinase encoding gene glk in the Escherichia coli genome; the knockout or interruption of the pyruvate oxidase encoding gene poxB is to use CRISPR / Cas9 technology to knock out or use CRISPR transposition technology to interrupt the pyruvate oxidase encoding gene poxB in the Escherichia coli genome.

8. The genetically engineered bacteria constructed by the construction method according to any one of claims 1 to 7.

9. Use of the genetically engineered bacteria according to claim 8 in the fermentation production of 2'-fucosyllactose.

10. The use according to claim 9, characterized in that: The seed liquid of the genetically engineered bacteria was inoculated into the fermentation medium and cultured until OD 600 The value is 0.6-0.8, and then IPTG inducer and lactose are added to the fermentation medium to ferment and synthesize 2′-fucosyllactose.

11. The use according to claim 10, characterized in that: The fermentation medium is a glycerol-containing medium; the addition amounts of the lactose and the IPTG inducer are 5-10 g / L and 0.1-0.3 mM respectively; the fermentation synthesis conditions are: fermentation at 28-30° C. and 200-220 rpm for 48-72 hours.

12. Use of the genetically engineered bacteria according to claim 8 in the preparation of a product containing 2′-fucosyllactose, characterized in that: The product is a food additive or infant food.

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