The method comprises the following steps: synthesizing 2apos from glucose and xylose; construction and application of genetically engineered bacterium of fucosyllactose

Abstract

The invention discloses construction and application of a genetically engineered bacterium for synthesizing 2 '-fucosyllactose from glucose and xylose, and belongs to the field of microbial genetic engineering and the field of biosynthesis. According to the invention, escherichia coli is taken as an original strain, and the productivity of engineering bacteria is improved through strategies such as reconstruction of a glucose transport pathway, knockout of a competitive bypass, elimination of a byproduct pathway, up-regulation of key enzymes in a de novo synthesis pathway, enhancement of xylose utilization, overexpression of efficient alpha 1, 2-fucosyltransferase and the like; glucose and xylose are used as substrates to realize efficient synthesis of lactose and 2 '-fucosyllactose. In a shake flask fermentation experiment process, the yield of the 2 '-fucosyllactose can reach 6.53 g / L under the condition that no exogenous lactose is added. Under the condition of batch feeding, the yield of 2 '-fucosyllactose in a 5L fermentation tank reaches 27.53 g / L, and the method has industrial production potential.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and biosynthesis, and specifically relates to the construction and application of a genetic engineering bacterium for synthesizing 2'-fucosyllactose from glucose and xylose. Background Art

[0002] Human milk oligosaccharides (HMOs), also known as human milk oligosaccharides, are abundant in breast milk. Among them, 2′-fucosyllactose (2′-FL) is one of the most abundant components in mature human milk, accounting for about 31% of the total oligosaccharides. Structurally, 2′-fucosyllactose is composed of L-fucose, D-galactose and D-glucose, expressed as Fucα1,2Galβ1,4Glc. The health benefits of 2′-fucosyllactose have been widely studied, including prebiotic effects, immune regulation, intestinal protection, antibacterial and antiviral effects, and brain development and cognitive enhancement. 2′-fucosyllactose is the first HMO to be commercially added to infant formula. Safety assessments have confirmed that 2′-fucosyllactose has no acute oral toxicity, genotoxicity, and subchronic toxicity. It has been recognized and approved as an effective infant formula ingredient by many authoritative agencies such as the U.S. Food and Drug Administration (FDA) and the European Union, paving the way for its use in infant formula and other nutritional products designed to mimic the benefits of human milk.

[0003] At present, the production of 2′-fucosyllactose mainly includes chemical synthesis, enzyme synthesis and engineered microbial fermentation. Among them, the engineered microbial fermentation method is widely used because of its convenient operation, low cost and mild reaction conditions. However, the engineered bacteria that produce 2′-fucosyllactose currently mainly use lactose or fucose as substrates, and need exogenous lactose or fucose supply, which has high raw material costs and reduces the economic benefits of 2′-fucosyllactose. To this end, Li et al. constructed a de novo biosynthetic pathway of 2′-FL by introducing a lactose production module, and efficiently produced 2′-FL using glycerol and glucose. Parschat et al. used sucrose as a raw material to efficiently produce 2′-FL. The strain can produce lactose intracellularly and further use it for the production of 2′-FL. Although these sugars reduce the production cost of 2′-FL to a certain extent compared with lactose and fucose, they still have the defect of high raw material prices. In addition, these methods also need to add antibiotics and inducers during the fermentation synthesis process, which further increases the production cost of 2′-FL.

[0004] Therefore, constructing a genetically engineered bacterium that can produce 2′-fucosyllactose using cheap sugar as a substrate without adding antibiotics and inducers is of great significance for reducing the production cost of 2′-fucosyllactose. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a construction and application of a genetically engineered bacterium for synthesizing 2′-fucosyllactose from glucose and xylose. The bacterium uses xylose and glucose as substrates and does not add lactose, antibiotics or inducers, thereby greatly reducing the production cost.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A lactose-producing genetically engineered bacterium using glucose and xylose as substrates, using Escherichia coli as the starting strain, knocking out the β-galactosidase gene lacZ on the Escherichia coli genome, and knocking out the glucose-specific transporter EIICB Glc The component encoding gene ptsG, the glucose transporter glf was integrated and expressed at the ptsG site, the glucose kinase gene glk was knocked out, the β-1,4-galactosyltransferase NmlgtB was integrated and expressed at the glk site, the UDP-glucose-6-dehydrogenase gene ugd was knocked out, the UDP-glucose-4-isomerase gene galE was integrated and expressed at the ugd site, and the arsenite / antimonate: H was knocked out + The antiporter gene arsB converts D-xylose to H + The symporter gene xylE was integrated into the arsB site to obtain a lactose-producing genetically engineered bacterium.

[0008] As a further technical solution, the glucose transporter glf is derived from Zymomonas mobilis, with the NCBI sequence number AAA27691.1 and the nucleotide sequence as SEQ ID NO.1; the glucose transporter glf is driven by a strong constitutive promoter P J23119 Start expression;

[0009] As a further technical solution, β-1,4-galactosyltransferase NmlgtB is derived from Neisseria meningitidis, with the NCBI sequence number AAC44085.1 and the nucleotide sequence as SEQ ID NO.2; β-1,4-galactosyltransferase NmlgtB is expressed by a strong constitutive promoter P J23119 Start expression;

[0010] As a further technical solution, the UDP-glucose-4-epimerase gene galE is derived from Escherichia coli K-12MG1655, the NCBI sequence number is NP_415280.3, and the nucleotide sequence is shown in SEQ ID NO.3; the UDP-glucose-4-epimerase gene galE is driven by a strong constitutive promoter P J23119Start expression;

[0011] As a further technical solution, D-xylose: H + The symporter gene xylE is derived from Escherichia coli BL21 (DE3), and its NCBI sequence number is NP_418445.1. Its nucleotide sequence is shown in SEQ ID NO.6. D-xylose: H + The symporter gene xylE is driven by a strong constitutive promoter P J23119 Start expression.

[0012] As a further technical solution, the Escherichia coli includes but is not limited to Escherichia coli BL21 (DE3), and may also be Escherichia coli K-12MG1655, Escherichia coli BW25113, Escherichia coli W3110, and the like.

[0013] A 2'-fucosyllactose producing genetically engineered bacterium using glucose and xylose as substrates is provided. The genetically engineered bacterium with high lactose production is used as a starting strain, the D-lactate dehydrogenase gene ldhA is knocked out, and α1,2-fucosyltransferase BKHT is integrated and expressed at the ldhA site.

[0014] As a further technical solution, α1,2-fucosyltransferase BKHT is derived from Helicobacter pylori 11S02629-2, with NCBI sequence number PAF50342.1, and its nucleotide sequence is shown in SEQ ID NO.5; α1,2-fucosyltransferase BKHT is expressed by a strong constitutive promoter P J23119 Start expression.

[0015] As a further technical solution, the UDP-glucose lipid carrier transferase gene wcaJ was knocked out.

[0016] As a further technical solution, a strong constitutive promoter P J23119 Replace the original promoters of the phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB and the GDP-mannose-6-dehydratase-GDP-fucose synthase gene cluster gmd-wcaG on the genome.

[0017] As a further technical solution, the strong constitutive promoter P J23119 The nucleotide sequence is shown in SEQ ID NO.4.

[0018] As a further technical solution, the β-galactosidase gene lacZ has the NCBI sequence number NP_414878.1;

[0019] As a further technical solution, the glucose-specific transporter EIICB GlcThe component encoding gene ptsG has the NCBI sequence number NP_415619.1;

[0020] As a further technical solution, the NCBI sequence number of the glucokinase gene glk is NP_416889.1;

[0021] As a further technical solution, the UDP-glucose-6-dehydrogenase gene ugd is in the NCBI sequence number NP_416532.1. As a further technical solution, arsenite / antimonate: H + The antiporter gene arsB has the NCBI sequence number NP_417959.4;

[0022] As a further technical solution, the D-lactate dehydrogenase gene ldhA has a NCBI sequence number of NP_415898.1;

[0023] As a further technical solution, the UDP-glucose lipid transferase encoding gene wcaJ has the NCBI sequence number NP_416551.1;

[0024] As a further technical solution, in the phosphomannose mutase-mannose-1-phosphate guanyl transferase gene cluster, the mannose 1-guanylate transferase encoding gene manC has a NCBI sequence number of NP_416553.1, and the phosphomannose encoding gene manB has a NCBI sequence number of NP_416552.1;

[0025] As a further technical solution, in the GDP-mannose-6-dehydratase-GDP-fucose synthase gene cluster, the GDP-D-mannose-4,6-dehydratase encoding gene gmd has a NCBI sequence number of NP_416557.1, and the GDP-fucose synthase encoding gene wcaG has a NCBI sequence number of NP_416556.1.

[0026] The lactose-producing genetically engineered bacteria is used in the fermentation production of lactose.

[0027] The 2'-fucosyllactose-producing genetically engineered bacteria is used in the fermentation production of 2'-fucosyllactose.

[0028] A method for synthesizing 2′-fucosyllactose using glucose and xylose as substrates, wherein the 2′-fucosyllactose-producing genetically engineered bacteria according to any one of claims 4 to 7 are fermented and cultured using glucose and xylose as substrates;

[0029] The fermentation was carried out in a shake flask, and the fermentation medium contained 20 g / L glucose and 10 g / L xylose. The process conditions of the shake flask fermentation culture were as follows: fermentation culture at 35-38°C until OD 600After the pH value reaches 0.6-0.8, continue culturing at 22-25℃ for at least 72h;

[0030] Alternatively, a fermentation tank is used for fermentation culture, and the initial fermentation tank culture medium contains 30 g / L xylose or 25 g / L xylose and 5 g / L glucose. The fermentation conditions of the fermentation tank are: fermentation culture at 35-38°C until the xylose is completely consumed, and then feed xylose is added to keep the final xylose concentration at 2-5 g / L, and culture is continued until OD 600 Reach about 15, then cool to 22-25°C, while continuing to add glucose as a substrate to make the final concentration of glucose 5-10g / L, and continue fermentation until the total duration is at least 72h; during the fermentation culture, the pH is maintained at 6.8±0.2 throughout the process, and the foam is controlled by adding a defoamer. The dissolved oxygen is controlled by adjusting the stirring speed (100-900rpm) and the ventilation volume (2-8vvm).

[0031] As a further technical solution, the glucose and xylose are derived from an enzymatic hydrolysis mixture of lignocellulose.

[0032] As a further technical solution, the fermentation medium of the genetically engineered bacteria producing 2′-fucosyllactose contains 20 g / L glucose and 10 g / L xylose;

[0033] As a further technical solution, the fermentation medium of the genetically engineered bacteria producing 2′-fucosyllactose also contains 5.0 g / L yeast extract, 4.0 g / L diammonium hydrogen phosphate, 13.5 g / L potassium dihydrogen phosphate, 1.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid and 10 ml / L trace metal elements; the trace metal elements include: 0.35 g / L manganese sulfate monohydrate, 10 g / L ferrous sulfate, 1.0 g / L anhydrous copper sulfate, 2.25 g / L zinc sulfate heptahydrate, 2.0 g / L calcium chloride dihydrate, 0.23 g / L sodium borate decahydrate and 0.11 g / L ammonium molybdate.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention introduces a method independent of PTS Glc NPTS for transfer systems Glc The production capacity of the engineered bacteria was improved by knocking out the competitive bypass, strengthening the promoter of key enzymes, overexpressing α1,2-fucosyltransferase, eliminating the by-product pathway, and other strategies. With glucose and xylose as substrates, the efficient synthesis of lactose and 2′-fucosyllactose was achieved, greatly reducing the cost of raw materials.

[0036] 2. Lignocellulosic raw materials are the most abundant renewable resources in the world. They can be used as raw materials for microbial fermentation to produce high-value-added biochemicals, and they do not compete with the global food supply. They are very promising biological production raw materials. Glucose, as a component of cellulose, is the most common monosaccharide obtained from lignocellulosic biomass. Hemicellulose with xylose as the main component accounts for about 20-40% of lignocellulosic biomass; after pretreatment and enzymatic hydrolysis, lignocellulose hydrolysate is a mixed sugar containing glucose and xylose. The development of lactose and 2′-fucosyllactose-producing engineered bacteria using glucose and xylose as substrates in the present invention has laid a foundation for the high-value utilization of lignocellulose and greatly reduced the raw material cost of lactose and 2′-fucosyllactose synthesis.

[0037] 3. The recombinant strain of the present invention is constitutively integrated and expressed, and does not require induction by an inducer or the addition of antibiotics, which further reduces the raw material cost of 2′-fucosyllactose production. In addition, it also avoids the contamination of the product by the addition of inducers and antibiotics, reduces the removal steps of inducers and antibiotics during product extraction and purification, and is more conducive to the industrial application of 2′-fucosyllactose.

[0038] In summary, the present invention constructs an efficient and reasonable de novo synthesis of 2′-fucosyllactose pathway in Escherichia coli BL21 (DE3). The recombinant Escherichia coli obtained by genetic engineering and metabolic engineering strategy design and transformation can use glucose and xylose as substrates. Under the condition of no exogenous lactose addition, the yield of 2′-fucosyllactose in shake flask fermentation reaches 6.53 g / L, and no antibiotics and inducers are required to be added during the entire fermentation process. The present invention has the potential for scaled-up production, commercial application value and good industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Metabolic pathway for 2′-fucosyllactose biosynthesis in recombinant Escherichia coli. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0042] In the present invention,

[0043] 1. The plasmids, endonucleases, PCR enzymes, column DNA extraction kits, DNA gel recovery kits and other commercial products used were operated according to the kit instructions. Colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition). The sequencing of plasmids and DNA products was completed by (Suzhou) GeneWeichi.

[0044] 2. Culture medium

[0045] (1) LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride.

[0046] (2) LB solid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 15 g / L agar powder.

[0047] (3) Shake flask fermentation medium: 20 g / L glucose, 10 g / L xylose, 5 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; the trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate.

[0048] (4) Fermentation medium A in the initial fermenter: 25 g / L xylose, 5 g / L glucose, 5 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; the trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate.

[0049] (5) Initial fermentation tank fermentation medium B: The difference from the primary fermentation tank fermentation medium A is that it does not contain glucose and the concentration of xylose is 30 g / L.

[0050] 3. Detection of 2′-fucosyllactose, lactose, glucose and xylose in fermentation broth

[0051] Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, and take the supernatant for HPLC determination.

[0052] HPLC detection conditions: by high performance liquid chromatography (HPLC) system (Waters e2695); chromatographic column: RezexROA-Organic acid H + (8%); detector: Waters 2414RIDetector differential detector; mobile phase: 5mM H2SO4; flow rate: 0.6mL / min; column temperature: 60℃; injection volume: 10μL.

[0053] 4. Plasmid

[0054] The dual-plasmid gene editing system pEcCpf1 / pcrEG has been published in the literature: Zhu X, Wu Y, Lv X. Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli[J]. ACS Synthetic Biology, 2022(5):11

[0055] 5. The strains constructed by the present invention are shown in Table 1

[0056] Table 1

[0057]

[0058] 6. In the present invention, the raw materials used are commercially available unless otherwise specified.

[0059] Example 1: Construction of lactose-producing strains

[0060] Escherichia coli uptakes glucose mainly through two pathways, namely the phosphotransferase system (PTS Glc ) and nonphosphotransferase system (NPTS Glc ). PTS of Escherichia coli Glc The system is responsible for the specific transport of glucose from the extracellular membrane into the cell, and in the process generates glucose-6-phosphate for intracellular metabolism, which is the main way of glucose transport. Glc After the system is inactivated, the galactose:H + NPTS consists of a cotransporter (encoded by the galP gene) and a glucose kinase (encoded by the glk gene). GlcIn addition, the glf glucose facilitator protein (encoded by the glf gene) from Zymomonas mobilis can transport glucose when the glucose concentration is high, which does not require the consumption of ATP and H + The synergistic participation of ions.

[0061] In the culture medium with mixed carbon sources, E. coli exhibits a specific carbon source utilization order, a phenomenon known as "Carbon catabolite repression" (CCR). Glucose is usually used first, while the expression of transporters and metabolic enzymes of secondary carbon sources such as xylose is inhibited, thus ensuring that E. coli first uses glucose, and only starts to use other carbon sources after it is exhausted.

[0062] Using Escherichia coli BL21 (DE3) as the starting strain, the pEcCpf1 / pcrEG gene editing system (derived from the CRISPR-Cpf1 system) was used to knock out the β-galactosidase gene lacZ; the glucose-specific transporter protease EIICB was knocked out Glc The component encodes the gene ptsG, and a strong constitutive promoter P is integrated and expressed at the ptsG locus. J23119 Expression of glucose transporter glf.

[0063] Efficient expression of β-1,4-galactosyltransferase (β-1,4-GalT) is essential for the synthesis of lactose. Therefore, the glucokinase gene glk was knocked out and a strong constitutive promoter P was integrated and expressed at the glk locus. J23119 The expressed β-1,4-galactosyltransferase NmlgtB is from Neisseria meningitidis.

[0064] In order to inhibit the competitive pathway of UDP-galactose degradation, the UDP-glucose-6-dehydrogenase gene ugd was knocked out. At the same time, in order to increase the production of UDP-galactose, a strong constitutive promoter P was integrated and expressed at the ugd locus. J23119 The UDP-glucose-4-epimerase gene galE was expressed to obtain the EC-BL-01 strain.

[0065] EC-BL-01 strain construction. Required primers are shown in Table 2.

[0066] For more information about the CRISPR / Cpf1 gene editing system, please refer to Zhu X, Wu Y, Lv X. Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichiacoli[J]. ACSS Synthetic Biology, 2022(5):11.

[0067] Table 2 Primers for construction of EC-BL-01 strain

[0068]

[0069]

[0070]

[0071]

[0072] The specific steps are as follows:

[0073] Step 1: Construction of BL21(DE3)ΔlacZ engineered bacteria

[0074] (1) pEcCpf1 was plasmidized and transformed into Escherichia coli BL21 (DE3). The single colony grown above was inoculated into a test tube containing 4 mL of LB medium containing kanamycin resistance and cultured for 12 h to prepare electrocompetent cells.

[0075] (2) Using the BL21 (DE3) genome as a template, primers lacZ-UH-F / R and lacZ-DH-F / R were used to amplify the upstream and downstream fragments of the lacZ gene, respectively, and then subjected to gel electrophoresis and gel recovery and purification; primers lacZ-ol-F and lacZ-ol-R were used to perform fusion PCR amplification of the above two gene fragments to obtain a complete template gene fragment.

[0076] (3) Using the original commercial plasmid pcrEG as a template, the pcrEG-lacZ plasmid was obtained using primers lacZ-N23-F / R loop P.

[0077] (4) Take BL21 (DE3) electroporation competent cells containing pEcCpf1 plasmid on ice, thaw, add 200ng of pcrEG-lacZ plasmid and 800ng of template DNA fragment, mix well and transfer to the slit of a fully pre-cooled 2mm electroporation cup, and let it stand on ice. Place the electroporation cup in the electroporator, electroporate at 2.5kV for about 0.5ms, and immediately add 1mL of fully pre-cooled LB medium, and culture at 37℃ and 200r / min for 2h. Then centrifuge at 3,500rpm for 3min, discard the supernatant, mix the cells and spread them all on the double-antibody plate (final concentration is 50μg / L kanamycin and 50μg / L spectinomycin), and culture them upside down in a 37℃ incubator for 12-24h.

[0078] (5) Use colony PCR to verify the grown single colonies, and the verification primers are lacZ-YZ-F and lacZ-YZ-R. If the verification band is shorter than the negative control band, the gene knockout may be successful. The colonies that have been successfully verified by colony PCR are amplified by PCR with primers lacZ-YZ-F / R and sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing.

[0079] (6) The above positive clone colonies were picked into a 4 mL LB liquid test tube (containing rhamnose at a final concentration of 10 mM and 4 μL Kan), and cultured at 37°C for 12 h to remove the pcrEG-lacZ plasmid. The recombinant strain that was successfully removed was then picked into a 4 mL LB liquid test tube (containing glucose at a final concentration of 5 g / L), and cultured at 37°C for 12 h to remove the pEcCpf1 plasmid. The recombinant E. coli BL21 (DE3) ΔlacZ engineered bacteria was obtained after successful removal of the pEcCpf1 plasmid.

[0080] Step 2, BL21(DE3)ΔlacZΔptsG::P J23119 -Construction of GLF engineering bacteria

[0081] (1) pEcCpf1 was plasmidized and transformed into Escherichia coli BL21 (DE3) ΔlacZ. The single colony grown above was inoculated into a test tube containing 4 mL of LB medium containing kanamycin resistance and cultured for 12 h to prepare electrocompetent cells.

[0082] (2) Using the BL21 (DE3) genome as a template, primers ptsG-UH-F / R and ptsG-DH-F / R were used to amplify the upstream and downstream fragments of the ptsG gene, respectively, and then subjected to gel electrophoresis and gel recovery purification; using the pCO-glf plasmid as a template, primers glf-F / R were used to amplify the glf gene fragment; primers ptsG-ol-F and ptsG-ol-R were used to perform fusion PCR amplification of the above three gene fragments to obtain a complete template gene fragment.

[0083] (3) Using the original commercial plasmid pcrEG as a template, the pcrEG-ptsG plasmid was obtained using primers ptsG-N23-F / R loop P.

[0084] (4) Take BL21(DE3)ΔlacZ electroporation competent cells containing pEcCpf1 plasmid on ice, thaw, add 200ng of pcrEG-ptsG plasmid and 800ng of template DNA fragment, mix well and transfer to the slit of a fully pre-cooled 2mm electroporation cup, and let it stand on ice. Place the electroporation cup in the electroporator, electroporate at 2.5kV for about 0.5ms, and immediately add 1mL of fully pre-cooled LB medium, and culture at 37℃ and 200r / min for 2h. Then centrifuge at 3,500rpm for 3min, discard the supernatant, mix the cells and spread them all on the double-antibody plate (final concentration is 50μg / L kanamycin and 50μg / L spectinomycin), and culture them upside down in a 37℃ incubator for 12-24h.

[0085] (5) Use colony PCR to verify the grown single colonies, and the internal verification primers are ptsG-YZ-F and glf-YZ-R. If there are bands in the internal verification, the gene integration may be successful. For the colonies that have been successfully verified by colony PCR, use the external verification primers ptsG-YZ-F / R to perform PCR colony verification, and use primers ptsG-YZ-F / R for PCR amplification and then send to Suzhou Genewise Biotechnology Co., Ltd. for sequencing.

[0086] (6) The above positive clone colonies were picked into a 4 mL LB liquid test tube (containing rhamnose at a final concentration of 10 mM and 4 μL Kan), and cultured at 37°C for 12 h to remove the pcrEG-ptsG plasmid. The recombinant strain that was successfully removed was then picked into a 4 mL LB liquid test tube (containing glucose at a final concentration of 5 g / L), and cultured at 37°C for 12 h to remove the pEcCpf1 plasmid. The recombinant E. coli BL21 (DE3) ΔlacZΔptsG::P was obtained after successful removal of the pEcCpf1 plasmid. J23119 -glf engineered bacteria.

[0087] Step 3, BL21(DE3)ΔlacZΔptsG::P J23119 -glfΔglk::P J23119 -Genome construction of NmlgtB

[0088] (1) The strain to be transformed, BL21(DE3)ΔlacZΔptsG::P containing the pEcCpf1 plasmid, was transformed into J23119 -glf were prepared into electroporation competent cells.

[0089] (2) Using the BL21 (DE3) genome as a template, primers glk-UH-F / R and glk-DH-F / R were used to amplify the upstream and downstream fragments of the glk gene, respectively, and then subjected to gel electrophoresis and gel recovery purification; using the pET-NmlgtB plasmid as a template, primers lgtB-F / R were used to amplify the lgtB gene fragment; primers glk-ol-F and glk-ol-R were used to perform fusion PCR amplification of the above three gene fragments to obtain a complete template gene fragment.

[0090] (3) Using the original commercial plasmid pcrEG as a template, the pcrEG-glk plasmid was obtained using primers glk-N23-F / R loop P.

[0091] (4) The pcrEG-glk plasmid and donor DNA fragment were electroporated into the competent cells of the host to be transformed, spread on LB plates containing kanamycin and spectinomycin, and cultured at 37°C for 24 h.

[0092] (5) Use colony PCR to verify the grown single colonies, and the internal verification primers are glk-YZ-F and lgtB-YZ-R. If there are bands in the internal verification, the gene integration may be successful. The colonies that have been successfully verified by colony PCR are verified by PCR colonies using external verification primers glk-YZ-F / R, and PCR amplification is performed with primers glk-YZ-F / R and sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing.

[0093] (6) The positive clone colonies were transferred to a 4 mL LB liquid test tube (containing a final concentration of 10 mM rhamnose and 4 μL Kan) and cultured at 37°C for 12 h to remove the pcrEG-glk plasmid. The recombinant strain that was successfully removed was transferred to a 4 mL LB liquid test tube (containing a final concentration of 5 g / L glucose) and cultured at 37°C for 12 h to remove the pEcCpf1 plasmid. The recombinant E. coli BL21 (DE3) ΔlacZΔptsG::P was obtained after successful removal of the pEcCpf1 plasmid. J23119 -glfΔglk::P J23119 -NmlgtB.

[0094] Step 4, BL21(DE3)ΔlacZΔptsG::P J23119 -glfΔglk::P J23119 -NmlgtBΔugd::P J23119 -GalE genome construction

[0095] (1) The strain to be transformed, BL21(DE3)ΔlacZΔptsG::P containing the pEcCpf1 plasmid, was transformed into J23119 -glfΔglk::P J23119-NmlgtB were prepared into electroporation competent cells.

[0096] (2) Using the BL21 (DE3) genome as a template, primers ugd-UH-F / R and ugd-DH-F / R were used to amplify the upstream and downstream fragments of the ugd gene, respectively, and then subjected to gel electrophoresis and gel recovery and purification; using the Escherichia coli K-12MG1655 genome as a template, primers galE-F / R were used to amplify the galE gene fragment; primers ugd-ol-F and ugd-ol-R were used to perform fusion PCR amplification of the above three gene fragments to obtain a complete template gene fragment.

[0097] (3) Using the original commercial plasmid pcrEG as a template, the pcrEG-ugd plasmid was obtained using primers ugd-N23-F / R loop P.

[0098] (4) The pcrEG-ugd plasmid and donor DNA fragment were electroporated into the competent cells of the host to be transformed, spread on LB plates containing kanamycin and spectinomycin, and cultured at 37°C for 24 h.

[0099] (5) Use colony PCR to verify the grown single colonies, and the internal verification primers are ugd-YZ-F and galE-YZ-R. If there are bands in the internal verification, the gene integration may be successful. The colonies that have been successfully verified by colony PCR are verified by PCR colonies using external verification primers ugd-YZ-F / R, and PCR amplification is performed with primers ugd-YZ-F / R and sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing.

[0100] (6) The above positive clone colonies were picked into a 4 mL LB liquid test tube (containing rhamnose at a final concentration of 10 mM and 4 μL Kan), and cultured at 37°C for 12 h to remove the pcrEG-ugd plasmid. The recombinant strain that was successfully removed was then picked into a 4 mL LB liquid test tube (containing glucose at a final concentration of 5 g / L), and cultured at 37°C for 12 h to remove the pEcCpf1 plasmid. The recombinant E. coli EC-BL-01 was obtained after successful removal of the pEcCpf1 plasmid.

[0101] Example 2: Strengthening xylose utilization and promoting lactose production

[0102] When xylose and glucose coexist in the external environment, the glucose effect inhibits xylose utilization. There are two xylose-specific transporters in Escherichia coli, one is the ATP-binding high-affinity transporter XylFGH (encoded by xylFGH), which consumes one molecule of ATP while transporting xylose; the other is the low-affinity transporter XylE (encoded by xylE), which is a H +Proton symporter, does not consume ATP. To enhance xylose utilization, the expression of xylE was enhanced in three ways based on the EC-BL-01 strain. The recombinant plasmid pCDFDuet-xylE was introduced under the control of the T7 promoter to obtain the EC-BL-02 strain; the strong constitutive promoter P J23119 Substitution code D-xylose: H + The original promoter of the synthase xylE gene was obtained to obtain the EC-BL-03 strain; the strong constitutive promoter P was integrated at the arsB site on the genome J23119 Controls the activation of the coding D-xylose:H + The xylE gene of the synthetase (the xylE gene comes from Escherichia coli BL21 (DE3), and during integration, the original xylE gene on Escherichia coli is still retained) was used to obtain the EC-BL-04 strain.

[0103] The primers used to construct strains EC-BL-02, EC-BL-03, and EC-BL-04 are shown in Table 3 ;

[0104] Table 3 Primers for strains EC-BL-02, EC-BL-03 and EC-BL-04

[0105]

[0106]

[0107] Example 3: Construction of 2′-fucosyllactose engineered strain

[0108] In order to further utilize xylose and glucose to synthesize 2′-fucosyllactose, the strong promoter P was integrated into the ldhA locus of the recombinant strain EC-BL-04 genome. J23119 The EC-BL-05 strain was obtained by controlling the activation of the α1,2-fucosyltransferase BKHT. The typical competitive pathway gene wcaJ encoding UDP-glucose lipid carrier transferase was knocked out to prevent the conversion of GDP-fucose to colanic acid, and the EC-BL-06 strain was obtained. In order to further improve the biosynthesis of 2′-FL, the present invention strengthens the synthesis of GDP-fucose and uses a strong constitutive promoter P J23119 The original promoter was replaced, and the gene clusters manC-manB and gmd-wcaG were strengthened respectively to obtain strains EC-BL-07 and EC-BL-08. The two gene clusters manC-manB and gmd-wcaG were strengthened at the same time to obtain strain EC-BL-09.

[0109] The primers for constructing EC-BL-05, EC-BL-06, EC-BL-07, EC-BL-08, and EC-BL-09 are shown in Table 4 ;

[0110] Table 4 Primers for constructing strains EC-BL-05, 06, 07, 08, and 09

[0111]

[0112]

[0113]

[0114] Example 4: Shake flask fermentation of lactose and 2′-fucosyllactose

[0115] The engineered bacteria EC-BL-01, EC-BL-02, EC-BL-03, EC-BL-04, EC-BL-05, EC-BL-06, EC-BL-07, EC-BL-08 and EC-BL-09 constructed in Examples 1-3 were inoculated into LB liquid medium without antibiotics, cultured overnight at 37°C, 200 rpm for 12 h to obtain seed solution, 0.5 mL of seed solution was inoculated into 25 mL of fermentation medium, and cultured at 37°C, 200 rpm until OD 600 When the pH value is 0.6-0.8, transfer to 25°C and continue culturing at 200 rpm for 72 h. Take 1 mL of the fermentation broth, centrifuge at 10,000 rpm for 10 min, and take the supernatant for HPLC determination.

[0116] The results are shown in Table 5. Under the shake flask fermentation culture conditions without the addition of antibiotics and inducers, the lactose production of 6.58 g / L and the 2′-fucosyllactose production of 6.53 g / L can be achieved after 72 h of shake flask culture.

[0117] Table 5 Detailed information of shake flask fermentation of E. coli in different engineering

[0118] Strain name 2′-FL(g / L) Lactose (g / L) Glucose (g / L) Xylose(g / L) <![CDATA[OD 600 ]]> EC-BL-01 / 4.74 / 0.69 12.16 EC-BL-02 / 5.95 / 0.44 8.79 EC-BL-03 / 4.60 / 0.31 9.87 EC-BL-04 / 6.58 / 0.35 10.78 EC-BL-05 3.92 3.09 3.28 0.32 9.37 EC-BL-06 4.30 2.46 3.15 0.36 9.26 EC-BL-07 5.66 1.18 0.68 0.54 8.88 EC-BL-08 5.15 1.52 1.16 0.44 8.70 EC-BL-09 6.53 0.63 1.14 0.50 8.60

[0119] Example 5: Optimization of conditions for industrial fermentation of EC-BL-09 to synthesize 2′-fucosyllactose

[0120] The strain EC-BL-09 was selected and a 5L fermenter was used to carry out a fed-batch fermentation experiment of 2′-fucosyllactose.

[0121] The strain EC-BL-09 was inoculated into 4 mL LB medium and cultured overnight at 37°C, 200 rpm for 12 h to obtain the primary seed solution. 1 mL of the primary seed solution was inoculated into 100 mL LB medium and cultured at 37°C, 200 rpm until the OD 600The secondary seed solution was 1.5, and 400 mL of the secondary seed solution was inoculated into a 5 L fermenter containing 2 L of initial fermenter medium A for fermentation. The initial temperature was maintained at 37 °C. The initial fermentation medium contained 25 g / L xylose and 5 g / L glucose as mixed carbon sources. When the 25 g / L xylose in the initial medium was completely consumed, feed xylose was started to maintain the final xylose concentration in the fermentation system at 2-5 g / L to meet cell growth. When OD 600 When the temperature reaches about 15, the temperature is lowered to 25°C, and glucose is added as a substrate to maintain the final glucose concentration in the fermentation system at 5-10 g / L, and the fermentation is continued until the total duration is 72 hours. During the fermentation process, the pH is maintained at 6.8±0.2, and the foam is controlled by adding a defoamer, and the dissolved oxygen is controlled by adjusting the stirring speed (100-900 rpm) and the ventilation volume (2-8 vvm).

[0122] After 72 h of fermentation, the yield of 2′-fucosyllactose reached 25.69 g / L, and the OD 600 The highest reached 78.

[0123] Example 6: Optimization of conditions for industrial fermentation of EC-BL-09 to synthesize 2′-fucosyllactose

[0124] The same as Example 5, except that the initial fermentation tank medium A is replaced by the initial fermentation tank medium B;

[0125] After 72 h of fermentation, the yield of 2′-fucosyllactose reached 27.53 g / L, and the OD 600 The highest reached 75.

[0126] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A lactose-producing genetically engineered bacterium using glucose and xylose as substrates, characterized in that: Using Escherichia coli as the starting strain, the β-galactosidase gene lacZ on the Escherichia coli genome was knocked out, and the glucose-specific transporter EIICB was knocked out Glc The component encoding gene ptsG, the glucose transporter glf was integrated and expressed at the ptsG site, the glucose kinase gene glk was knocked out, the β-1,4-galactosyltransferase NmlgtB was integrated and expressed at the glk site, the UDP-glucose-6-dehydrogenase gene ugd was knocked out, the UDP-glucose-4-isomerase gene galE was integrated and expressed at the ugd site, and the arsenite / antimonate: H was knocked out + The antiporter gene arsB converts D-xylose to H + The symporter gene xylE was integrated into the arsB site to obtain a lactose-producing genetically engineered bacterium.

2. The lactose-producing genetically engineered bacteria using glucose and xylose as substrates according to claim 1, characterized in that: The glucose transporter glf is derived from Zymomonas mobilis, and its nucleotide sequence is shown in SEQ ID NO.1; the glucose transporter glf is driven by a strong constitutive promoter P J23119 Start expression; β-1,4-galactosyltransferase NmlgtB is derived from Neisseria meningitidis, and its nucleotide sequence is shown in SEQ ID NO.2; β-1,4-galactosyltransferase NmlgtB is expressed by a strong constitutive promoter P J23119 Start expression; The UDP-glucose-4-epimerase gene galE is derived from Escherichia coli K-12MG1655 (Escherichiacoli), and its nucleotide sequence is shown in SEQ ID NO.3; the UDP-glucose-4-epimerase gene galE is driven by a strong constitutive promoter P J23119 Start expression; D-Xylose: H + The symporter gene xylE is derived from Escherichia coli BL21 (DE3), and its nucleotide sequence is shown in SEQ ID NO.

6. + The symporter gene xylE is driven by a strong constitutive promoter P J23119 Start expression.

3. The lactose-producing genetically engineered bacteria using glucose and xylose as substrates according to claim 1, characterized in that: The Escherichia coli includes one or more of Escherichia coli BL21 (DE3), Escherichia coli K-12MG1655, Escherichia coli BW25113, and Escherichia coli W3110.

4. A genetically engineered bacterium that produces 2′-fucosyllactose using glucose and xylose as substrates, characterized in that: The high lactose-producing genetically engineered bacteria according to any one of claims 1 to 3 are used as the starting strain, the D-lactate dehydrogenase gene ldhA is knocked out, and the α1,2-fucosyltransferase BKHT is integrated and expressed at the ldhA site.

5. The genetically engineered bacteria producing 2′-fucosyllactose using glucose and xylose as substrates according to claim 4, characterized in that: α1,2-fucosyltransferase BKHT is derived from Helicobacter pylori 11S02629-2, with the NCBI sequence number PAF50342.1, and its nucleotide sequence is shown in SEQ ID NO.5; α1,2-fucosyltransferase BKHT is expressed by a strong constitutive promoter P J23119 Start expression.

6. The genetically engineered bacteria producing 2′-fucosyllactose using glucose and xylose as substrates according to claim 4, characterized in that: Knockout of the UDP-glucose lipid carrier transferase gene wcaJ.

7. The genetically engineered bacteria producing 2′-fucosyllactose using glucose and xylose as substrates according to claim 4, characterized in that: Using a strong constitutive promoter P J23119 Replace the original promoters of the phosphomannose mutase-mannose-1-phosphate guanylyltransferase gene cluster manC-manB and the GDP-mannose-6-dehydratase-GDP-fucose synthase gene cluster gmd-wcaG on the genome; Strong constitutive promoter P J23119 The nucleotide sequence is shown in SEQ ID NO.

4.

8. Use of the lactose-producing genetically engineered bacteria according to any one of claims 1 to 3 in the fermentation production of lactose.

9. Use of the 2'-fucosyllactose-producing genetically engineered bacteria according to any one of claims 4 to 7 in the fermentation production of 2'-fucosyllactose.

10. A method for synthesizing 2′-fucosyllactose using glucose and xylose as substrates, characterized in that: The 2′-fucosyllactose-producing genetically engineered bacteria according to any one of claims 4 to 7 are fermented and cultured using glucose and xylose as substrates; The fermentation was carried out in a shake flask, and the shake flask medium contained 20 g / L glucose and 10 g / L xylose. The process conditions of the shake flask fermentation culture were: fermentation culture at 35-38°C until OD 600 After the pH value reaches 0.6-0.8, continue culturing at 22-25℃ for at least 72h; Alternatively, a fermenter is used for batch feeding culture, the initial fermenter medium contains 30 g / L xylose or 25 g / L xylose and 5 g / L glucose, and the fermentation culture conditions of the fermenter are: after fermentation culture at 35-38°C until xylose is completely consumed, feeding xylose to keep the final xylose concentration at 2-5 g / L, and continuing to culture until OD 600 Reach about 15, then cool to 22-25°C, continue to add glucose as a substrate to make the final concentration of glucose 5-10 g / L, and continue fermentation until the total duration is at least 72 hours; during the fermentation process, the pH is maintained at 6.8±0.2, and a defoamer is added, the stirring speed is adjusted to 100-900 rpm, and the ventilation volume is adjusted to 2-8 vvm.

Images