Construction method and application of genetically engineered bacterium for producing lactoyl-N-neotetraose

Abstract

The invention discloses a construction method and application of genetically engineered bacteria for producing lactoyl-N-neotetraose, and belongs to metabolic engineering and food fermentation technologies. A gene lacZ, a gene wecB, a gene ushA and a gene setA in escherichia coli BL21 (DE3) are knocked out, a gene SmSET is integrated in situ, expression of beta-1, 3-acetyl glucosamine transferase and beta-1, 4-galactosyl transferase is regulated and controlled, efficient synthesis of lactoyl-N-neotetraose is achieved, and the capacity of producing LNnT in a shake flask is improved to 1.85 g / L from initial 0.79 g / L; in a 3L fermentation tank, the yield of the LNnT reaches 39.85 g / L. The recombinant escherichia coli is used for preparing the LNnT, so that the method has the advantages of simple culture medium, cheap substrate, fast strain growth, genetic stability, high expression level and the like, and has remarkable industrial production potential.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a genetically engineered bacteria producing lacto-N-neotetraose and its application, belonging to the field of metabolic engineering and food fermentation technology. BACKGROUND

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after lactose and lipids, with a concentration of 20-25 g / L in colostrum and 5-15 g / L in mature milk. HMOs are formed by β-1, 3 / 4 / 6 glycosidic bonds between glucose, galactose, fucose, N-acetylglucosamine and sialic acid, and more than 200 different structures have been identified, which can be divided into neutral (such as LNnT, 2'-FL) and acidic (such as 3'-SL, 6'-SL) two categories. HMOs have key physiological functions such as regulating infant intestinal flora, inhibiting pathogen adhesion and enhancing immune response. Lacto-N-neotetraose (LNnT), as one of the core components of HMOs, is a tetrasaccharide structure formed by glucose, galactose and N-acetylglucosamine through specific glycosidic bonds. Studies have shown that lacto-N-neotetraose can not only selectively promote the proliferation of probiotics such as bifidobacterium, but also block pathogen infection by simulating host cell surface receptors, so it has important application potential in infant nutrition, functional food and pharmaceutical fields.

[0003] Currently, the synthesis pathways of lacto-N-neotetraose mainly include chemical synthesis, enzyme catalysis and microbial metabolic engineering. Chemical synthesis relies on multi-step protection-deprotection reactions, which has problems such as complicated steps, low yield and environmental pollution; although enzyme method can achieve stereospecific synthesis, it needs high-purity enzyme preparations and expensive substrates (such as uridine diphosphate sugar), which restricts its large-scale application. The engineering strain represented by Escherichia coli has a clear genetic background, wide substrate utilization and low culture cost, so it becomes a very potential synthesis chassis. By precisely regulating the genetic elements of microorganisms and reconstructing the metabolic network, researchers can design a cell factory that can efficiently synthesize lacto-N-neotetraose. However, in the field of microbial metabolic synthesis of target products, although this technology has many advantages, there are still some obvious defects in the existing technology. First, endogenous metabolic competition leads to low precursor supply efficiency and carbon flow distribution imbalance, which makes the optimization of lacto-N-neotetraose synthesis pathway face great challenges and makes it difficult to accurately improve the yield of products. Second, the lack of product-specific transport system causes intracellular accumulation and enzyme activity feedback inhibition, which limits the further accumulation of products and reduces the fermentation yield. Third, the catalytic efficiency of natural glycosyltransferase is insufficient, which affects the efficiency of glycosylation modification and the yield of products. These problems to a large extent restrict the large-scale industrial application of Escherichia coli in the metabolic synthesis of lacto-N-neotetraose.

[0004] The present application aims to utilize synthetic biology means to construct an E. coli cell factory to achieve the synthesis of lactyl-N-neotetraose. In view of the problems of insufficient precursor supply and carbon flow allocation imbalance caused by endogenous metabolic competition, modular expression of metabolic pathways is carried out, a multi-plasmid expression system is designed, the expression level of key genes is increased, and CRISPR / Cas12 gene editing technology is used to precisely knock out the bypass genes in the host cells, so as to optimize the cell metabolic flow and realize the optimization of global metabolic pathways. In order to solve the problems of intracellular accumulation and feedback inhibition caused by the lack of product-specific transport system, a gene encoding a product-specific efflux protein is integrated on the E. coli genome through genetic engineering means. At the same time, in order to solve the problem of insufficient catalytic efficiency of natural glycosyltransferase, mutants of β-1, 3-acetylglucosamine transferase and β-1, 4-galactosyltransferase are screened by using directed evolution technology, which significantly improves the activity of the rate-limiting enzyme. In addition, the RBS sequence upstream of the β-1, 4-galactosyltransferase gene is optimized, and a high-expression element is screened from a random mutation library to enhance the expression of the enzyme. This innovative method opens up a new path for the large-scale production of lactyl-N-neotetraose, and has significant theoretical research value and social and economic benefits, with a broad market prospect. SUMMARY

[0005] In view of the above problems of the prior art, the present application provides a method for constructing a genetically engineered bacterium producing lactyl-N-neotetraose and an application, aiming to solve the problem of low yield of lactyl-N-neotetraose synthesized by the existing microbial method.

[0006] The first technical solution provided by the present application is a genetically engineered bacterium producing lactyl-N-neotetraose, wherein the genetically engineered bacterium knocks out the β-galactosidase gene lacZ, the UDP-N-acetylglucosamine-2-epimerase gene wecB, and the sugar efflux transporter protein A gene setA, and in-situ integrates the sugar efflux transporter protein gene SmSET, and expresses the UTP-glucose-1-phosphate uridylyltransferase gene galU, the UDP-glucose-4-epimerase gene galE, the β-1, 3-acetylglucosamine transferase gene lgtA, and the β-1, 4-galactosyltransferase gene lgtB.

[0007] In some embodiments, the genetically engineered bacterium also knocks out the UDP-sugar hydrolase gene ushA.

[0008] In some embodiments, the Gene ID of the β-galactoside hydrolase gene lacZ is 945006, the Gene ID of the UDP-N-acetylglucosamine-2-epimerase gene wecB is 944789, the Gene ID of the sugar efflux transporter A gene setA is 944793, the nucleotide sequence of the sugar efflux transporter gene SmSET is shown in SEQ ID NO. 1, and the Gene ID of the UDP-sugar hydrolase gene ushA is 947331.

[0009] In some embodiments, both the β-1, 3-acetylglucosamine transferase and the β-1, 4-galactosyltransferase genes include wild types and mutants, and both the wild type gene lgtA and the wild type gene lgtB are derived from Neisseria meningitidis, and the nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0010] In some embodiments, the original ribosome binding site (RBS) upstream of the β-1, 4-galactosyltransferase gene lgtB is replaced by a preset sequence RBS-64, and the sequence of RBS-64 is shown in SEQ ID NO. 4.

[0011] In some embodiments, the mutant of the β-1, 3-acetylglucosamine transferase is obtained by mutating the glutamine at position 55 of the wild type β-1, 3-acetylglucosamine transferase to alanine, and the nucleotide sequence of the mutant Q55A is shown in SEQ ID NO. 5.

[0012] In some embodiments, the mutant of the β-1, 4-galactosyltransferase is obtained by mutating the histidine at position 179 and the isoleucine at position 225 of the wild type β-1, 4-galactosyltransferase to alanine and leucine, respectively, and the nucleotide sequences of the mutants H179A and I225L are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively.

[0013] In some embodiments, the β-1, 4-galactosyltransferase gene lgtB mutant I225L upstream contains any one of 34 ribosome binding sites obtained by random mutation, and the RBS sequences are shown in SEQ ID NO. 8 to SEQ ID NO. 41, preferably, the RBS sequence is shown in SEQ ID NO. 17 (RBS-10) or SEQ ID NO. 29 (RBS-22).

[0014] In some embodiments, both the UTP-glucose-1-phosphate uridylyltransferase gene galU and the UDP-glucose-4-epimerase gene galE are derived from Escherichia coli K12 MG655.

[0015] In some embodiments, the gene ID of UTP-glucose-1-phosphate uridylyltransferase gene galU is 945730, and the gene ID of UDP-glucose-4-epimerase gene galE is 945354.

[0016] In some embodiments, the genetically engineered bacterium uses Escherichia coli as the host.

[0017] In some embodiments, the genetically engineered bacterium uses pETDuet-1, pRSFDuet-1, pCDFDuet-1 or pCOLADuet-1 vector to express genes galU, galE, lgtA and / or lgtB.

[0018] In some embodiments, the genetically engineered bacterium uses pETDuet-1 to express galU and galE, and pRSFDuet-1 vector to express genes lgtA and lgtB.

[0019] In some embodiments, the genetically engineered bacterium uses pETDuet-1 to express galU and galE, and pCOLADuet-1 vector to express genes lgtA and lgtB.

[0020] In some embodiments, the genetically engineered bacterium uses pETDuet-1 to express galU and galE, and pCDFDuet-1 vector to express genes lgtA and lgtB.

[0021] In some embodiments, the genetically engineered bacterium uses pETDuet-1 to express galU and galE, and pCDFDuet-1 vector to express genes lgtA and lgtB mutant H179A.

[0022] In some embodiments, the genetically engineered bacterium uses pETDuet-1 to express galU and galE, and pCDFDuet-1 vector to express genes lgtA and lgtB mutant I225L.

[0023] In some embodiments, the genetically engineered bacterium uses pETDuet-1 to express galU and galE, and pCDFDuet-1 vector to express genes lgtA and lgtB mutant I225L, and the lgtB mutant I225L upstream comprises a ribosome binding site RBS-10 designed by genetic engineering.

[0024] In some embodiments, the genetically engineered bacteria utilize pETDuet-1 to express galU and galE, utilize pCDFDuet-1 vector to express mutant Q55A of gene lgtA and mutant I225L of gene lgtB, and the mutant I225L of lgtB comprises a ribosome binding site RBS-10 upstream.

[0025] The second technical solution provided by the present application is a method for producing lactyl-N-neotetraose, which uses the genetically engineered bacteria of the first technical solution as a fermentation strain to produce lactyl-N-neotetraose in a fermentation system containing lactose, with glycerol as a carbon source and IPTG as an inducer.

[0026] In some embodiments, the concentration of glycerol in the fermentation system is 10-30 g / L.

[0027] In some embodiments, the fermentation system contains 20-30 g / L of glycerol, 10-15 g / L of potassium dihydrogen phosphate, 1-2 g / L of citric acid, 3-5 g / L of dihydrogen phosphate, 1-2 g / L of magnesium sulfate heptahydrate, and 8-10 mL / L of trace metal solution.

[0028] In some embodiments, the trace metal solution contains 8-10 g / L of ferrous sulfate, 0.5-1.0 g / L of copper sulfate pentahydrate, 0.2-0.5 g / L of manganese sulfate pentahydrate, 0.02-0.05 g / L of borax, 0.1-0.2 g / L of ammonium molybdate tetrahydrate, 1-2 g / L of calcium chloride dihydrate, 2-2.5 g / L of zinc sulfate heptahydrate, and 5 mol / L of hydrochloric acid.

[0029] In some embodiments, the genetically engineered bacteria are cultured in the fermentation system to an OD 600 of 0.6±0.1, and then 0.4-0.5 mM of IPTG and 5-10 g / L of lactose are added, and the culture is induced at 20-30°C for not less than 70 h.

[0030] In some embodiments, the genetically engineered bacteria are cultured in the fermentation system to an OD 600 of 20±3, and then 0.4-0.5 mM of IPTG and 5-10 g / L of lactose are added, and the culture is induced at 20-30°C, with the dissolved oxygen in the fermentation system maintained at 20-50% and the pH maintained at 6.5-7.0.

[0031] In some embodiments, glycerol is added after the initial glycerol in the reaction system is consumed, so that the concentration of glycerol can maintain the growth and metabolism of the bacteria.

[0032] In some embodiments, lactose is added after the initial lactose is consumed, so that the concentration of lactose is maintained at 5-10 g / L.

[0033] In some embodiments, the fermentation time is no less than 72h.

[0034] The third technical solution provided by the present application is the application of the genetically engineered bacteria of the first technical solution or the method of the second technical solution in the production of lacto-N-neotetraose, lacto-N-neotetraose-containing products or lacto-N-triose-containing products.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The present application realizes the high-efficiency synthesis of lacto-N-neotetraose in recombinant E. coli by knocking out the beta-galactosidase gene lacZ, the UDP-N-acetylglucosamine-2-epimerase gene wecB and the sugar efflux transporter A gene setA in E. coli BL21(DE3) and in-situ integrating the sugar efflux transporter gene SmSET and the UDP-sugar hydrolase gene ushA, and regulating the expression of the beta-1, 3-acetylglucosamine transferase gene lgtA and the beta-1, 4-galactosyltransferase gene lgtB required for de novo synthesis of LNTII and LNnT. The present application uses recombinant E. coli to prepare LNnT, which has the advantages of simple culture medium, cheap substrate, fast growth of strains, genetic stability and high expression level, and has significant industrial production potential.

[0037] Under the condition of shake flask fermentation culture, the ability of the genetically engineered bacteria constructed by the present application to produce LNnT is improved from the initial 0.79g / L to 1.85g / L; under the condition of 3L fermenter culture, the yield of LNnT reaches 39.85g / L, laying a foundation for the industrial production of lacto-N-neotetraose. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a metabolic pathway diagram of lacto-N-neotetraose.

[0039] Figure 2 It is the fed-batch fermentation of strain ZBASU-55 in a 3L fermenter.

[0040] Figure 3 It is the HPLC detection result of LNnT. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explanation of the present application and are not used to limit the present application.

[0042] In the following examples, the plasmids, PCR reagents, restriction enzymes, plasmid extraction kits, DNA gel recovery kits and the like used are commercial products, and the specific operations are carried out according to the kit instructions.

[0043] The embodiments of the present application are not limited thereto, and other experimental operations and process parameters not mentioned are performed according to conventional techniques.

[0044] The vectors pETDuet-1, pRSFDuet-1, pCDFDuet-1 and pCOLADuet-1 were purchased from Addgene.

[0045] The sequencing of DNA products and plasmids was completed by Tianlin Biotechnology (Wuxi) Co., Ltd.

[0046] Preparation of E. coli competence: kit from Shanghai Shengong Bioengineering Co., Ltd.

[0047] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0048] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0049] Fermentation medium: glycerol 20 g / L, potassium dihydrogen phosphate 13.5 g / L, citric acid 1.7 g / L, dihydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 1.4 g / L, trace metal solution 10 mL / L (ferrous sulfate 10 g / L, calcium chloride dihydrate 2.0 g / L, zinc sulfate heptahydrate 2.2 g / L, manganese sulfate pentahydrate 0.5 g / L, copper sulfate pentahydrate 1.0 g / L, borax 0.02 g / L, ammonium molybdate tetrahydrate 0.1 g / L), pH 6.8.

[0050] Fermentation medium: glycerol 20 g / L, potassium dihydrogen phosphate 13.5 g / L, citric acid 1.7 g / L, dihydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 1.4 g / L, yeast extract 5 g / L, trace metal solution 10 mL / L (ferrous sulfate 10 g / L, calcium chloride dihydrate 2.0 g / L, zinc sulfate heptahydrate 2.2 g / L, manganese sulfate pentahydrate 0.5 g / L, copper sulfate pentahydrate 1.0 g / L, borax 0.02 g / L, ammonium molybdate tetrahydrate 0.1 g / L), pH 6.8.

[0051] Method for determining LNnT:

[0052] HPLC was used for determination: 1 mL of fermentation broth was boiled at 100℃ for 10 min, centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm membrane. The amount of lactyl-N-neotetrasaccharide produced and the amount of lactose and glycerol consumed were determined by HPLC. HPLC detection conditions: differential refractive index detector; Rezex ROA-organic acid column (Phenomenex, USA), column temperature 50℃; mobile phase 5 mM H2SO4 aqueous solution, flow rate 0.6 mL / min; injection volume 10 μL.

[0053] The shake-flask fermentation conditions for the strains in the following examples were as follows: A single colony of the engineered strain was inoculated into LB liquid medium and cultured at 37°C and 200 rpm with shaking for 12 h to obtain a seed culture; the seed culture was then inoculated into 25 mL of fermentation medium at an inoculation rate of 4% (v / v) and cultured at 37°C and 200 rpm in shake flasks until OD reached. 600 The concentration was 0.6; IPTG was added to a final concentration of 0.4 mM, and lactose was added to a final concentration of 10 g / L. The mixture was induced and cultured at 25°C and 200 rpm for 72 h.

[0054] Example 1: Construction of de novo synthetic pathway and screening of expression vectors with different copy numbers of key enzyme genes

[0055] The specific steps for constructing de novo synthetic pathway expression vectors are as follows (the primer sequences involved are shown in Table 1):

[0056] (1) Obtaining galU and galE fragments: Using the genome of Escherichia coli K12 as a template, PCR amplification was performed using primers galU-F / R and galE-F / R, and DNA was recovered by gel to obtain galU and galE gene fragments.

[0057] (2) Using pETDuet-1 as a template, the vector backbone sequence was amplified using primers V1-F / R and V2-F / R. According to the In-Fusion cloning technology, the galU fragment was inserted between the Nco I / Not I restriction sites of the above vector, and the galE fragment was inserted between the Bgl II / Xho I restriction sites of the above vector. Positive clones were screened and sequenced to finally obtain the recombinant plasmid pET-galUE.

[0058] (3) Obtaining of lgtA and lgtB gene fragments: lgtA and lgtB gene sequences derived from Neisseria meningitidis were synthesized by Tianlin Biotech (Shanghai) Co., Ltd. (the nucleotide sequence of lgtA is shown as SEQ ID NO. 1, and the nucleotide sequence of lgtB is shown as SEQ ID NO. 2), and the synthesized target gene fragments were connected by a seamless cloning kit (Nanjing Novogene Bioinformatics Technology Co., Ltd.) between the BamH I / Sal I and Bgl II / Xho I enzyme digestion sites of vectors pRSFDuet-1, pCDFDuet-1 and pCOLADuet-1, respectively, to obtain plasmids pRSF-lgtAB, pCDF-lgtAB and pCOL-lgtAB.

[0059] (4) Replacement of the RBS sequence upstream of the lgtB gene: the vector backbone sequence was amplified using primers RBS-64-F / R with pRSF-lgtAB, pCDF-lgtAB and pCOL-lgtAB plasmids as templates, respectively, and the DNA was recovered by gel recovery. The product was digested with restriction enzyme Dpn I to remove excess circular templates. The linear fragments were subjected to recombination reaction using One Step Cloning Kit (Vazyme) kit, followed by transformation into E. coli DH5α and overnight culture. Single colonies on the plate were picked into 5 mL of LB medium, and after expansion culture, the plasmid was extracted and sequenced. The successfully constructed plasmids were named pRSF-lgtAB64, pCDF-lgtAB64 and pCOL-lgtAB64.

[0060] Table 1 Primers for plasmid construction

[0061]

[0062] (5) The plasmids pET-galUE and pRSF-lgtAB64, pCDF-lgtAB64 and pCOL-lgtAB64 obtained in the above steps were combined and transformed into E. coli BL21 (DE3) ΔlacZ ΔwecB to obtain three different engineered bacteria (see Table 2), which are designated as ZB-1, ZB-2 and ZB-3. Under shake flask conditions, the optimal strain ZB-2 produced the highest amount of product LNnT after 72 h of fermentation, reaching 0.79 g / L, indicating that there is a nonlinear optimization relationship between the plasmid copy number and the gene expression level, and the medium copy vector pCDFDuet-1 achieves the optimal point of metabolic balance in LNnT synthesis.

[0063] Table 2 Detailed information of strains

[0064]

[0065] Example 2: Screening of β-1, 4-galactosyltransferase gene lgtB mutants

[0066] (1) Obtaining of lgtB mutants H179A, I225L: Primers H179A-F / H179A-R and I225L-F / I225L (see Table 3 for primer sequences) were used to amplify the target fragments from the plasmid pCDF-lgtAB64 by PCR, respectively. The PCR products were digested with Dpn I to remove the excess circular plasmids. The linear fragments were then subjected to recombination reaction using One Step Cloning Kit (Vazyme), followed by transformation into E. coli DH5a and overnight culture. Single colonies were picked from the plates and inoculated into 5 mL LB medium for expansion culture. The plasmids were extracted and sequenced, and the successfully constructed plasmids were named as pCDF-lgtAB64179 and pCDF-lgtAB64225, respectively.

[0067] Table 3. Primers for plasmid construction

[0068]

[0069] (2) The expression vectors pET-galUE in Example 3 and the expression vectors of lgtA and lgtB in (1) above were co-transformed into E. coli BL21(DE3) A lacZ A wecB, respectively, to obtain two different engineered bacteria (see Table 4), which are denoted as ZB-2179 and ZB-2225. The fermentation results showed that, compared with lgtB wild type and mutant H179A, mutant I225L had higher efficiency in the conversion production of LNnT.

[0070] Table 4. Detailed information of strains

[0071]

[0072] Example 3: Construction of LNnT high-yield strain based on transport engineering and blocking of competitive pathways and fermentation verification

[0073] In order to solve the problem of inhibiting synthesis and precursor UDP-sugar degradation caused by intracellular accumulation of lactyl-N-neotetraose, the yield of lactyl-N-neotetraose is improved by transport engineering and blocking byproduct pathways. The sugar efflux transporter A gene setA of the engineering bacteria is knocked out by using the CRISPR-Cas12 gene editing system, and the E. coli BL21(DE3)AlacZAwecBAseta is obtained. In addition, the sugar efflux transporter gene SmSET is integrated at the setA site, and the E. coli BL21(DE3)AlacZAwecBAseta::SmSET is obtained. The UDP-sugar hydrolase gene ushA is knocked out based on the E. coli BL21(DE3)AlacZAwecBAseta::SmSET, and the E. coli BL21(DE3)AlacZAwecBAseta::SmSETAushA is obtained.

[0074] The metabolic pathway of lactyl-N-neotetraose is shown in Figure 1 The specific steps of gene knockout and integration are as follows (the primer sequences involved are shown in Table 5):

[0075] (1) Taking the knockout of setA and the integration of SmSET as an example, the specific target gRNA (23bp) of the target gene is found through https: / / chopchop.cbu.uib.no / , the upstream and downstream primers of setA-gRNA-F / gRNA-R are used for PCR amplification with pcrEG plasmid as a template, and the amplification product is digested with restriction endonuclease Dpn I to remove the excess circular pcrEG plasmid. Then the amplification product is transformed into E. coli DH5a competent cells, the plasmid is extracted, and the primers gRNA-PF / gRNA-PR are used for sequencing identification, and the successfully constructed knockout plasmid is named as pcrEG-setA.

[0076] (2) The homologous arm sequence fragments are amplified by using the upstream homologous arm primers setA-US-F / setA-US-R and the downstream homologous arm primers setA-DS-F / setA-DS-R, respectively, with the E. coli BL21(DE3)AlacZAwecB genome as a template; the SmSET whole fragment is amplified by using the primers SmSET-MS-F / SmSET-MS-R with pCDF-SmSET as a template, and the product is purified and recovered, and then the three fragments are connected by using the primers setA-US-F / setA-DS-R by SOE-PCR method to obtain the gene homologous repair template.

[0077] (3) Take pEcCpf1 plasmid and E. coli BL21(DE3) AlacZ AwecB electrocompetent cells, place on ice for 5 min, then thaw the competent cells. Take 5 μL plasmid and add to 100 μL competent cells, mix gently. Incubate on ice for 30 min, then heat shock at 42°C for 90 s, immediately place on ice for 5 min. Add 1 mL LB medium, incubate at 42°C, 200 rpm for 40 min. Take 50 μL concentrated bacterial solution and evenly spread on LB plate (containing kanamycin), incubate at 37°C overnight until E. coli BL21(DE3) AlacZ AwecB / pEcCpf1 single colonies grow.

[0078] (4) Pick E. coli BL21(DE3) AlacZ AwecB / pEcCpf1 single colonies in LB medium, and add L-arabinose at a final concentration of 10 mM to induce expression of the lambda-red system. When the OD 600 reaches 0.6-0.8, prepare E. coli BL21(DE3) AlacZ AwecB / pEcCpf1 electrocompetent cells.

[0079] (5) Electroporate 500 ng of the target plasmid pcrEG-setA with setA-specific target gRNA (23 bp) constructed in step (1) and 1000 ng of the homologous repair template constructed in step (2) into the E. coli BL21(DE3) AlacZ AwecB / pEcCpf1 competent cells prepared in step (4), and spread on LB plate (kanamycin and streptomycin), incubate at 37°C for 36 h. Use primers SmSET-IF / SmSET-IR to perform colony PCR on single colonies grown on the plate to screen for positive transformants.

[0080] (6) Eliminate pcrEG-setA and pEcCpf1 plasmids from the verified single colonies. Inoculate single colonies in LB medium containing 50 μg / mL kanamycin and 10 mM rhamnose, incubate at 37°C, 200 r / min overnight. The next day, streak the culture on a plate containing 50 μg / mL kanamycin and incubate at 37°C overnight. Pick single colonies on an LB plate containing 50 μg / mL kanamycin and 100 μg / mL streptomycin. Single colonies sensitive to streptomycin and not sensitive to kanamycin have successfully eliminated the pcrEG-setA plasmid.

[0081] (7) The single colony successfully eliminating the pcrEG-setA plasmid was transferred to LB liquid medium containing 5 g / L glucose and cultured at 37°C, 200 r / min for 12-16 h, then the bacterial solution was streaked on the plate containing 5 g / L glucose and 10 g / L sucrose and incubated at 37°C overnight. The screening was carried out on the LB plate added with 50 ug / mL kanamycin. The colony sensitive to kanamycin was successfully eliminated pEcCpf1 plasmid.

[0082] (8) The knockout operation of genes setA and ushA refers to the operation described above.

[0083] Table 5 Gene editing primers

[0084]

[0085] The plasmids pET-galUE and pCDF-lgtAB64225 obtained in Example 2 were co-transferred into the E. coli deficient strain obtained in the above step to obtain three different engineering bacteria (see Table 6), which are represented as ZBA, ZBAS and ZBASU, respectively. The fermentation results show that compared with the starting strain, the knockout of gene setA only makes the LNnT yield increase by 6.2%, the knockout of setA and the integration of SmSET make the LNnT yield increase by 9.8%, and on this basis, the knockout of ushA makes the LNnT yield increase by 12.3%.

[0086] Table 6 Strain detailed information

[0087]

[0088]

[0089] Example 4: Construction of β-1, 4-galactosyltransferase gene lgtB regulated by different RBS

[0090] By randomly mutating the RBS-64 of the expression plasmid pCDF-lgtAB64225 itself, 34 RBS sequences with different translation intensities were obtained (see Table 7 for RBS sequences). In this embodiment, the translation intensity of β-1, 4-galactosyltransferase was regulated by different RBS to construct and screen the engineering strain for efficiently producing lactosyl-N-neotetraose.

[0091] Table 7 RBS sequences

[0092]

[0093]

[0094] Random mutation was performed on the RBS-64 of plasmid pCDF-lgtAB64225, and the RBS mutation step of the expression vector was as follows (the primer sequences involved are shown in Table 8):

[0095] (1) The vector skeleton sequence was amplified using primers RBS64-F / R and the pCDF-lgtAB64225 plasmid as a template, and the DNA was recovered by gel recovery. The product was digested by restriction endonuclease Dpn I to remove the excess circular template. The linear fragment was subjected to recombination reaction using One Step Cloning Kit (Vazyme) kit, and then transformed into E. coli DH5a and cultured overnight. A single colony on the plate was picked into 4 mL of LB medium, and after expansion culture, the plasmid was extracted and sequenced to obtain 34 plasmids with different RBS sequences. The successfully constructed plasmids were named pCDF-lgtAB1225-pCDF-lgtAB34225.

[0096] Table 8 Primer sequences of RBS

[0097]

[0098] (2) The successfully constructed plasmid in the above step was transformed into the host E. coli BL21(DE3)AlacZAwecBAselA: :SmSETAUshA containing pET-galUE, and the obtained engineering strain is shown in Table 9. The obtained engineering strain was subjected to shake flask fermentation to produce LNnT, and the yield results are shown in Table 9. The fermentation results showed that when the RBS sequence was optimized to RBS-10 in Table 7, the yield reached 1.57 g / L.

[0099] Table 9 Detailed information of strains

[0100]

[0101]

[0102]

[0103] Example 5: Screening of lgtA mutant of β-1, 3-acetylglucosamine transferase gene

[0104] In the process of metabolic synthesis of lacto-N-neotetraose by E. coli, the role of lgtA is to synthesize lacto-N-triose II, a precursor of lacto-N-neotetraose, using UDP-GlcNAc and lactose. Therefore, in order to determine the mutation effect of lgtA, lgtA was constructed on pET-22b(+) plasmid alone, and the specific steps were as follows (the primer sequences involved are shown in Table 10):

[0105] (1) The lgtA fragment was obtained by PCR amplification using primers lgtA-F / R with pCDF-lgtAB10225 as the template, and the DNA was recovered from the gel.

[0106] (2) The vector backbone sequence was amplified using primers V-F / R with pET-22b(+) as the template. According to the In-Fusion cloning technology, the fragment lgtA was inserted between the Nde I / Xho I enzyme digestion sites of the above-mentioned vector. Positive clones were screened and sequenced, and finally the recombinant plasmid pET-lgtA was obtained.

[0107] Table 10 Primers for plasmid construction

[0108]

[0109] (3) Design of lgtA mutants: upload the protein sequence of wild-type lgtA through https: / / evcouplings.org / to predict possible single-point forward mutations, and obtain 12 mutants, namely A25S, W31Y, Q55A, Q66N, G88C, G88A, G89R, I110M, P163L, G182H, G182Y, and S230K.

[0110] (4) Take mutant A25S as an example: use primers A25S-F / R (see Table 11 for the sequences of the primers involved) to perform PCR amplification with pET-lgtA plasmid as the template. The amplification product was digested with restriction enzyme Dpn I to remove excess circular plasmid. The linear fragment was subjected to recombination reaction using One Step Cloning Kit (Vazyme) kit, and then transformed into E. coli DH5a and cultured overnight. Single colonies on the plate were picked into 5 mL of LB medium, and the plasmid was extracted and sequenced after expansion culture. The successfully constructed plasmid was named pET-lgtA(A25S).

[0111] (5) The remaining mutant operations are all referred to the above operations.

[0112] Table 11 lgtA mutant primers

[0113]

[0114]

[0115] The successfully constructed plasmids in the above steps were transformed into the host E. coli BL21(DE3)AlacZAwecBAselA::SmSETAushA, and the obtained engineering strains are shown in Table 12. The obtained engineering strains were used to produce LNTII by shake flask fermentation, and the yield results are shown in Table 12.

[0116] Table 12 Strain detailed information

[0117]

[0118]

[0119] The fermentation results showed that when the glutamine at the 55th position of lgtA was mutated to alanine, the β-1, 3-acetylglucosamine transferase could more efficiently synthesize lacto-N-triose. Therefore, the point was mutated on the vector pCDF-lgtAB10225 to verify its role in the complete LNnT synthesis pathway, and the mutation steps were as follows (the primer sequences involved are shown in Table 11):

[0120] (1) Using primers Q55A-F / R, the vector backbone sequence was amplified using the pCDF-lgtAB10225 plasmid as the template, the DNA was recovered by gel, and the product was digested with restriction endonuclease Dpn I to remove the excess circular template. The linear fragment was subjected to recombination reaction using One Step Cloning Kit (Vazyme) kit, followed by transformation into E. coli DH5a and overnight culture, and a single colony on the plate was picked into 4 mL of LB medium, and after expansion culture, the plasmid was extracted and sequenced, and the successfully constructed plasmid was named pCDF-lgtA(Q55A)B10225.

[0121] (2) The successfully constructed plasmid in the above step was transformed into the host E. coli BL21(DE3)AlacZAwecBAselA: :SmSETAUshA containing pET-galUE, and the obtained engineering strain is shown in Table 13. The obtained engineering bacteria were subjected to shake flask fermentation to produce LNnT, and the yield results are shown in Table 13.

[0122] Table 13 Strain detailed information

[0123]

[0124] Example 6: Fed-batch fermentation in a 3L fermenter to produce lacto-N-neotetraose

[0125] In order to prepare high-yield LNnT, high-density fed-batch fermentation was carried out in a 3L fermenter using the recombinant bacteria ZBASU-Q55A.

[0126] Fermentation conditions: fermentation medium, inoculum size 5% (v / v), culture temperature before induction 37°C, and OD 600The IPTG was added to induce the protein expression so that the concentration of the protein in the fermentation system was 0.4 mmol / L, the initial concentration of lactose was 10 g / L, and the induction fermentation temperature was 25℃. The pH of the tank was controlled to be constant at 6.80 by using NH4OH during the whole fermentation. In order to maintain the growth of the bacteria and the synthesis of LNnT, 750 g / L of glycerol (containing 20 g / L of MgSO4·7H2OH and 25 g / L of NH4Cl) was added to supplement the carbon source after the initial glycerol was consumed, the glycerol concentration in the fermentation system was maintained at a low concentration level (glycerol was used for the growth and metabolism of the bacteria, and the concentration was about 0 g / L) by pH feedback regulation (the flow rate was set to be 10 mL / h) until the end of the fermentation, 250 g / L of lactose was manually added after the initial lactose was consumed, and the final concentration of lactose in the fermentation system was maintained at 10±2 g / L, and lactose was continuously added until the end of the fermentation if the lactose was consumed to a low concentration during the fermentation. The system was cascade controlled during the fermentation, and the dissolved oxygen in the tank was 30±5% by adjusting the rotation speed, the aeration amount and the oxygen.

[0127] The fermentation was sampled at regular time intervals, and the OD of the bacteria was measured 600 , 1 mL of the fermentation liquid was boiled for 10 min to completely break the cells, centrifuged at 12000 r / min for 10 min, the supernatant was filtered by a 0.22 μm membrane, and the production amount of LNnT Figure 3 ) and the consumption amounts of lactose and glycerol Figure 2 ) were detected by using HPLC during the fermentation. The results showed that during the fermentation (the total fermentation time was 94.5 hours), the concentration of the product LNnT reached the maximum at 89.5 h, and the maximum concentration was 39.85 g / L.

[0128] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A genetically engineered bacterium producing lactosyl-N-neotetraose, characterized by, The genetically engineered bacteria knock out the beta-galactosidase gene lacZ, the UDP-N-acetylglucosamine-2-epimerase gene wecB, the sugar efflux transporter A gene setA, and integrate the sugar efflux transporter gene SmSET in situ, and express the UTP-glucose-1-phosphate uridylyltransferase gene galU, the UDP-glucose-4-epimerase gene galE, the beta-1, 3-acetylglucosamine transferase gene lgtA, and the beta-1, 4-galactosyltransferase gene lgtB, and the genetically engineered bacteria also knock out the UDP-sugar hydrolase gene ushA. 2.The genetically engineered bacteria according to claim 1, characterized in that, The beta-1, 3-acetylglucosamine transferase and the beta-1, 4-galactosyltransferase gene both include wild type and mutant, and the wild type of the gene lgtA and the wild type of the gene lgtB are both derived from Neisseria meningitidis, and the nucleotide sequences are shown in SEQ ID NO. 2 and 3 respectively. 3.The genetically engineered bacteria according to claim 2, characterized in that, The mutant of the beta-1, 3-acetylglucosamine transferase is obtained by mutating the glutamine at the 55th position of the wild type of the beta-1, 3-acetylglucosamine transferase to alanine, and is Q55A; The mutant of the beta-1, 4-galactosyltransferase is obtained by mutating the histidine at the 179th position and the isoleucine at the 225th position of the wild type of the beta-1, 4-galactosyltransferase to alanine and leucine respectively, and is H179A and I225L. 4.The genetically engineered bacteria according to claim 1, characterized in that, The original ribosome binding site upstream of the beta-1, 4-galactosyltransferase gene lgtB is replaced by the nucleotide sequence shown in SEQ ID NO. 4, SEQ ID NO. 17 or SEQ ID NO.

29. 5.The genetically engineered bacterium of claim 1, characterized in that, The Gene ID of the beta-galactosidase gene lacZ is 945006, the Gene ID of the UDP-N-acetylglucosamine-2-epimerase gene wecB is 944789, the Gene ID of the sugar efflux transporter A gene setA is 944793, the nucleotide sequence of the sugar efflux transporter gene SmSET is shown in SEQ ID NO. 1, the Gene ID of the UTP-glucose-1-phosphate uridylyltransferase gene galU is 945730, the Gene ID of the UDP-glucose-4-epimerase gene galE is 945354, and the Gene ID of the UDP-sugar hydrolase gene ushA is 947331. 6.The genetically engineered bacterium of claim 1, characterized in that, The genetically engineered bacteria use Escherichia coli as the host; and the genetically engineered bacteria use pETDuet-1, pRSFDuet-1, pCDFDuet-1 or pCOLADuet-1 vector to express the genes galU, galE, lgtA and / or lgtB.

7. A method for producing lactosyl-N-neotetraose, characterized by, The genetically engineered bacteria of any one of claims 1-6 are used as fermentation strains to produce lacto-N-neotetraose in a lactose-containing fermentation system with glycerol as the carbon source at a concentration of 10-30 g / L and IPTG as the inducer.

8. The method of claim 7, wherein, The genetically engineered bacteria are cultured in a fermentation system to OD 600 0.6±0.1, and then IPTG with a final concentration of 0.4-0.5 mM and lactose with a final concentration of 5-10 g / L are added, and the culture is carried out at 20-30 °C for not less than 70 h. or the genetically engineered bacteria are cultured in a fermentation system to OD 600 is 20±3, IPTG with a final concentration of 0.4-0.5 mM and lactose with a final concentration of 5-10 g / L are added, and the culture is induced at 20-30 °C, with the dissolved oxygen in the fermentation system maintained at 20-50% and the pH maintained at 6.5-7.

0.

9. The method of claim 8, wherein, Glycerol was added when the initial glycerol was consumed in the reaction system; lactose was added when the initial lactose was consumed to maintain the lactose concentration at 5-10 g / L.

10. Use of the genetically engineered bacteria of any one of claims 1-6 or the method of any one of claims 7-9 in the production of lacto-N-neotetraose, a product containing lacto-N-neotetraose, or a product containing lacto-N-triose.

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