Recombinant escherichia coli for improving production of lacto-n-tetraose and construction and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant escherichia coli for improving the yield of lactose-N-tetrasaccharide, construction and application thereof. The present application uses the technology of AI intelligent auxiliary regulation of metabolic flow, combines with optimization of metabolic pathways, and performs specific genetic modification on escherichia coli, so as to significantly improve the synthesis efficiency and yield of recombinant escherichia coli for producing lactose-N-tetrasaccharide (LNT), reduce raw material consumption and byproduct generation, and thus effectively reduce the production cost. The present application can accelerate the development of strains and process optimization, and shorten the development cycle in combination with AI technology. The present application realizes the efficient industrial production of LNT through genetic engineering modification and optimization of process conditions, and meets the growing demand of the market.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant Escherichia coli for improving the yield of lacto-N-tetraose as well as a construction and application thereof. BACKGROUND

[0002] Human milkoligosaccharides (HMOs for short) are the second most abundant component in human milk after fat and lactose, and are unique and irreplaceable components in human milk. Lacto-N-tetraose (LNT for short) is one of the important HMOs in human milk. LNT has a variety of potential health-promoting effects, including promoting the colonization of beneficial flora, preventing pathogen adhesion and antiviral functions. It has been approved by the European Food Safety Authority (EFSA), the European Union (EU) and the US Food and Drug Administration (FDA) to be added to infant formula as a nutritional fortifier.

[0003] At present, the production of LNT mainly includes chemical synthesis, enzymatic synthesis and microbial fermentation (Choi et al., 2019). Enzymatic synthesis often requires the addition of expensive precursors, but biosynthesis can use inexpensive renewable substrates, and has the characteristics of cleanness, greenness and high efficiency. Compared with conventional chemical synthesis and enzymatic synthesis, biosynthesis has the advantages of simple process, simple metabolic pathway and less by-products, and is more suitable for large-scale industrialization. At present, the synthesis of HMOs has been realized in various microorganisms such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis and Corynebacterium glutamicum. Most of the production strains are genetically engineered bacteria. Lactose is converted into lacto-N-triose II (LNT II) under the action of β-1,3-N-acetylglucosamine transferase, and LNT II is the precursor for the synthesis of LNT. Galactose is connected to LNT II by β-1,3 bond, and then LNT is synthesized. Therefore, the construction of engineering strains is usually through the introduction of β-1,3-N-acetylglucosamine transferase and specific β-1,3-galactosyltransferase to complete two consecutive enzymatic reactions in the cell, so as to realize the synthesis of LNT. The engineering strains for efficient synthesis of HMOs usually have three characteristics: they can effectively absorb the precursors required for the synthesis of HMOs such as lactose, they can produce activated nucleotide-sugar donors (such as guanosine diphosphate fucose), and they can use glycosyltransferases (such as fucosyltransferase) to connect monosaccharides to lactose.

[0004] Currently, the production of LNT by microbial fermentation is limited by several factors: 1. Low substrate utilization rate, cannot be fully utilized, and more intermediate products accumulate. For example, Zhang Tao's team at Jiangnan University overexpressed lgtA and WbgO in E. coli, and regulated the expression of galE, galT and galK in the synthesis pathway, while knocking out the lacZ gene, achieving an LNT yield of 31.56 g / L, but the byproduct LNT II accounted for more than 15%. In terms of regulations, the LNT produced by Glycom in Denmark, Jennewein in Germany and Inbiose in Belgium obtained GRAS certification from the US FDA in 2019, 2021 and 2023 respectively. Among them, Glycom requires that the LNT content be not less than 70%, and the LNT II be not more than 10%; Jennewein requires that the LNT content be not less than 75%, and the LNT II be not more than 5%; Inbiose requires that the LNT content be not less than 80%, and the LNT II be not more than 5%. Therefore, improving the utilization efficiency of microorganisms to substrates and reducing the generation of byproducts have a gain effect on improving the production of microbial LNT; 2. Enzyme activity and stability: the activity and stability of key enzymes may not be sufficient, which needs to be optimized through protein engineering; 3. Influence of metabolic flux: the influence of metabolic bypass on yield; the recombinant strain needs to be genetically engineered to improve yield and tolerance, especially to construct more stable recombinant genome engineering bacteria instead of plasmid bacteria, reduce the use of antibiotics, which is beneficial to reduce the cost of production and simplify the purification process; 4. Influence of fermentation process: fine regulation of fermentation process is needed to achieve efficient production; 5. Downstream purification: the extraction and purification process of LNT may be complex and costly, and efficient separation technology is needed. At present, the production of LNT by microbial fermentation is still mainly based on overexpression of UTP supply and LNT synthesis related genes, fine tuning of LNT pathway related genes, and blocking of UDP-galactose related competitive pathways, in order to reduce the production of byproducts and reduce the cost of industrial purification. At present, most of the enzyme screening is based on genome and structural database using bioinformatics tools for sequence analysis and function prediction to identify potential target enzymes. Then, through homology modeling and function prediction, the structure and activity of the enzyme are further understood. Then, the screened genes are cloned and expressed to determine the enzyme activity in a suitable host to verify the catalytic ability and substrate specificity. This prediction method may not be accurate enough, especially for unknown or new enzymes, and the predicted function and structure may be biased. Experimental verification is complex: gene cloning and expression, enzyme activity determination and other experimental steps are tedious, time-consuming and require professional skills. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies in the prior art and provide a recombinant E. coli for improving the yield of lactose-N-tetrasaccharide.

[0006] To achieve the above object, the technical scheme adopted by the present application is:

[0007] In the first aspect, the present application provides a recombinant Escherichia coli for improving the yield of lactose-N-tetrasaccharide, wherein the recombinant Escherichia coli is a chassis strain of Escherichia coli, and is genetically modified according to any one of the following (1)-(3):

[0008] (1) knocking out any one of 6-phosphofructokinase gene pfkA, succinyl-CoA synthetase gene sucCD, transporter-encoding gene proP and transketolase gene tktA;

[0009] (2) knocking out succinyl-CoA synthetase gene sucCD and transporter-encoding gene proP;

[0010] (3) knocking out succinyl-CoA synthetase gene sucCD and transketolase gene tktA.

[0011] In the present application, pfkA, sucCD, proP and tktA are knocked out in the genome of the chassis strain, the metabolic competition branch is eliminated, the consumption of intermediate products such as F-6-P and G-6-P in the synthesis of LNT is reduced, and the carbon pathway in the strain is adjusted. By combining and superimposing the genes related to the side branch metabolic pathway, the flow of glc-1-p and GlcN-6-P to G-6-P and F-6-P is strengthened, the accumulation of precursor substances is increased, and the yield of LNT is further improved.

[0012] Preferably, the chassis strain is constructed by using Escherichia coli E. coli BL21 star (DE3) as the initial strain, sequentially superimposing the knockout of β-galactosidase gene lacZ, fucose isomerase gene fucI, L-fucose kinase gene fucK, UDP glycolipid carrier transferase gene wcaJ, UDP-glucose dehydrogenase gene ugD and glucosamine-6-phosphate deaminase gene nagB by using gene editing technology, and inserting single copy β-1,3-N-acetylglucosamine amino transferase gene lgtA and UDP-galactose-4-epimerase gene galE at lacA site and β-1,3-galactosyltransferase gene wbdO at wzxC site.

[0013] The chassis strain used in the present application contains T7 polymerase, which is beneficial to the high-efficiency expression of the target gene after genetic modification by IPTG induction in the present application.

[0014] In a second aspect, the present application provides a method for constructing the recombinant E. coli for improving the production of lacto-N-tetraose, comprising the following steps: on the basis of the chassis strain, at least one of 6-phosphofructokinase gene pfkA, succinyl-CoA synthetase gene sucCD, transketolase gene tktA and transporter-encoding gene proP is knocked out by using pEcCas / pEcgRNA system through homologous recombination.

[0015] The pEcCas / pEcgRNA system is used to realize gene editing. When the target gene homologous recombination fragment exists, the Cas9 protein first cuts the genomic DNA specifically on the target gene characteristic sequence under the guidance of sgRNA, and then the integration of the homologous fragment is realized through the action of λ-Red recombination protein, so as to achieve the purpose of gene editing.

[0016] In a third aspect, the present application provides the application of the recombinant E. coli for improving the production of lacto-N-tetraose in the production of lacto-N-tetraose.

[0017] In a fourth aspect, the present application provides a method for producing lacto-N-tetraose by using the recombinant E. coli for improving the production of lacto-N-tetraose, comprising the following steps:

[0018] (a) inoculating the recombinant E. coli for improving the production of lacto-N-tetraose into a seed culture medium to activate;

[0019] (b) inoculating the activated bacterial solution into a fermentation culture medium, adding an inducer to perform fermentation culture, and producing lacto-N-tetraose.

[0020] Preferably, the seed culture medium is LB liquid culture medium.

[0021] Preferably, the fermentation culture medium formula is: Na2HPO4·12H2O 15-20 g / L, KH2PO4 2-5 g / L, NH4Cl 1-3 g / L, (NH4)2HPO4 1-3 g / L, trisodium citrate 2H2O 1-3 g / L, CaCl2·2H2O 0.01-0.03 g / L, Triton-X 100 0.1-1 mL, glycerol 20-40 g / L, yeast extract powder 1-5 g / L, and tryptone 10-25 g / L.

[0022] More preferably, the fermentation medium formula is: Na2HPO4·12H2O 17.9 g / L, KH2PO4 3 g / L, NH4Cl 2 g / L, (NH4)2HPO4 1 g / L, trisodium citrate 2H2O 2.2 g / L, CaCl2·2H2O 0.015 g / L, Triton-X100 0.3 mL, glycerol 30 g / L, yeast extract 2 g / L, tryptone 15 g / L.

[0023] Preferably, the inducer includes IPTG and lactose.

[0024] Preferably, the fermentation culture conditions are: 26-30℃, 180-300 rpm culture for 70-100 h.

[0025] The beneficial effects of the present application are:

[0026] The present application collects data on metabolites, enzymes, genes and reactions by combining the metabolic network pathways of the strain, and constructs a metabolic network model with metabolites as nodes and reactions as edges, and performs network topology analysis to identify key metabolites and reactions to find key enzymes. The supply of nucleotide sugar donors is crucial for microbial synthesis of HMO. The present application systematically studies the enhancement of UDP-Gal supply or the weakening of the side branch metabolic pathway to indirectly improve the LNT productivity. In terms of efflux engineering, the intracellular accumulation of precursor LNTri II is enhanced and the synthesis of LNT is improved. Using AI intelligent auxiliary regulation of metabolic flow technology, the metabolic pathway of Escherichia coli is optimized by gene editing means to improve the efficiency of recombinant Escherichia coli in producing lactose-N-tetrasaccharide (LNT). By optimizing the metabolic pathway, the synthesis efficiency and yield of LNT are significantly improved, the raw material consumption and byproduct generation are reduced, thereby effectively reducing the production cost.

[0027] The present application optimizes the metabolic flow to the target product by CRISPR-Cas9 and other technologies. By intelligently regulating the metabolic pathways of the side branch of the strain, the substrate flow to LNT biosynthesis is increased, not only improving the stability and consistency of the production process, but also enhancing the flexibility of the process, enabling it to quickly adapt to different production conditions and demand changes. Through data-driven decision support, combined with post-experimental verification, through multiple parallel shake flask tests, the fermentation parameters such as stable medium composition, temperature, pH and induction conditions are reduced to a minimum error. The engineered bacteria provided by the present application optimize the metabolic pathway, helping to achieve efficient industrial production of LNT and meet the growing demand of the market. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0029] The Genbank IDs of the genes involved in the present application are as follows:

[0030] lacZ (Genbank ID: ACT42197.1), fucI (Genbank ID: ACT44469.1), fucK (Genbank ID: ACT44470.1), wcaJ (Genbank ID: ACT43804.1), ugD (Genbank ID: ACT43781.1), nagB (Genbank ID: ACT42511.1), lgtA (Genbank ID, CAX50885.1), galE (Genbank ID, NP_415280.3), wbdO (Genbank ID, AAV34525.1), pfkA (Genbank ID, ACT45594.1), sucC (Genbank ID, ACT42562.1), sucD (Genbank ID, ACT42563.1), proP (Genbank ID, ACT45772.1) and tktA (Genbank ID, ACT44582.1).

[0031] The primers used in the embodiments of the present application and the N20 sequence are shown in Table 1:

[0032] Table 1

[0033]

[0034]

[0035]

[0036]

[0037] Example 1: Metabolic network analysis and construction of an optimized model

[0038] The present application uses iML1515 as an initial model, and uses Cobrapy to construct a new whole genome metabolic model. By adding enzyme-catalyzed reactions for generating LNT and LNTtri and their gene-protein-reaction (GPR) associations in the model, it is ensured that the model can simulate the production of LNT.

[0039] Then, using the Flux Variability Scanning based on Enforced Objective Flux (FVSEOF) algorithm, LNT is used as the target product, and the key metabolic reactions that need to be enhanced are identified, so as to optimize the synthesis path of LNT.

[0040] After obtaining the results of the FVSEOF algorithm, the metabolic reactions essential for LNT production are selected, including the generation of glucose-1-phosphate, the synthesis of UDP-glucose precursor, the generation of UDP-glucose, the generation of UDP-galactose, the generation of N-acetylglucosamine, the generation of UDP-N-acetylglucosamine, the synthesis of LNT, the generation of xylulose-5-phosphate, and other related metabolic reactions, and the key enzymes and their encoding genes of these reactions are determined according to the GPR correlation.

[0041] After a large number of screening studies, the present application finally screens four key genes: 6-phosphofructokinase gene pfkA, succinyl-CoA synthase gene sucCD, transport protein encoding gene proP, and transketolase gene tktA. Subsequently, these genes are further edited, and the influence of the edited genes on the production of LNT is verified.

[0042] Example 2: Construction of chassis strain

[0043] E. coli BL21 star (DE3) is used as the initial strain (purchased from Beyotime), and gene editing technology is used to knockout the β-galactosidase encoding gene lacZ, the fucose isomerase gene fucI, the L-fucose kinase gene fucK, the UDP sugar lipid carrier transferase gene wcaJ, the UDP-glucose dehydrogenase gene ugD, the glucosamine-6-phosphate deaminase gene nagB, insert a single copy of the β-1, 3-N-acetylglucosamine amino transferase gene lgtA and the UDP-galactose-4-epimerase gene galE at the lacA site, and insert the β-1, 3-galactosyltransferase gene wbdO at the wzxC site (lgtA and wbdO are synthesized from Huada), to obtain E. coli BL21 star (DE3) ΔlacZ ΔfucIK ΔwcaJ ΔugD ΔnagB ΔlacA::lgtA-galE ΔwzxC::wbdO, which is the chassis strain of the present application.

[0044] The specific construction method of the chassis strain is as follows:

[0045] (1) E. coli BL21 star (DE3) strain is used as the initial strain, and pEcCas plasmid containing Cas9 and λ-Red recombinase (purchased from addgene) is used as the tool plasmid to prepare E. coli BL21 star (DE3) competent cells and transform the pEcCas plasmid into the cells to obtain E. coli BL21 star (DE3) containing the pCas9 plasmid.

[0046] (2) Design the N20 sequence of lacZ gene (lacZ-N20), use Gibson assembly method to assemble it with pEcgRNA (purchased from addgene) containing sgRNA sequence to construct pEcgRNA-N20 plasmid; obtain pEcgRNA-N20 positive plasmid by heat shock transformation, and screen on the medium containing spectinomycin spec.

[0047] (3) Clone the fragments lacZ-up and lacZ-Do from E. coli BL21 star (DE3) with primers lacZ-up-F / R, lacZ-Do-F / R, respectively, then use lacZ-up and lacZ-Do as templates, and lacZ-up-F / lacZ-Do-R as primers to prepare the fusion Donor DNA fragment (lacZ-Donor), then transform pEcgRNA-N20 positive plasmid and Donor DNA into E. coli BL21 star (DE3) strain containing pCas9 plasmid; verify the strain successfully knocked out lacZ gene by PCR, and confirm by sequencing.

[0048] (4) Eliminate plasmids through culture and screening steps to ensure that the strain only retains the desired gene editing, and finally obtain the recombinant E. coli for improving the production of lacto-N-tetraose, named E. coli BL21 star (DE3)ΔlacZ.

[0049] (5) Refer to the gene knockout method of steps (2)-(4), design the corresponding primers, and on the basis of E. coli BL21 star (DE3)ΔlacZ, knock out fucIK (fucI and fucK combined knockout), wcaJ, ugD and nagB in turn, to obtain recombinant E. coli E. coli BL21 star (DE3)ΔlacZΔfucIKΔwcaJΔugDΔnagB.

[0050] (6) Insert single copy β-1, 3-N-acetylglucosamine amino transferase gene lgtA and UDP-galactose-4-epimerase gene galE at lacA site:

[0051] Synthetic plasmid lgtA-galE was synthesized by Huada Lihe, T7-lgtA fragment 1 was obtained from synthetic plasmid lgtA-galE by designing primers T7-F and lgtA-T7-R, lgtA-galE fragment 2 was obtained from synthetic plasmid lgtA-galE by designing primers lgtA-F and pSPIN-galE-R, and fusion Donor DNA fragment T7-lgtA-galE-Donor was prepared by taking T7-lgtA fragment 1 and lgtA-galE fragment 2 as templates by designing primers T7-F and pSPIN-galE-R;

[0052] Linearized vector pSPIN-lacA was obtained by using restriction endonuclease to cut pSPIN (purchased from addgene) containing lacA-N32 specific sequence;

[0053] Plasmid pSPIN-lacA-T7-lgtA-galE was obtained by Gibson assembly method to assemble and construct linearized vector pSPIN-lacA and T7-lgtA-galE-Donor, and E. coli BL21 star (DE3)ΔlacZΔfucIKΔwcaJΔugDΔnagBΔlacA::lgtA-galE was obtained by transforming the recombinant E. coli E. coli BL21 star (DE3)ΔlacZΔfucIKΔwcaJΔugDΔnagB obtained in step (5).

[0054] (7) Inserting β-1, 3-galactosyltransferase gene wbdO at wzxC site: in the method, the final plasmid was transformed into E. coli BL21 star (DE3)ΔlacZΔfucIKΔwcaJΔugDΔnagBΔlacA::lgtA-galE obtained in step (6), and finally, strain E. coli BL21 star (DE3)ΔlacZΔfucIKΔwcaJΔugDΔnagBΔlacA::lgtA-galEΔwzxC::wbdO was obtained, which is hereinafter referred to as LJ2.

[0055] Example 3:

[0056] An embodiment of the recombinant E. coli for improving the yield of lacto-N-tetraose is provided, wherein the recombinant E. coli is based on E. coli LJ2, and the following genetic modification is performed: knocking out 6-phosphofructokinase gene pfkA.

[0057] The construction method of the recombinant E. coli for improving the yield of lacto-N-tetraose is as follows:

[0058] (1) Take LJ2 strain as the starting strain, take pEcCas plasmid containing Cas9 and lambda-Red recombinase (purchased from addgene) as the tool plasmid, prepare LJ2 competent cells, and transform the pEcCas plasmid into the cells to obtain the LJ2 strain containing the pCas9 plasmid.

[0059] (2) Design the N20 sequence of the pfkA gene (pfkA-N20), use Gibson assembly method to assemble it with pEcgRNA (purchased from addgene) containing sgRNA sequence to obtain pEcgRNA-N20 plasmid; obtain pEcgRNA-N20 positive plasmid by heat shock transformation, and screen on the culture medium containing spectinomycin spec.

[0060] (3) Clone the fragments pfkA-up and pfkA-Do from Escherichia coli LJ2 by using primers pfkA-up-F / R and pfkA-Do-F / R respectively; then take pfkA-up and pfkA-Do as templates, and take pfkA-up-F / pfkA-Do-R as primers to prepare the fusion Donor DNA fragment (pfkA-Donor); then transform the pEcgRNA-N20 positive plasmid and the Donor DNA fragment into the LJ2 strain containing the pCas9 plasmid; verify the strain in which the pfkA gene is successfully knocked out by PCR, and perform sequencing confirmation.

[0061] (4) Eliminate the plasmid through the culture and screening steps to ensure that the strain only retains the required gene editing, and finally obtain the recombinant Escherichia coli for improving the yield of lacto-N-tetraose, named LJ2ΔpfkA.

[0062] Example 4:

[0063] An embodiment of the recombinant Escherichia coli for improving the yield of lacto-N-tetraose; the recombinant Escherichia coli takes Escherichia coli LJ2 as the chassis strain, and is genetically modified as follows: the succinyl-CoA synthetase gene sucCD is knocked out.

[0064] The construction method of the recombinant Escherichia coli for improving the yield of lacto-N-tetraose is as follows:

[0065] (1) Take LJ2 strain as the starting strain, take pEcCas plasmid containing Cas9 and lambda-Red recombinase (purchased from addgene) as the tool plasmid, prepare LJ2 competent cells, and transform the pEcCas plasmid into the cells to obtain the LJ2 strain containing the pCas9 plasmid.

[0066] (2) Design the N20 sequence of sucCD gene (sucCD-N20), and use Gibson assembly method to assemble it with pEcgRNA containing sgRNA sequence (purchased from addgene) to construct pEcgRNA-N20 plasmid; obtain pEcgRNA-N20 positive plasmid by heat shock transformation, and screen on the medium containing spectinomycin spec.

[0067] (3) Clone the sucCD-up and sucCD-Do fragments from E. coli LJ2 using sucCD-up-F / R and sucCD-Do-F / R as primers, respectively; then use sucCD-up and sucCD-Do as templates, and sucCD-up-F / sucCD-Do-R as primers to prepare the fusion Donor DNA fragment (sucCD-Donor), and then transform the pEcgRNA-N20 positive plasmid and the Donor DNA fragment into the LJ2 strain containing the pCas9 plasmid; verify the strain with sucCD gene successfully knocked out by PCR, and confirm by sequencing.

[0068] (4) Eliminate plasmids through culture and screening steps to ensure that the strain only retains the desired gene editing, and finally obtain the recombinant E. coli for improving the yield of lacto-N-tetraose, named LJ2ΔsucCD.

[0069] Example 5:

[0070] An embodiment of the recombinant E. coli for improving the yield of lacto-N-tetraose; the recombinant E. coli takes E. coli LJ2 as the chassis strain, and is genetically modified as follows: the transport protein coding gene proP is knocked out.

[0071] The construction method of the recombinant E. coli for improving the yield of lacto-N-tetraose is:

[0072] (1) Take LJ2 strain as the starting strain, and take pEcCas plasmid (purchased from addgene) containing Cas9 and lambda-Red recombinase as the tool plasmid, prepare LJ2 competent cells, and transform the pEcCas plasmid into the cells to obtain the LJ2 strain containing the pCas9 plasmid.

[0073] (2) Design the N20 sequence of proP gene (proP-N20), and use Gibson assembly method to assemble it with pEcgRNA containing sgRNA sequence (purchased from addgene) to construct pEcgRNA-N20 plasmid; obtain pEcgRNA-N20 positive plasmid by heat shock transformation, and screen on the medium containing spectinomycin spec.

[0074] (3) proP-up and proP-Do were cloned from E. coli LJ2 using primers proP-up-F / R, respectively; then proP-up and proP-Do were used as templates, and primers proP-up-F / proP-Do-R were used to prepare a fusion Donor DNA fragment (proP-Donor); subsequently, the pEcgRNA-N20 positive plasmid and the Donor DNA fragment were transformed into the LJ2 strain containing the pCas9 plasmid; the strain with enhanced proP gene expression was verified by PCR and sequencing.

[0075] (4) The plasmid was eliminated through the culture and screening steps to ensure that the strain only retained the desired gene editing, and finally the recombinant E. coli with enhanced lacto-N-tetraose yield was obtained, which was named LJ2ΔproP.

[0076] Example 6:

[0077] An embodiment of the recombinant E. coli with enhanced lacto-N-tetraose yield according to the present application; the recombinant E. coli takes E. coli LJ2 as the chassis strain and is genetically modified as follows: the transketolase gene tktA is knocked out.

[0078] The construction method of the recombinant E. coli with enhanced lacto-N-tetraose yield is as follows:

[0079] (1) The LJ2 strain was used as the starting strain, and the pEcCas plasmid (purchased from addgene) containing Cas9 and lambda-Red recombinase was used as the tool plasmid to prepare LJ2 competent cells and transform the pEcCas plasmid into the cells to obtain the LJ2 strain containing the pCas9 plasmid.

[0080] (2) The N20 sequence of the tktA gene (tktA-N20) was designed, and Gibson assembly was used to assemble it with the pEcgRNA (purchased from addgene) containing the sgRNA sequence to construct the pEcgRNA-N20 plasmid; the pEcgRNA-N20 positive plasmid was obtained by heat shock transformation, and was screened on the culture medium containing spectinomycin (spec).

[0081] (3) The tktA-up and tktA-Do fragments were cloned from the E. coli LJ2 strain using tktA-up-F / R and tktA-Do-F / R as primers, respectively; then the tktA-Donor fusion DNA fragment was prepared using tktA-up and tktA-Do as templates and tktA-up-F / tktA-Do-R as primers, and subsequently, the pEcgRNA-N20 positive plasmid and the Donor DNA fragment were transformed into the LJ2 strain containing the pCas9 plasmid; the strain with the successfully knocked-out tktA gene was verified by PCR and sequencing.

[0082] (4) The plasmid was eliminated through the culture and screening steps to ensure that the strain only retained the desired gene editing, and finally the recombinant E. coli for improving the production of lacto-N-tetraose was obtained, which was named LJ2ΔtktA.

[0083] Example 7:

[0084] In one embodiment of the recombinant E. coli for improving the production of lacto-N-tetraose, the recombinant E. coli is based on the LJ2ΔsucCD strain, and the following genetic modification is performed: the transport protein coding gene proP is knocked out.

[0085] The construction method of the recombinant E. coli for improving the production of lacto-N-tetraose is as follows:

[0086] (1) The LJ2ΔsucCD strain was used as the starting strain, and the pEcCas plasmid (purchased from addgene) containing Cas9 and lambda-Red recombinase was used as the tool plasmid to prepare the LJ2ΔsucCD competent cells and transform the pEcCas plasmid into the cells to obtain the LJ2ΔsucCD strain containing the pCas9 plasmid.

[0087] (2) The N20 sequence of the proP gene (proP-N20) was designed, and the Gibson assembly method was used to assemble it with the pEcgRNA (purchased from addgene) containing the sgRNA sequence to construct the pEcgRNA-N20 plasmid; the pEcgRNA-N20 positive plasmid was obtained by heat shock transformation, and was screened on the culture medium containing spectinomycin spec.

[0088] (3) The proP-Donor fusion DNA fragment was prepared according to step (3) of Example 5, and subsequently, the pEcgRNA-N20 positive plasmid and the Donor DNA fragment were transformed into the LJ2ΔsucCD strain containing the pCas9 plasmid; the strain with the successfully knocked-out proP gene was verified by PCR and sequencing.

[0089] (4) Through the culture and screening steps, the plasmid is eliminated to ensure that the strain only retains the required gene editing, and finally the recombinant Escherichia coli for improving the production of lacto-N-tetraose is obtained, which is named LJ2ΔsucCDΔproP.

[0090] Example 8:

[0091] An embodiment of the recombinant Escherichia coli for improving the production of lacto-N-tetraose; the recombinant Escherichia coli takes the LJ2ΔsucCD strain as a chassis strain, and is genetically modified as follows: knocking out the transketolase gene tktA.

[0092] The construction method of the recombinant Escherichia coli for improving the production of lacto-N-tetraose is:

[0093] (1) Taking the LJ2ΔsucCD strain as a starting strain, taking the pEcCas plasmid containing Cas9 and lambda-Red recombinase (purchased from addgene) as a tool plasmid, preparing LJ2ΔsucCD competent cells and transforming the pEcCas plasmid into the cells to obtain the LJ2ΔsucCD strain containing the pCas9 plasmid.

[0094] (2) Designing the N20 sequence of the tktA gene (tktA-N20), and using the Gibson assembly method to assemble and construct the pEcgRNA-N20 plasmid with the pEcgRNA containing the sgRNA sequence (purchased from addgene); through heat shock transformation, the pEcgRNA-N20 positive plasmid is obtained, and is screened on the culture medium containing spectinomycin spec.

[0095] (3) The fusion Donor DNA fragment (tktA-Donor) is prepared according to step (3) of Example 6, and then the pEcgRNA-N20 positive plasmid and the Donor DNA fragment are transformed into the LJ2ΔsucCD strain containing the pCas9 plasmid; the strain in which the tktA gene is successfully knocked out is verified by PCR, and sequencing is performed to confirm.

[0096] (4) Through the culture and screening steps, the plasmid is eliminated to ensure that the strain only retains the required gene editing, and finally the recombinant Escherichia coli for improving the production of lacto-N-tetraose is obtained, which is named LJ2ΔsucCDΔtktA.

[0097] Example 9:

[0098] An embodiment of the method for producing lacto-N-tetraose by using the recombinant Escherichia coli for improving the production of lacto-N-tetraose; the recombinant Escherichia coli is subjected to shake flask fermentation, which comprises the following steps:

[0099] (1) Strain activation: single colonies of recombinant E. coli were picked from solid screening plates and inoculated into 5 mL LB medium containing the corresponding antibiotic, and incubated at 37°C for 10-20 h;

[0100] (2) Preparation of primary seed: the above overnight culture was inoculated into a 250 mL flask containing 50 mL LB medium at a 1% inoculation amount, and incubated at 37°C until OD 600 =0.4~1.2.

[0101] LB liquid medium (g / L) formula: sodium chloride 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, pH 7.2-7.4; sterilized at 121°C for 20 min.

[0102] (3) The primary seed liquid was inoculated into a 500 mL flask containing 100 mL fermentation medium at a 1-3% inoculation amount, and incubated at 37°C, 220 rpm until OD 600 =0.6-0.8 (the OD 600 value at this time point), and then 0.1-0.5 mM IPTG and 7 g / L lactose were added for induction, and the culture was transferred into a 28°C, 250 rpm incubator for transformation synthesis of the induced product, and the fermentation was completed after 90 h.

[0103] Shaking flask fermentation medium formula: fermentation medium (g / L) components are Na2HPO4·12H2O 17.9 g / L, KH2PO4 3 g / L, NH4Cl 2 g / L, (NH4)2HPO4 1 g / L, trisodium citrate 2H2O 2.2 g / L, CaCl2·2H2O 0.015 g / L, Triton-X 100 0.3 mL, glycerol 30 g / L, yeast extract powder 2 g / L, tryptone 15 g / L. Trace elements, vitamin B1, and MgSO4·7H2O were added before inoculation.

[0104] Trace element formula: sodium nitrilotriacetate 6.87 g / L, ferric ammonium citrate 2.8 g / L, zinc sulfate heptahydrate 0.45 g / L, CoCl2•6H2O 0.1 g / L, manganese chloride tetrahydrate 0.5 g / L, CuCl2•6H2O 0.05 g / L, boric acid 0.1 g / L, Na2MoO4•2H2O 0.1 g / L.

[0105] Reagent preparation:

[0106] 20% lactose preparation: weigh 20 g of lactose and dissolve in 80 mL of pure water, heat in a microwave oven to dissolve, and then dilute to 100 mL. After inoculation, about 5 hours, OD600 When = 0.6-0.8, the final concentration of the shake flask fermentation medium is 7 g / L (3.5 mL is added to 100 mL of the medium).

[0107] IPTG (0.1M stock solution) preparation: 0.238 g of IPTG was dissolved in 8 mL of deionized water, and after complete dissolution, the volume was adjusted to 10 mL. After sterilization with a sterile filter membrane (0.22 µm) in a clean bench, the solution was stored at -20℃ for standby use. After inoculation, about 5 hours, OD 600 When = 0.6-0.8, the final concentration of the shake flask fermentation medium is 0.2 mM (200 µL is added to 100 mL of the medium).

[0108] 50% glucose preparation: 50 g of glucose was dissolved in 80 mL of pure water in a microwave oven, and after dissolution, the volume was adjusted to 100 mL. The final concentration of the shake flask fermentation medium before inoculation was 5 g / L (1 mL was added to 100 mL of the medium).

[0109] 10% MgSO4•7H2O preparation: 10 g of MgSO4•7H2O was dissolved in 80 mL of pure water, and after dissolution, the volume was adjusted to 100 mL. The final concentration of the shake flask fermentation medium before inoculation was 1 g / L (1 mL was added to 100 mL of the medium).

[0110] VB1 (thiamine hydrochloride 10 mg / mL) preparation: the final concentration of the shake flask fermentation medium before inoculation was 20 mg / L (200 µL was added to 100 mL of the medium).

[0111] Comparative Example 1

[0112] A comparative example of the recombinant E. coli for improving the yield of lacto-N-tetraose; the recombinant E. coli takes the LJ2ΔsucCD strain as the chassis strain, and is genetically modified as follows: the 6-phosphofructokinase gene pfkA is knocked out.

[0113] The construction method of the recombinant E. coli for improving the yield of lacto-N-tetraose is as follows:

[0114] (1) Taking the LJ2ΔsucCD strain as the starting strain, and taking the pEcCas plasmid (purchased from addgene) containing Cas9 and λ-Red recombinase as the tool plasmid, the LJ2ΔsucCD competent cells were prepared and the pEcCas plasmid was transformed into the cells to obtain the LJ2ΔsucCD strain containing the pCas9 plasmid.

[0115] (2) Design the N20 sequence of pfkA gene (pfkA-N20), use Gibson assembly method to assemble it with pEcgRNA (purchased from addgene) containing sgRNA sequence to construct pEcgRNA-N20 plasmid; obtain pEcgRNA-N20 positive plasmid by heat shock transformation, and screen on the culture medium containing spectinomycin spec.

[0116] (3) Prepare the fusion Donor DNA fragment (pfkA-Donor) as in step (3) of Example 3, then transform the pEcgRNA-N20 positive plasmid and the Donor DNA fragment into the LJ2ΔsucCD strain containing the pCas9 plasmid; verify the strain with the pfkA gene successfully knocked out by PCR, and perform sequencing confirmation.

[0117] (4) Eliminate plasmids through culture and screening steps to ensure that the strain only retains the desired gene editing, and finally obtain the recombinant Escherichia coli for improving the production of lacto-N-tetraose, named LJ2ΔsucCDΔpfkA.

[0118] Test Example 1

[0119] This test example detects the content of LNT II and LNT of the fermentation broth obtained by fermenting the recombinant Escherichia coli of Examples 3-8 and Comparative Example 1 according to the fermentation method of Example 9 (the fermentation broth obtained by fermenting the LJ2 as a control strain is compared).

[0120] Fermentation broth pretreatment:

[0121] Take 1 mL of fermentation broth, boil in boiling water for 5 min, centrifuge at 12000 rpm for 5 min, take the supernatant, filter twice with a 0.22 μm filter membrane, and store for HPLC detection of LNT level.

[0122] HPLC detection:

[0123] Instrument model: Shimadzu LC-16-high performance liquid chromatograph;

[0124] Detection conditions: chromatographic column: hilic, detector: differential detector; mobile phase: 75% acetonitrile; flow rate: 1 mL / min, column temperature: 40°C.

[0125] The results are shown in Tables 2-3.

[0126] Table 2 (unit: g / L)

[0127]

[0128] From the above table, the efficiency of the control strain LJ2 for producing LNT in a shake flask is 5.65 g / L, and the shake flask yield is 0.0785 g / L / h. In comparison, the recombinant E. coli of the present application for fermenting to produce LNT has a significantly improved LNT fermentation yield. Among them, the engineered bacteria of Example 4 knock out the succinyl-CoA synthetase gene sucCD, and the LNT yield is significantly improved, with a yield of 8.81 g / L and a shake flask yield of 0.1224 g / L / h, which is increased by nearly 55.92% compared with the control; the recombinant strains obtained by knocking out the 6-phosphofructokinase gene pfkA, the transporter coding gene proP and the transketolase gene tktA of Example 3, Example 5 and Example 6, respectively, also have improved LNT yield, with a yield of 8 g / L, 7.99 g / L and 7.63 g / L, respectively, and a shake flask yield of 0.111 g / L, 0.111 g / L and 0.106 g / L, respectively, which is increased by 41.4%, 41.4% and 35.03% compared with the control. In addition, the yield of LNT II produced by Example 3 and 5 is 2.04 g / L and 1.88 g / L, respectively, while the yield of the by-product LNT II produced by the control strain LJ2 in a shake flask fermentation is 2.05 g / L. Therefore, knocking out the transporter coding gene proP or the 6-phosphofructokinase gene pfkA not only improves the efficiency of producing LNT, but also reduces the generation of by-products. This shows that the recombinant strain obtained by knocking out the metabolic flow pathway related enzyme in the present application has excellent fermentation production of LNT.

[0129] Table 3 (unit: g / L)

[0130]

[0131] From the above table, the efficiency of the control strain LJ2 for producing LNT in a shake flask is 5.65 g / L, and the shake flask yield is 0.0785 g / L / h. In comparison, the recombinant E. coli of the present application for fermenting to produce LNT has a significantly improved LNT fermentation yield. Among them, the engineered bacteria of Example 4 knock out the succinyl-CoA synthetase gene sucCD, and the LNT yield is significantly improved, with a yield of 8.81 g / L and a shake flask yield of 0.1224 g / L / h, which is increased by nearly 55.92% compared with the control; the recombinant strains obtained by knocking out the 6-phosphofructokinase gene pfkA, the transporter coding gene proP and the transketolase gene tktA of Example 3, Example 5 and Example 6, respectively, also have improved LNT yield, with a yield of 8 g / L, 7.99 g / L and 7.63 g / L, respectively, and a shake flask yield of 0.111 g / L, 0.111 g / L and 0.106 g / L, respectively, which is increased by 41.4%, 41.4% and 35.03% compared with the control. In addition, the yield of LNT II produced by Example 3 and 5 is 2.04 g / L and 1.88 g / L, respectively, while the yield of the by-product LNT II produced by the control strain LJ2 in a shake flask fermentation is 2.05 g / L. Therefore, knocking out the transporter coding gene proP or the 6-phosphofructokinase gene pfkA not only improves the efficiency of producing LNT, but also reduces the generation of by-products. This shows that the recombinant strain obtained by knocking out the metabolic flow pathway related enzyme in the present application has excellent fermentation production of LNT.

[0132] The LNT production of the comparative example 1 strain with sucCD and pfkA double knockout is lower than that of the control strain, and the LNT II production is higher. This shows that single gene knockout is effective, but does not mean that any combination of knockout genes can have an additive effect. This is caused by the complexity of the LNT production pathway of the strain and the interaction between genes. Combination of knockout genes may not have a positive additive effect, but may also cause a reverse effect, resulting in a decline in effect, which has a certain risk and cannot be routinely predicted.

[0133] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not a limitation on the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A recombinant Escherichia coli for improving production of lacto-N-tetraose, characterized by, The recombinant E. coli is a chassis strain, and is genetically modified according to any one of (1) and (2) as follows: (1) knocking out any one of 6-phosphofructokinase gene pfkA, succinyl-CoA synthetase gene sucCD, transporter-encoding gene proP, and transketolase gene tktA; (2) knocking out succinyl-CoA synthetase gene sucCD and transporter-encoding gene proP; The chassis strain is constructed by using gene editing technology to sequentially stack, based on E. coli BL21 star (DE3) as an initial strain, knockout of beta-galactosidase gene lacZ, fucose isomerase gene fucI, L-fucose kinase gene fucK, UDP glycolipid carrier transferase gene wcaJ, UDP-glucose dehydrogenase gene ugD, and glucosamine-6-phosphate deaminase gene nagB, while inserting single copy beta-1, 3-N-acetylglucosamine amino transferase gene lgtA and UDP-galactose-4-epimerase gene galE at lacA site, and inserting beta-1, 3-galactosyltransferase gene wbdO at wzxC site.

2. The method for constructing a recombinant E. coli that produces increased amounts of lacto-N-tetraose according to claim 1, wherein, The method comprises the following steps: on the basis of the chassis strain, any one of 6-phosphofructokinase gene pfkA, succinyl-CoA synthetase gene sucCD, transketolase gene tktA, and transporter-encoding gene proP is knocked out by homologous recombination using a pEcCas / pEcgRNA system; or, on the basis of the chassis strain, succinyl-CoA synthetase gene sucCD and transporter-encoding gene proP are knocked out by homologous recombination using a pEcCas / pEcgRNA system.

3. The recombinant E. coli for improving the yield of lacto-N-tetraose in claim 1 is applied in the production of lacto-N-tetraose.

4. A method for producing lacto-N-tetraose using the recombinant Escherichia coli for increasing production of lacto-N-tetraose according to claim 1, characterized by, The method comprises the following steps: (a) inoculating the recombinant E. coli for improving the yield of lacto-N-tetraose into a seed culture medium for activation; (b) inoculating the activated bacterial solution into a fermentation culture medium, adding an inducer for fermentation culture, and producing lacto-N-tetraose; The seed culture medium is LB liquid culture medium; The fermentation culture medium formula is: Na2HPO4·12H2O 15-20 g / L, KH2PO4 2-5 g / L, NH4Cl 1-3 g / L, (NH4)2HPO4 1-3 g / L, trisodium citrate 2H2O 1-3 g / L, CaCl2·2H2O 0.01-0.03 g / L, Triton-X100 0.1-1 mL, glycerol 20-40 g / L, yeast extract powder 1-5 g / L, and tryptone 10-25 g / L; The inducer comprises IPTG and lactose; The fermentation culture conditions are: 26-30°C, 180-300 rpm for 70-100 h.

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