A genetically engineered strain for producing 2′-fucosylated lactose without exogenous lactose addition and its application.

Abstract

This invention discloses a genetically engineered strain capable of producing 2′-fucosylated lactose without exogenous lactose addition and its applications, belonging to the field of genetic engineering technology. This invention systematically reconstructs an engineered strain capable of de novo synthesis of 2′-FL from glucose. By using plasmids with different copy numbers to coordinate the expression intensity of each module, precise balance of metabolic flux is achieved. Furthermore, by reconstructing the glucose transport pathway, the bottleneck of non-phosphorylated glucose precursor supply is solved, effectively coordinating the carbon flux allocation between lactose synthesis and central metabolism, avoiding metabolic imbalance and resource waste. Further, by knocking out… RSS The gene was modified to enhance the strain's resistance to stress, and the promoters of the key genes glf and glk were replaced, enabling the engineered strain to produce 13.1 g / L of 2′-FL in shake-flask fermentation and 51.9 g / L in a 3 L bioreactor with glucose as the sole carbon source after 70 h of fermentation.

Description

Technical Field

[0001] This invention relates to a genetically engineered strain that produces 2′-fucosylated lactose without the addition of exogenous lactose and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] 2′-Fucosyllactose (2′-FL) is one of the most abundant fucosylated human milk oligosaccharides (HMOs) in breast milk, with the structure Fuc-α1,2-Gal-β1,4-Glc. Numerous studies have shown that 2′-FL not only possesses significant prebiotic activity, selectively promoting the proliferation of beneficial bacteria in the infant gut, but also plays an irreplaceable and crucial role in regulating immune system function, directly inhibiting pathogen adhesion to intestinal epithelial cells, and supporting nervous system development. Given its clear health benefits, 2′-FL has been approved by major regulatory agencies such as the National Health Commission of China (NHC), the U.S. Food and Drug Administration (FDA), and the European Food Safety Authority (EFSA) for legal use as a nutritional additive in infant formula and other foods for special medical purposes. Therefore, developing efficient, economical, and industrially viable 2′-FL manufacturing technologies has become a research hotspot in both synthetic biology and food science.

[0003] Currently, the preparation of 2′-FL mainly relies on three technical routes: chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis involves complex multi-step protection and deprotection reactions, is cumbersome, and may use harmful reagents and produce difficult-to-separate byproducts, posing challenges to food safety. Enzymatic synthesis typically relies on purified fucosyltransferases and requires the direct supply of the expensive high-energy nucleotide precursor GDP-fucose and its acceptor lactose. Although the product has high specificity, the substrate cost is extremely high, severely limiting the economic feasibility of its large-scale application. In contrast, microbial fermentation utilizes metabolically engineered cell factories (such as E. coli and yeast) to de novo synthesize 2′-FL based on inexpensive carbon sources (such as glucose and glycerol) through intracellularly reconstructed metabolic pathways. This method has comprehensive advantages such as short production cycle, low cost, readily available raw materials, and ease of process scale-up, and is considered the most promising strategy for achieving large-scale production of 2′-FL.

[0004] In traditional microbial fermentation strategies, based on the supply method of GDP-fucose, two main pathways can be identified: "rescue synthesis" and "de novo synthesis." The former requires the exogenous addition of L-fucose or GDP-fucose as a precursor to the culture medium, resulting in high costs. The latter involves the introduction or enhancement of enzymes such as mannose isomerase and GDP-mannose dehydratase. Gmd ) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase ( WcaG By using key enzymes such as fructose and phosphodiesterase, and starting from the central metabolite (fructose-6-phosphate), an endogenous GDP-fucose synthesis module can be constructed, thereby significantly reducing the synthesis cost of the fucose fraction. However, regardless of the route taken, most existing industrial research schemes suffer from a fundamental bottleneck: they must rely on exogenously added lactose as the acceptor for fucose. This significantly increases raw material costs and process complexity. Furthermore, the low transmembrane transport efficiency of exogenous lactose, the potential for unnecessary metabolic diversion, and the osmotic pressure challenges posed by high lactose concentrations all fundamentally limit further improvements in the final yield, production intensity, and overall process economics of 2′-FL.

[0005] To overcome this bottleneck, recent research has begun exploring completely de novo biosynthetic routes that do not rely on exogenous lactose. For example, studies have reconstructed the endogenous pathway from glucose to lactose in Bacillus subtilis and achieved a 2′-FL yield of 30.1 g / L by balancing precursor supply through dynamic regulation strategies. However, the above-mentioned fermentation strategies using glucose as a carbon source still have the following drawbacks: First, the endogenous lactose synthesis capacity is insufficient. Although engineered strains introduce lactose synthesis modules, the efficiency of intracellular lactose self-synthesis is low, making it difficult to meet the acceptor requirements for efficient 2′-FL synthesis. Second, the carbon metabolic flux allocation is not precise, and the rational allocation of phosphorylated glucose is needed for 2′-FL synthesis in Escherichia coli with glucose as the sole carbon source. Therefore, developing a completely de novo synthesis technology with glucose as the sole carbon source that is simpler in process, has a more singular carbon source, and can achieve higher yields remains an urgent need and of great value for promoting the low-cost industrial production of 2′-FL. This invention aims to provide a novel metabolic engineering solution against this background. Summary of the Invention

[0006] The technical problem to be solved by this invention is that the existing microbial synthesis of 2′-fucosyllactose (2-FL) generally requires the addition of exogenous lactose as a key precursor, which leads to high raw material costs, complex fermentation processes, and the product purity and yield being easily affected by lactose residues or degradation.

[0007] To address the aforementioned technical challenges and achieve efficient 2′-FL synthesis using inexpensive glucose as the sole carbon source, this invention provides a novel metabolic engineering strategy. The core of this strategy lies in: 1) constructing an efficient endogenous lactose synthesis pathway within engineered bacteria to replace exogenous addition; 2) systematically modifying glucose uptake and initial metabolism to provide sufficient non-phosphorylated glucose substrate for lactose synthesis; and 3) coordinating central carbon metabolism and cofactor regeneration to enhance the supply of precursors and energy required for 2′-FL synthesis.

[0008] The first objective of this invention is to provide a genetically engineered bacterium that knocks out the pathway gene competing for the 2′-fucosylation precursor and overexpresses the genes of modules (a) to (d) below: (a) Lactose autosynthesis module: gene encoding β-1,4-galactosyltransferase GalTpm1141 ; (b) NPTS glucose uptake and phosphorylation module: genes encoding glucose-promoting proteins glf Gene encoding glucokinase glk ; (c) Central carbon metabolism coordination module: gene encoding glucose-6-phosphate isomerase pgi Genes encoding inorganic pyrophosphatase ppa ; phosphoglucosuricase gene pgm UTP-glucose-1-phosphate uridine transferase gene galU UDP-glucose 4-epiisomerase gene galE ; (d) Cofactor and nucleotide precursor supply module: gene encoding glucose-6-phosphate dehydrogenase zwf Gene encoding guanylate kinase gsk .

[0009] In one embodiment, the genetically engineered bacteria are Escherichia coli BL21 as the starting strain.

[0010] In one embodiment, the pathway genes competing for the 2′-fucosylated lactose synthesis precursor include β -Galactosidase gene lacZ galactoside O - Acetyltransferase gene lacA The gene for glucose-1-phosphatase agp UDP-glucose 6-dehydrogenase gene ugd Quinone protein glucose dehydrogenase gene gcd Glycoexport protein gene setA EIICB Glc protein gene ptsG . In one embodiment, the genetically engineered bacteria also knocked out the gene encoding the RpoS protease aptamer protein. rssB (nucleotide sequence as shown in SEQ ID NO.11) to enhance the strain's stress tolerance.

[0011] In one embodiment, the genetically engineered bacteria also use a moderately strong constitutive promoter P. J23119 Regulatory genes glf The expression, and P T7 Promoter regulation glk Gene expression.

[0012] In one implementation, the deletion is knocked out using a CRISPR / Cas9 system.

[0013] In one embodiment, the high-copy plasmid includes, but is not limited to, pRSFDuet-1; the medium-copy plasmid includes, but is not limited to, pETDuet-1; and the low-copy plasmid includes, but is not limited to, pCDFDuet-1.

[0014] In one embodiment, the genetically engineered bacteria are expressed using a high-copy plasmid. β-1,4- galactosyltransferase gene GalTpm1141 phosphoglucosuricase gene pgm UTP-glucose-1-phosphate uridine transferase gene galU UDP-glucose 4-epiisomerase gene galE Gene; and expressed glucose-6-phosphate isomerase using a medium copy plasmid. pgi Fucosyltransferase BKHT, and glucokinase gene expressed in a low-copy plasmid. glk, glucose facilitated protein gene glf glucose-6-phosphate dehydrogenase gene zwf Inorganic pyrophosphatase gene ppa guanylate kinase gene gsk .

[0015] In one embodiment, the genetically engineered bacteria are produced by knocking out... lacZ , lacA , agp , ugd , gcd , setA , ptsG The gene strain BL21(DE3)Δ lacZ Δ lacA Δ agp Δ ugd Δ gcd Δ setA Δ ptsG Δ rssBBased on this, the gene was expressed using the high-copy plasmid pRSFDuet-1. GalTpm1141 , pgm , galU, galE It is expressed using the medium copy plasmid pETDuet-1. pgi BKHT, and expressed the gene using the low-copy plasmid pCDFDuet-1. glk , glf , zwf , ppa and gsk .

[0016] In one embodiment, the β -Galactosidase gene lacZ The nucleotide sequence is as shown in the NCBI accession number: NC_000913.3 (363231..366305); the galactoside O - Acetyltransferase gene lacA For example, NCBI accession number: NC_000913.3 (361249..361860); the glucose-1-phosphatase gene. agp For example, NCBI accession number: NC_000913.3 (1065585..1066826); the UDP-glucose 6-dehydrogenase gene. ugd For example, NCBI accession number: NC_000913.3 (2098447..2099613); the quinone protein glucose dehydrogenase gene. gcd For example, NCBI accession number: NC_000913.3 (138835..141225); the glycoprotein described. setA For example, NCBI accession number: NC_000913.3 (77621..78799); the EIICB Glc protein gene ptsG For example, NCBI login number: NC_000913.3 (1157869..1159302).

[0017] In one embodiment, the glucose-facilitated protein gene glf The nucleotide sequence is as shown in SEQ ID NO.1; the guanylate kinase gene. gsk The nucleotide sequence is as shown in SEQ ID NO.2; the phosphoglucosuric enzyme gene. pgm The nucleotide sequence is as shown in SEQ ID NO.3; the UTP-glucose-1-phosphate uridine transferase gene. galU The nucleotide sequence is as shown in SEQ ID NO.4; the UDP-glucose 4-epiisomerase gene. galE The nucleotide sequence is as shown in SEQ ID NO. 5; the glucose-6-phosphate dehydrogenase gene.zwf The nucleotide sequence is as shown in SEQ ID NO. 6; the inorganic pyrophosphatase gene. ppa The nucleotide sequence is as shown in SEQ ID NO.7; the glucose-6-phosphate isomerase gene. pgi The nucleotide sequence is as shown in SEQ ID NO. 8; β- 1,4-Galactosyltransferase gene GalTpm1141 The nucleotide sequence is as shown in SEQ ID NO.9.

[0018] In one implementation, the promoter P J23119 The nucleotide sequence is shown in SEQ ID NO.10.

[0019] The present invention also provides a method for producing 2′-fucosylated lactose by fermentation culture using the above-mentioned genetically engineered bacteria, the method comprising the following steps: (1) The genetically engineered bacteria are inoculated into a culture medium and cultured to obtain a bacterial solution; (2) Transfer the above bacterial culture to the fermentation medium and ferment to produce 2′-fucosylated lactose.

[0020] In one embodiment, the culture medium in step (1) includes LB culture medium.

[0021] In one embodiment, the culture conditions in step (1) are 8h to 12h, 7℃, and 200 rpm.

[0022] In one embodiment, the bacterial OD during transfer in step (2) 600 The range is 1 to 3.

[0023] In one embodiment, the fermentation medium in step (2) comprises: 10-30 g / L glucose, 3-4 g / L yeast extract, 0.25-1 g / L peptone, 10-15 g / L potassium dihydrogen phosphate, 4-6 g / L diammonium hydrogen phosphate, 1-2 g / L citric acid, 1-2 g / L magnesium sulfate heptahydrate, and 7-10 mL / L trace element solution. The trace element solution includes 8-12 g / L ferric citrate(III), 1-2 g / L zinc sulfate heptahydrate, 0.5-2 g / L copper sulfate pentahydrate, 0.1-0.5 g / L manganese sulfate monohydrate, 0.1-0.3 g / L sodium tetraborate decahydrate, 0.05-0.2 g / L ammonium heptamolybdate, and 1-3 g / L calcium chloride.

[0024] In one embodiment, the fermentation medium comprises: 30.00 g / L glucose, 5.00 g / L yeast extract, 0.50 g / L peptone, 13.50 g / L potassium dihydrogen phosphate, 4.00 g / L diammonium hydrogen phosphate, 1.70 g / L citric acid, 1.44 g / L magnesium sulfate heptahydrate, and 10 mL / L trace element solution. The trace element solution includes 10.00 g / L ferric citrate(III), 2.25 g / L zinc sulfate heptahydrate, 1.00 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.25 g / L sodium tetraborate decahydrate, 0.17 g / L ammonium heptamolybdate, and 2.00 g / L calcium chloride.

[0025] In one embodiment, the fermentation temperature in step (2) is 20-30°C, preferably 25°C.

[0026] In one embodiment, step (2) involves controlling the cell OD before fermentation production. 600 The preferred value is 20-30, OD 600 It is 25.

[0027] In one embodiment, the fermentation process in step (2) is further induced by an inducing agent; the inducing agent is IPTG.

[0028] In one embodiment, the final concentration of the inducer is 0.05–0.4 mM, preferably 0.1 mM.

[0029] In one embodiment, after the inducer is added, the fermentation culture time is not less than 40 h, preferably 72 h.

[0030] In one embodiment, a feed culture medium is added during fermentation production in step (2) to maintain a carbon source concentration of 5-10 g / L.

[0031] In one embodiment, the feed culture medium contains: 400-800 g / L glucose, 15-30 g / L MgSO4·7H2O, 2-8 g / L yeast extract, and 1-5 g / L peptone.

[0032] In one embodiment, the fed culture medium contains: 600 g / L glucose, 22.02 g / L MgSO4·7H2O, 5.76 g / L yeast extract, and 2.88 g / L peptone.

[0033] In one embodiment, the genetically engineered bacteria are cultured in LB medium for 10 hours at 337°C and 200 rpm to obtain a seed culture. The seed culture is then added at 5-10% to a fermentation medium containing a carbon source and cultured until the OD reaches its limit. 600 Add IPTG to a concentration of 0.05–0.4 mM in the reaction system, and incubate for at least 72 hours.

[0034] In one embodiment, the genetically engineered bacteria are inoculated into LB medium and cultured overnight at 37°C for 12 h to prepare a fermentation seed culture. Then, the seed culture is inoculated at a 1% inoculation rate into a fermentation medium containing glucose as a carbon source. When the OD... 600 Once the concentration reaches 1, add IPTG at a final concentration of 0.05 mM as an inducer and continue fermentation at 25°C for 72 h.

[0035] In one embodiment, the genetically engineered bacteria are cultured at 30–40°C and 200–220 rpm to obtain a seed culture. The seed culture is then added to a 3 L fermentation medium at a concentration of 5–10% and cultured until the OD reaches its limit. 600 Add IPTG to a concentration of 15–25 mM, bringing the final IPTG concentration to 0.05–0.4 mM. Induce culture for at least 50 h.

[0036] In one embodiment, the genetically engineered bacteria are cultured at 37°C and 200 rpm for 12 h to obtain a seed culture. The seed culture is then added at 10% to a 3 L fermentation medium and cultured until the OD reaches the target value. 600 Add IPTG to a concentration of 25, bringing the final concentration of IPTG to 0.1 mM. Induce culture for at least 40 h.

[0037] In one embodiment, the carbon source is glucose.

[0038] This invention also provides the application of the genetically engineered bacteria or the method in the preparation of 2′-fucosylated lactose or products containing 2′-fucosylated lactose in the food, chemical, and pharmaceutical fields.

[0039] Beneficial effects: (1) This invention blocks lactose (knockout) lacZ, lacA uridine-5′-bisphosphate-glucose (UDP-Glc) competitive pathway (knockout) ugd, gcd and invalid loops (knockout) agp ), and weaken extracellular transport of precursors and products (knockout) setA Simultaneously, knocking out EIICB, a key component of the glucose phosphotransferase system (PTS). Glc Protein-coding genes ptsGThis is to decouple glucose uptake from phosphorylation. Further, by knocking out... rssB The gene stabilizes the global stress response factor RpoS, enhancing the strain's stress tolerance in the later stages of fermentation. Based on this, [the following text is incomplete and requires further context: "…"] glf strong P T7 The promoter was replaced with a moderate-strength constitutive promoter P. J23119 To alleviate cytotoxicity caused by membrane protein overexpression, a balance between glucose uptake and phosphorylation expression was achieved. A modular engineering strategy was employed, systematically dividing the de novo synthesis pathway of 2′-fucosylated lactose (2′-FL) into a lactose de novo synthesis module, a GDP-fucosylated de novo synthesis module, a precursor supply and carbon flow balance module, and a glucose supply module. Plasmids with different copy numbers (pRSFDuet, pETDuet, pCDFDuet / pACYC) were used to coordinate the expression levels of each module. Through system optimization, the optimal plasmid combinations were determined to be: pRSFDuet-GalTpm1141-pgm-galE-galU, pETDuet-pgi-BKHT, and pCDFDuet-glf-glk-zwf-ppa-gsk, which resulted in a 2′-fucosylated lactose yield of 13.1 g / L at the shake-flask fermentation level.

[0040] (2) By systematically optimizing glucose uptake and intracellular utilization, in the knockout ptsG Based on this, exogenous glucose-facilitating proteins (GFAs) were introduced. glf ) and enhance glucokinase ( glk By expressing the gene, a highly efficient non-PTS glucose supply system was constructed. This strategy significantly improved the strain's ability to transport glucose and convert it into key phosphorylation precursors, providing sufficient carbon flux for the co-synthesis of lactose and GDP-fucose, and is one of the key steps to achieve high yield.

[0041] (3) The present invention was also validated in a 3 L bioreactor with high cell density fermentation. Without the need for exogenous addition of lactose or fucose precursors, and using glucose as the sole carbon source, the yield of 2′-fucosylated lactose reached 51.9 g / L after 70 h of fermentation, setting a new production record under the same raw material strategy, demonstrating the excellent industrial application potential of this engineered strain and process route. Attached Figure Description

[0042] Figure 1 This is the metabolic pathway for the synthesis of 2′-fucosylated lactose.

[0043] Figure 2 A graph showing the yield of 2′-fucosylated lactose under different module combinations. Figure 3Graph showing the yield of 2′-fucosylated lactose under different promoter combinations Figure 4 The figure shows the fermentation yield of 2′-fucosylated lactose in a 3L bioreactor. Detailed Implementation

[0044] Culture medium: LB medium: 5.00 g / L yeast extract, 10.00 g / L peptone and 10.00 g / L sodium chloride.

[0045] LB solid medium: 5.00 g / L yeast extract, 10.00 g / L peptone, 10.00 g / L sodium chloride, and 20 g / L agar powder.

[0046] Fermentation medium: 30.00 g / L glucose, 5.00 g / L yeast extract, 0.50 g / L peptone, 13.50 g / L potassium dihydrogen phosphate, 4.00 g / L diammonium hydrogen phosphate, 1.70 g / L citric acid, 1.44 g / L magnesium sulfate heptahydrate, and 10 mL / L trace element solution; Trace element solution: 10.00 g / L ferric citrate (III), 2.25 g / L zinc sulfate heptahydrate, 1.00 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium tetraborate decahydrate, 0.17 g / L ammonium heptamolybdate, and 2.00 g / L calcium chloride.

[0047] Feeding medium: 600 g / L glucose, 22.02 g / L MgSO4·7H2O, 5.76 g / L yeast extract, 2.88 g / L peptone.

[0048] The fermentation broth treatment method and the detection conditions for 2′-fucosyllactose described in this embodiment of the invention are as follows: The sample preparation and analysis methods are as follows: The fermentation broth sample is boiled at 100°C for 15 minutes, and then centrifuged at 12000 rpm for 15 minutes using a high-speed centrifuge. The supernatant after centrifugation is filtered through a 0.22 μm filter membrane as the sample to be tested. 2′-fucosyllactose is detected by high-performance liquid chromatography (HPLC): differential refractive index detector; chromatographic column: Aminex HPX-87H (300 × 7.8 mm); column temperature: 50°C; mobile phase: 5 mM H2SO4 aqueous solution; flow rate: 0.5 mL / min; injection volume: 10 μL.

[0049] Example 1: Construction of gene knockout chassis cells 1. Construction of knockout plasmids (with setA (Using the knockout as an example for illustration) N20 replacement: using sgRNAcas9 software in setA The predicted N20 sequence was selected from the gene, and primers were designed (Table 1). Using the original pTargetF as a template, the N20 sequence on the template was replaced by PCR substitution. The PCR product was transformed into E. coli JM109, and plasmids were extracted and sequenced for verification. Successfully verified pTargetF... setA Store at -20℃ for later use.

[0050] setA Preparation of homologous arms: Using the genome of the strain to be knocked out as a template, using... setA- up-F / R and [[ID=9I]]setA- The down-F / R primer pair amplifies the knockout... setA The required upstream and downstream homologous arms were obtained, and the two DNA fragments were purified and recovered. Further utilization... setA- up-F / setA- down-R primer pairs were used to perform overlap PCR. setA The upstream and downstream homologous arms are fused into one fragment, and the DNA fragment is purified and recovered.

[0051] setA Homologous arm integrated into pTarget- setA Construct the complete knockout plasmid pTarget-Δ setA Using a seamless cloning kit (Nanjing Novizan Life Sciences Co., Ltd.) setA Homologous arm connected to carrier pTarget- setA The ligation product was transferred into *E. coli* JM109 via EcoRI and HiNdIII restriction sites, and the plasmid was extracted and sequenced for verification. The successfully verified pTarget-Δ setA Store at -20℃ for later use.

[0052] Other genes lacA , lac Z, ugd , gcd , ptsG , agp The method for knocking out plasmids is the same as above (the primers involved in constructing knockout plasmids are shown in Table 1).

[0053] Table 1 Primer sequences for gene knockout

[0054] 2. Knockout of Escherichia coli BL21(DE3) genome genes (with setA (The knockout is used as an explanation) Transform the pcas9 plasmid into BL21(DE3) and incubate at 30°C for 16-24 h. Select single clones and transfer them to 10 mL LB medium (containing kanamycin) and incubate at 30°C and 200 rpm for 12-16 h.

[0055] Prepare competent cells containing the pcas9 plasmid and knock them out. setA Genes, the specific steps are as follows: (1) Pick cells from a culture tube or strewn plate (containing Cas9 plasmid) stored at -80℃ and transfer them to 10 mL LB medium (containing kanamycin). Incubate at 30℃ and 200 rpm for approximately 12-16 hours. Transfer to sterile LB medium (containing kanamycin) at an inoculum size of 1%. When cells grow to OD... 600 When the OD value was 0.1, arabinose was added to a final concentration of 10 mmol / L to induce λ-Red-mediated homologous recombination. 600 Collect bacterial cells when the bacterial concentration reaches 0.4-0.6 to prepare competent cells.

[0056] (2) PTarget-Δ setA The knockout plasmid was added to approximately 100 μL of competent cells containing the pCas9 plasmid and electroporated (1 / 2 mm cuvette, 2.5 kV). Immediately after electroporation, 1 mL of pre-chilled LB broth was added to suspend the cells, and the cells were incubated at 30 °C for 1–2 h. The cells were then plated onto LB agar containing kanamycin and spectinomycin and cultured overnight at 30 °C. PCR verification was performed. lacZ Knockout effect.

[0057] pTarget-Δ setA Elimination: Identify the correct pCas9 and pTarget-Δ content. setA Positive clones were inoculated into LB medium containing kanamycin and IPTG (0.5 mmol / L) and cultured for 8–16 h to remove pTarget-Δ. setA Gene knockout strain BL21(DE3)Δ was obtained. setA。

[0058] 3. lacA , lacZ , ugd , gcd , agp , ptsG gene knockout In the aforementioned constructed strain BL21(DE3)Δ setA Based on this, knock out genes lacA , lacZ ,ugd , gcd , agp , ptsG The operation strategy is the same as the aforementioned knockout. setA Similarly, strain BL21(DE3)Δ was ultimately obtained by knocking out the pathway gene that competes with 2′-fucosylation precursor synthesis. lacZ Δ lacA Δ ugd Δ gcd Δ setA Δ ptsG Δ agp It was named BE5.

[0059] Based on BE5, construct a knockout rssb The engineered bacterium (with the nucleotide sequence shown in SEQ ID NO.11) is named BE6.

[0060] Example 2: Construction and screening of engineered host bacteria for efficient production of 2′-fucosylated lactose 1. Plasmid construction (1) with Galtpm1141 Using gene sequences as templates, with 4-1 Galtpm1141 -F / R are primers used for PCR amplification. Galtpm1141 Gene fragment (shown in SEQ ID NO.9), gel-recovered DNA fragment; (2) By using 2 × Phanta Max Master Mix premixed enzyme (Nanjing Novizan Biotechnology Co., Ltd.) and employing the MEGAWHOP method to... Galtpm1141 gene fragment Recombinant ligation was performed with pRSFDuet-1, and the ligation product was transformed into E. coli JM109. Plasmids were extracted and sequenced for verification. Successful verification yielded pRSFDuet-1. Galtpm1141 .

[0061] (3) Using the genome of Escherichia coli K12 as a template pgm- F / R are primers, and PCR amplification is performed. pgm Gene fragments (nucleotide sequences as shown in SEQ ID NO.3), DNA fragments recovered via gel extraction. galU Using gene sequences as templates, galU -F / R are primers used for PCR amplification. galU Gene fragments (nucleotide sequences as shown in SEQ ID NO.4), DNA fragments recovered via gel extraction. galE Using gene sequences as templates, galE -F / R are primers used for PCR amplification. galE Gene fragments (nucleotide sequences as shown in SEQ ID NO.5), gel recovery of DNA fragments; further utilization 1-1 Galtpm1141-F / galE -R primer pair was used to perform overlap PCR. pgm, galE and galU DNA fragments are fused into a single fragment, and the DNA fragment is purified and recovered.

[0062] (4) Use 2 × Phanta Max Master Mix premixed enzyme (Nanjing Novizan Biotechnology Co., Ltd.) and employ the MEGAWHOP method to mix the enzymes. pgm, galE and galU The fusion fragment and pRSFDuet - Galtpm1141 Recombinant ligation was performed, the ligation product was transformed into E. coli JM109, plasmid was extracted and sent for sequencing verification, and pRSFDuet was successfully obtained. Galtpm1141-pgm-galU-galE .

[0063] plasmid pETDuet- pgi -BKHT- zwf-gsk , pCDFDuet- glf-glk-zwf-ppa-gsk, pCDFDuet - glf-glk-pgi pCDFDuet -glf-glk-ppa-gsk, pETDuet -Pgi -BKHT- zwf, pCDFDuet -glf-glk- ppa-gsk pETDuet -Pgi-zwf -BKHT, pCDFDuet-glf-glk-ppa-gsk, pETDuet -ppa-pgi- BKHT, pRSFDue t-GalTpm1141-pgm-galE-ppa-galU ,pRSFDue t-pgm-galE-galU- GalTpm1141 , pRSFDue t-GalTpm1141-pgm-galE-galU ,pRSFDue t-GalTpm1141-galE-galU ,pRSFDuet- GalTpm1141-pgm-galU The construction method is the same as above (see Table 2).

[0064] Table 2 Primers for overexpression plasmid construction

[0065] 2. Construction of an engineered host bacterium for efficient production of 2′-fucosylated lactose and optimization screening based on modular combinations of different plasmid copy numbers. The 15 overexpression plasmids constructed in step 1 were combined and matched to obtain a total of 9 combinations: (1) pCDFDuet -glf-glk-ppa-gsk,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi-zwf -BKHT; (2) pCDFDuet -glf-glk-ppa-gsk-zwf ,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi -BKHT; (3) pCDFDuet -glf-glk-ppa ,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi- zwf-gsk -BKHT; ⑷pCDFDuet -glf-glk-ppa-gsk ,pRSFDuet- GalTpm1141-pgm-galE-galU-zwf, pETDuet- Pgi -BKHT; ⑸pCDFDuet -glf-glk -ppa-gsk ,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi -BKHT -zwf; ⑹pCDFDuet -glf-glk-ppa-zwf-gsk ,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi -BKHT; (7) pCDFDuet -glf-glk-zwf-ppa-gsk ,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi -BKHT; (8) pCDFDuet -glf-glk-ppa ,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi -BKHT -zwf-gsk; ⑼pCDFDuet -glf-glk-zwf-ppa ,pRSFDuet- GalTpm1141-pgm-galE-galU, pETDuet- Pgi -BKHT -gsk; The recombinant plasmids of the above 9 combinations were electroporated into the knockout strain BL21(DE3)Δ of Example 1. lacZ Δ lacA Δ ugd Δ gcd Δ setA Δ ptsG Δ agp Nine different engineered bacteria were obtained and named BE5-1, BE5-2, BE5-3, BE5-4, BE5-5, BE5-6, BE5-7, BE5-8, and BE5-9, respectively.

[0066] The constructed strain was cultured in LB medium at 37°C and 200 rpm for 10 h to obtain a seed culture. The seed culture was then added at 5-10% to a fermentation medium containing a carbon source and cultured until the OD reached the target value. 600 IPTG was added to the reaction mixture to a concentration of 0.05 mM, and fermentation continued at 25°C for 72 h. The results showed that the 2′-fucosylated lactose yields of the nine engineered strains were 2.23 g / L, 4.41 g / L, 0.87 g / L, 1.27 g / L, 4.95 g / L, 4.34 g / L, 7.11 g / L, 3.36 g / L, and 0.79 g / L, respectively. These strains contained the plasmid pCDFDuet. -glf-glk-zwf-ppa-gsk pRSFDuet- GalTpm1141-pgm-galE-galU and pETDuet- Pgi -BKHT's engineered strain BE5-7 yielded 7.11 g / L of 2′-fucosylated lactose. Figure 2 ).

[0067] Table 3. Detailed information on each genetically engineered strain and the yield of fermentation products.

[0068] Example 3: Key Genes glf and glk promoter replacement 1. Plasmid construction (1) with glf Using gene sequences as templates, P J23119 -glf F / R are primers, and PCR amplification is performed. P J23119 glf Gene fragments, gel-recovered DNA fragments; (2) By using 2 × Phanta Max Master Mix premixed enzyme (Nanjing Novizan Biotechnology Co., Ltd.) and employing the MEGAWHOP method to... P J23119 glf Gene fragments and pCDFDuet -glk-zwf-ppa-gsk Recombinant ligation was performed, the ligation product was transformed into E. coli JM109, plasmid was extracted and sent for sequencing verification, and pCDFDuet was obtained after successful verification. - P J23119 glf-glk-zwf-ppa-gsk .

[0069] plasmid pCDFDuet -P J23119 glf-P T7 glk-zwf-ppa-gsk pCDFDuet -P J23110 glf-P T7 glk- zwf-ppa-gsk, pCDFDuet -P J23119 glf-P J23119 glk-zwf-ppa-gsk pCDFDuet -P J23119 glf- P J23110 glk-zwf-ppa-gsk、 pCDFDuet -P J23110 glf-P J23119 glk-zwf-ppa-gsk、 pCDFDuet - P J23110 glf-P J23110 glk-zwf-ppa-gsk、 pCDFDuet -P T7 glf-P J23119 glk-zwf-ppa-gsk、 pCDFDuet - P T7 glf-P J23110 glk-zwf-ppa-gsk The construction methods for the eight plasmids are the same as above (see Table 4).

[0070] Table 4. Promoter Sequence

[0071] pRSFDuet- GalTpm1141-pgm-galE-galU and pETDuet- Pgi The -BKHT plasmid and any one of the eight plasmids mentioned above were co-transformed into the strain BE6 constructed in Example 1. The resulting recombinant bacteria were fermented according to the method in Example 2, and the yield of 2′-fucosylated lactose after 72 h of fermentation was detected. The results showed that the strain contained plasmid pCDFDuet... - P J23119 glf-glk-zwf-ppa-gsk、 pRSFDuet- GalTpm1141-pgm-galE-galU and pETDuet- PgiThe strain BE6-5, containing the -BKHT plasmid, produced 13.1 g / L of 2′-fucosylated lactose. Figure 3 ).

[0072] Table 4. Detailed information and fermentation product yields of each genetically engineered strain.

[0073] Example 3: Fermentation of 2′-fucosylated lactose by a high-efficiency production strain in a 3 L bioreactor In a 3 L bioreactor (Applikon), 2′-fucosylated lactose was prepared by culturing the genetically engineered strain BE6-5 constructed in Example 2 in LB medium at 37°C and 200–220 rpm to prepare a seed culture. This seed culture was then inoculated at a rate of 10% into a 1 L working volume fermentation medium. The bioreactor parameters were set as follows: culture temperature 37°C, dissolved oxygen maintained at 30%, and pH set to 6.8. When OD... 600 When the temperature reached 25°C, the culture temperature was set to 25°C, and IPTG was added to a final concentration of 0.1 mM. The pH was adjusted with 50% ammonia to maintain the pH of the culture medium at 6.8. Feeding was initiated when the glucose concentration in the fermentation system fell below 5 g / L, adding feed medium to a 3 L bioreactor to maintain a glucose concentration of 5-15 g / L. 2′-fucosylated lactose was periodically sampled and tested during fermentation. After 70 hours of fermentation, strain BE6-5 achieved a maximum 2′-fucosylated lactose yield of 51.9 g / L. Figure 4 ).

[0074] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered bacterium, characterized in that, Genes competing for the 2′-fucosylated lactose precursor synthesis pathway were knocked out, and genes in modules (a) to (d) were overexpressed: (a) Lactose autosynthesis module: gene encoding β-1,4-galactosyltransferase GalTpm1141 ; (b) NPTS glucose uptake and phosphorylation module: genes encoding glucose-promoting proteins glf Gene encoding glucokinase glk ; (c) Central carbon metabolism coordination module: gene encoding glucose-6-phosphate isomerase pgi Genes encoding inorganic pyrophosphatase ppa ; phosphoglucosuricase gene pgm UTP-glucose-1-phosphate uridine transferase gene galU UDP-glucose 4-epiisomerase gene galE ; (d) Cofactor and nucleotide precursor supply module: gene encoding glucose-6-phosphate dehydrogenase zwf Gene encoding guanylate kinase gsk .

2. The genetically engineered bacterium according to claim 1, characterized in that, The pathway genes competing for the 2′-fucosylated lactose precursor synthesis include β -Galactosidase gene lacZ galactoside O - Acetyltransferase gene lacA The gene for glucose-1-phosphatase agp UDP-glucose 6-dehydrogenase gene ugd Quinone protein glucose dehydrogenase gene gcd Glycoexport protein gene setA EIICB Glc protein gene ptsG .

3. The genetically engineered bacteria according to claim 1 or 2, characterized in that, The gene encoding the RpoS protease aptamer protein was also knocked out. rssB .

4. The genetically engineered bacteria according to any one of claims 1 to 3, characterized in that, Using a moderately strong constitutive promoter P J23119 Regulatory genes glf The expression, and P T7 Promoter regulation glk Gene expression.

5. The genetically engineered bacteria according to any one of claims 1 to 4, characterized in that, Expression with high copy plasmid β-1,4- galactosyltransferase gene GalTpm1141 phosphoglucosuricase gene pgm UTP-glucose-1-phosphate uridine transferase gene galU UDP-glucose 4-epiisomerase gene galE Gene; and expressed glucose-6-phosphate isomerase using a medium copy plasmid. pgi Fucosyltransferase BKHT, and glucokinase gene expressed in a low-copy plasmid. glk, glucose facilitated protein gene glf glucose-6-phosphate dehydrogenase gene zwf Inorganic pyrophosphatase gene ppa guanylate kinase gene gsk .

6. The genetically engineered bacterium according to claim 5, characterized in that, The high-copy plasmids include, but are not limited to, pRSFDuet-1; the medium-copy plasmids include, but are not limited to, pETDuet-1; and the low-copy plasmids include, but are not limited to, pCDFDuet-1.

7. The genetically engineered bacterium according to claim 1, characterized in that, It was knocked out lacZ , lacA , agp , ugd , gcd , setA , ptsG and rssB Based on the gene in E. coli BL21(DE3), the gene was expressed using the high-copy plasmid pRSFDuet-1. GalTpm1141 , pgm , galU, galE It is expressed using the medium copy plasmid pETDuet-1. pgi BKHT, and expressed the gene using the low-copy plasmid pCDFDuet-1. glk , glf , zwf , ppa and gsk .

8. A method for preparing 2′-fucosylated lactose by fermentation, characterized in that, include: The genetically engineered bacteria described in any one of claims 1 to 7 are cultured in a medium with glucose as the carbon source, and the 2′-fucosylated lactose in the fermentation broth is collected.

9. The method according to claim 8, characterized in that, During the fermentation process, additional feed is added to maintain a carbon source concentration of 5-10 g / L in the fermentation system.

10. The genetically engineered bacteria according to any one of claims 1 to 7 or the method according to any one of claims 8 to 9 are applicable to the food, chemical, and pharmaceutical fields.

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