Antibiotic-free genetically engineered bacteria for efficient production of fucosylated lactose and application thereof

Abstract

This invention relates to antibiotic-free genetically engineered bacteria for the efficient production of fucoidan-based lactose and its applications, belonging to the fields of metabolic engineering and food fermentation. This invention utilizes CRISPR / Cas9 gene editing technology to modify chassis microorganisms. Through strategies such as reconstructing the 2'-FL and 3-FL synthesis pathways in lactose-synthesizing strains, regulating central carbon metabolism and weakening byproduct pathways, upregulating key enzymes in the de novo synthesis pathway, removing repressor protein inhibition, and enhancing extracellular export of the product, antibiotic-free genetically engineered bacteria and efficient fucoidan synthesis are achieved. The recombinant *E. coli* obtained by this invention can efficiently synthesize fucoidan-based lactose using glycerol and glucose. No antibiotics are added during fermentation, the product is safe and harmless, production costs are low, and the product yield is high, demonstrating strong socio-economic benefits and broad market development prospects.

Description

Technical Field

[0001] This invention relates to antibiotic-free genetically engineered bacteria for the efficient production of fucoidan and their applications, belonging to the fields of metabolic engineering and food fermentation. Background Technology

[0002] Producing high-value-added products from inexpensive biomass through green bioprocesses is an important way to achieve carbon neutrality and an environmentally friendly economy. 2'-Fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are the most abundant neutral fucoidyllactose (FL) secreted in breast milk, accounting for approximately 35% of the total human milk oligosaccharides (HMOs). The beneficial properties of FL (such as maintaining intestinal ecological balance, resisting pathogen adhesion, immune regulation, and promoting nervous system development and repair) have led to considerable interest in its potential applications in nutrition, health care, and pharmaceuticals.

[0003] The development of green, efficient, and safe chassis microorganisms is key to solving the large-scale production and application of HMOs. With the advancement of metabolic engineering and synthetic biology, many model microorganisms have been discovered as potential microbial cell factories for HMO production. Model microorganisms such as *Escherichia coli*, *Saccharomyces cerevisiae*, *Yersinia lipolytica*, and *Bacillus subtilis* have been successfully used for the de novo biosynthesis of 2'-FL. Currently, 3'-FL producing strains are commonly found in *Escherichia coli*, but reports are few and yields are low. It has been reported that under anaerobic and microaerobic conditions, glycerol- or glucose-based catabolism at high concentrations can lead to the production of various byproducts (acetic acid, lactic acid, formic acid, etc.). The presence of byproducts slows or stops strain growth, resulting in a significant decrease or complete cessation of the production rate of the target compound. Therefore, eliminating these byproduct pathways is particularly important in FL production. Furthermore, the production of fucoidan-based lactose requires an exogenous supply of lactose, which is expensive. Developing new lactose synthesis technologies in microorganisms can solve the problem of high lactose costs in the efficient biosynthesis of FL.

[0004] Starting with host bacterial selection, strategies such as metabolic pathway reconstruction, weakening of competing pathways, cofactor and energy regeneration, and metabolic flux optimization can be used to construct industrially competitive FL-producing strains. However, the construction of most FL strains is based on plasmid gene overexpression. The development of plasmid-based cell factories carries risks of genetic instability and antibiotic addition during fermentation. Therefore, using antibiotic-free and inducing agent-free *E. coli* to prepare FL has clearly become a green, safe, and commercially viable production strategy. Previously, Researchers introduced de novo and salvage pathways for 2'-FL synthesis into the chromosome of *E. coli* strain JM109, producing 20.28 ± 0.83 g / L of 2'-FL in antibiotic-free fed-batch fermentation. Parschat et al. developed a 2'-FL-producing strain using sucrose as a substrate only, which intracellularly produced lactose for 2'-FL production without the addition of antibiotics. Apart from these, no other antibiotic-free or inducer-free 2'-FL-producing strains have been reported.

[0005] This invention aims to utilize synthetic biology techniques to construct antibiotic-free genetically engineered bacteria and achieve efficient synthesis of fucosyl lactose through strategies such as reconstructing the 2'-FL and 3-FL synthetic pathways in lactose-synthesizing strains, regulating central carbon metabolism and weakening byproduct pathways, upregulating key enzymes in de novo synthesis pathways, removing repressor protein inhibition, and enhancing extracellular export of products. This research method provides strain safety and substrate flexibility for the industrial use of FL, enriches and develops the technical research on microbial metabolic regulation, provides new methods and cases for reconstructing microbial metabolic networks and improving carbon economy, and offers new ideas for the rational design and construction of next-generation microbial cell factories. It has significant value in both theoretical and practical applications. Summary of the Invention

[0006] [Technical Issues]

[0007] Existing technologies for synthesizing fucoidan are not very efficient. Plasmid-type strains have risks of genetic instability and antibiotic addition. Receptor lactose and inducers are expensive. It is impossible to provide strains for safe and efficient production of fucoidan, nor can it provide a low-cost and environmentally friendly method for preparing fucoidan.

[0008] [Technical Solution]

[0009] To address the problems of expensive lactose and inducers, genetic instability of plasmid strains, and the risk of antibiotic addition during the synthesis of fucoidosyllactose, this invention provides a genetically engineered bacterium for the efficient production of lactose and fucoidosyllactose, and its construction method.

[0010] This invention relates to a genetically engineered bacterium, BP6, which can produce lactose using low-cost carbon sources (glycerol and glucose), obtained in the previous stage. BP6 is based on strain BZWNAPAL, with the glucose-specific transport protease EⅡABC knocked out. GlcThe components encode the genes crr and ptsG, and SetA and Glf are integrated at the crr and ptsG sites, respectively; the UDP-glucose-6-dehydrogenase gene ugd is knocked out, and the UDP-glucose-4-epimerase gene GalE is integrated at the ugd gene site; the glucokinase gene Glk is knocked out, and the β-1,4-galactosyltransferase NmlgtB derived from Neisseria meningitidis is integrated at the Glk site of the glucokinase gene.

[0011] The strain BZWNAPAL is BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlon, and its construction method has been disclosed in patent document with publication number CN114480240A.

[0012] The genetically engineered bacteria provided in this invention are based on strain BP6, with the ubiquinone-dependent pyruvate dehydrogenase gene poxB knocked out, and the α-1,2-fucosyltransferase gene HpfutC and the α-1,3-fucosyltransferase gene HpM32 integrated at the poxB site; the phosphoacetyltransferase and acetate kinase gene cluster pta-ackA knocked out, and the phosphomannose mutase and mannose-1-phosguanine succinate gene cluster manC-manB integrated at the pta-ackA site; the formate lyase gene pflB knocked out, and the GDP-mannose-6-dehydrogenase and GDP-fucose synthase gene cluster gmd-wcaG integrated at the pflB site; the D-lactate dehydrogenase gene ldhA knocked out, and the mannose-6-phosphoisomerase gene manA integrated at the ldhA site; and the lactose operon repressor protein lacI knocked out.

[0013] In one embodiment, the α-1,2-fucosyltransferase gene HpfutC is derived from Helicobacter pylori ATCC 26695, and the α-1,3-fucosyltransferase gene HpM32 is derived from Helicobacter pylori NCTC 11639, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0014] In one embodiment, the β-galactosidase gene lacZ has a Gene ID of 945006, the UDP-glucose lipotransferase gene wcaJ has a Gene ID of 946583, the GDP-mannose-mannosyl hydrolase gene nudD has a Gene ID of 946559, the 6-phosphofructokinase-1 gene pfkA has a Gene ID of 948412, the protease gene lon has a Gene ID of 945085, the glucose-specific transport protease genes crr and ptsG have Gene IDs of 946880 and 945651, respectively, the UDP-glucose-6-dehydrogenase gene ugd has a Gene ID of 946571, the UDP-glucose-4-epimerase gene GalE has a Gene ID of 945354, the glucokinase gene Glk has a Gene ID of 946858, and the nucleotide sequence of the β-1,4-galactosyltransferase NmlgtB derived from Neisseria meningitidis is shown in SEQ ID NO. 3.

[0015] In one embodiment, the α-1,2-fucosyltransferase gene HpfutC, the α-1,3-fucosyltransferase gene HpM32, the gene clusters manC-manB, gmd-wcaG, and the mannose-6-phosphate isomerase gene manA are all expressed using the T7 promoter.

[0016] In one implementation, the strong promoter T7 is used to replace the self-promoters of the genes encoding manC, manB, gmd-wcaG, and manA in the E. coli genome.

[0017] In one embodiment, the ubiquinone-dependent pyruvate dehydrogenase gene poxB has a Gene ID of 946132, the phosphoacetyltransferase gene pta has a Gene ID of 946778, the acetate kinase gene ackA has a Gene ID of 946775, the D-lactate dehydrogenase gene ldhA has a Gene ID of 946315, the formate lyase gene pflB has a Gene ID of 945514, the lactose operon repressor protein lacI has a Gene ID of 945007, the mannose-6-phosphate isomerase gene manA has a Gene ID of 944840, the phosphomannose mutase gene manB has a Gene ID of 946574, the mannose-1-phosphate guanine syltransferase gene manC has a Gene ID of 946580, the GDP-mannose-6-dehydrogenase gene gmd has a Gene ID of 946562, and the GDP-fucose synthase gene wcaG has a Gene ID of 946563.

[0018] A second object of the present invention is to provide the use of the recombinant Escherichia coli in the production of 2'-fucosylated lactose and / or 3-fucosylated lactose.

[0019] In one embodiment, the recombinant Escherichia coli is used as the fermentation strain to produce 2'-fucosylated lactose and / or 3-fucosylated lactose in a fermentation system with glycerol and glucose as carbon sources.

[0020] In one embodiment, 2'-fucosylated lactose and / or 3-fucosylated lactose are produced by fermentation in a shake flask or fermenter.

[0021] In one embodiment, the recombinant Escherichia coli is inoculated into a shake-flask fermentation medium, and glucose with a final concentration of 8 g / L is added at the beginning of fermentation. The mixture is then cultured at 30–40°C and 150–250 rpm for 72 h.

[0022] In one embodiment, a fermentation medium is added to a fermenter, and the recombinant Escherichia coli described in BP10-3 and BP11-3 are inoculated for fermentation.

[0023] In one embodiment, the concentration of glycerol in the fermenter is 10–40 g / L.

[0024] In one embodiment, the fermentation system contains 20-30 g / L glycerol, 5-10 g / L glucose, 10-15 g / L potassium dihydrogen phosphate, 1-2 g / L citric acid, 3-5 g / L diammonium hydrogen phosphate, 1-2 g / L magnesium sulfate heptahydrate, 8-10 g / L yeast extract, and 8-10 mL / L trace metal solution.

[0025] In one embodiment, the trace metal solution contains 8-10 g / L of triferric citrate, 2-3 g / L of magnesium sulfate heptahydrate, 0.5-1.0 g / L of copper sulfate pentahydrate, 0.2-0.5 g / L of manganese sulfate monohydrate, 0.2-0.5 g / L of borax, 0.1-0.2 g / L of ammonium molybdate, and 1-2 g / L of calcium chloride dihydrate.

[0026] In one embodiment, the genetically engineered bacteria are cultured at 20–40°C, maintaining dissolved oxygen at 30±5% and pH at 6.5–7.0 in the fermentation system.

[0027] In one embodiment, glycerol is added after the initial glycerol in the reaction system has been consumed, so that the concentration of glycerol is sufficient to maintain the cell growth and metabolism.

[0028] In one embodiment, glucose is added after the initial glucose is consumed to maintain its concentration at 10±0.5g / L.

[0029] In one implementation, the fermentation time is not less than 70 hours.

[0030] Preferably, the fermentation time is 100 hours.

[0031] The beneficial effects of this invention are:

[0032] This invention introduces the biosynthetic pathway of fucoidan into lactose-producing bacteria. By weakening the byproduct pathway, regulating central carbon metabolism, upregulating key enzymes in the de novo synthesis pathway, removing the repression of repressor proteins, and enhancing the extracellular export of the product, it achieves the construction of antibiotic-free strains and the efficient synthesis of fucoidan without the addition of exogenous lactose. Under shake-flask fermentation conditions without the addition of antibiotics and inducers, the genetically engineered bacteria constructed in this application achieve production capacities of 4.36 and 3.23 g / L for 2'-FL and 3-FL, respectively; under 3L fermenter cultivation conditions, the yields of 2'-FL and 3-FL reach 40.44 and 30.42 g / L, respectively, laying the foundation for the industrial production of fucoidan. Attached Figure Description

[0033] Figure 1 A schematic diagram of the metabolic process for producing fucoidosyl lactose from glucose and glycerol as substrates.

[0034] Figure 2 A comparison of the yields of engineered bacteria that were introduced to weaken the byproduct pathway and incorporate the fucoidosyl lactose pathway.

[0035] Figure 3 To investigate the effect of the GDP-L-fucose pathway on fucoidan production in engineered bacteria.

[0036] Figure 4 The relative transcription levels of the GDP-L-fucose module gene in strains BP10-3 and BP10-4 are given.

[0037] Figure 5 Feed the BP10-3 strain into a 3L fermenter for batch fermentation.

[0038] Figure 6 Feed the BP11-3 strain into a 3L fermenter for batch fermentation. Detailed Implementation

[0039] The following examples and accompanying drawings further illustrate the specific implementation of the present invention. The plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products, and the specific operations are performed in accordance with the kit instructions.

[0040] The embodiments of the present invention are not limited thereto; other unspecified experimental operations and process parameters shall be carried out in accordance with conventional techniques.

[0041] The vectors pCas9 and pTargetF were purchased from Addgene.

[0042] The sequencing of DNA products and plasmids was completed by Tianlin Biotechnology (Wuxi) Co., Ltd.

[0043] Preparation of competent Escherichia coli cells: reagent kit from Shanghai Sangon Biotech Co., Ltd.

[0044] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0045] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar powder.

[0046] Fermentation medium: glycerol 30 g / L, potassium dihydrogen phosphate 13.5 g / L, citric acid 1.7 g / L, diammonium hydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 1.4 g / L, yeast extract 10 g / L, trace metal solution 10 mL / L (ferric citrate 10 g / L, magnesium sulfate heptahydrate 2.25 g / L, copper sulfate pentahydrate 1.0 g / L, manganese sulfate monohydrate 0.35 g / L, borax 0.23 g / L, ammonium molybdate 0.11 g / L, calcium chloride dihydrate 2.0 g / L), pH 6.8.

[0047] Determination methods for 2'-FL, 3-FL and GDP-L-fucose:

[0048] 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 fucoidosyl lactose produced and the amount of glucose and glycerol consumed were determined by HPLC. HPLC detection conditions: differential refractive index detector; Rezex ROA-organic acid (Phenomenex, USA) column, column temperature 50℃; mobile phase 0.005 mol / L H2SO4 aqueous solution, flow rate 0.6 mL / min; injection volume 10 μL. HPLC detection conditions for GDP-L-fucose: UV detector; detection wavelength 254 nm; Inertsil ODS-SP column (GLSciences, Kyoto, Japan); mobile phase A was 20 mM triethylamine-glacial acetic acid aqueous solution (pH 6.0), and mobile phase B was acetonitrile solution; gradient elution; flow rate 0.6 mL / min; injection volume 10 μL.

[0049] 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 in a shake flask at 37°C and 200 rpm for 12 h to obtain a seed culture; the seed culture was then inoculated into 50 mL of fermentation medium at an inoculation rate of 3% (v / v) and cultured in a shake flask at 37°C and 200 rpm until OD reached. 600 The concentration was 0.6, and glucose was added to a final concentration of 8 g / L. The mixture was then induced and cultured at 25°C and 200 rpm for 72 h.

[0050] Example 1: Constructing a fucosylated lactose-free strain by removing the inhibition of repressor proteins and glycolysis byproducts.

[0051] Fucosyllactose synthesis requires an exogenous supply of lactose. Previously, a genetically engineered bacterium capable of producing lactose using low-cost carbon sources (glycerol and glucose) was constructed. Building upon this, to prevent metabolic spillover from the glycolysis pathway, the byproduct pathway was weakened and a fucosyllactose synthesis pathway was introduced. Using *E. coli* BP6 as the starting strain, the *poxB* gene was knocked out using the CRISPR-Cas9 gene editing system, and a double-copy α-1,2 / 3-fucosyltransferase gene, *HpfutC / HpM32*, was integrated at this site; *pta-ackA* was knocked out, and *manC-manB* was integrated at this site; *pflB* was knocked out, and *gmd-wcaG* was integrated at this site; *ldhA* was knocked out, and *manA* was integrated at this site; finally, the repressor protein gene, *lacI*, was knocked out. The metabolic pathway of fucosyllactose using glycerol and glucose as substrates in the lactose-producing strain is as follows: Figure 1 As shown, the specific steps for gene knockout and integration are as follows (the primer sequences involved are shown in Table 1):

[0052] (1) Taking the α-1,2-fucosyltransferase gene HpfutC, which is a knockout of poxB and integrated into the chromosome in a double tandem configuration, as an example, the specific target gRNA (20 bp) of the poxB gene was searched through http: / / www.regenome.net / cas-offinder. Using poxB-gRNA-F / gRNA-R primers, PCR amplification was performed with pTargetF plasmid (Addgene: #62226) as a template. The amplified product was digested with the restriction endonuclease Dpn I to remove the redundant circular plasmid pTargetF. The amplified product was then transformed into E. coli DH5α competent cells, and the plasmid was extracted and identified by sequencing. The successfully constructed knockout plasmid was named pTargetF-poxB.

[0053] (2) Using the genome of Escherichia coli BP6 strain as a template, three sequence fragments were amplified using the upstream homologous arm primer poxB-US-F / poxB-US-R, the midstream homologous arm primer 2HpfutC-MS-F / 2HpfutC-MS-R, and the downstream homologous arm primer poxB-DS-F / poxB-DS-R, respectively. After purification and recovery of the products, the three fragments were ligated using the SOE-PCR method with primers poxB-US-F / poxB-DS-R to obtain the gene homologous repair template.

[0054] (3) Take pCas9 plasmid (Addgene:#62225) and E. coli BP6 electroporation competent cells. After placing them on ice for 5 min, thaw the competent cells. Add 10 μL of plasmid to 100 μL of competent cells and mix gently. Transfer the plasmid and electroporation competent cells into a pre-chilled electroporation cuvette and electroporate at 2.5 kV for 5 ms. Immediately after electroporation, add pre-chilled liquid LB and mix gently by pipetting. Transfer the culture medium containing plasmid and competent cells to a new centrifuge tube for expansion culture for 1.5 h. Centrifuge at 6000 r / min for 2 min, discard the supernatant, spread the bacterial cells on LB agar plates containing kanamycin, and incubate overnight at 30℃.

[0055] (4) Single colonies of *E. coli* BP6 / pCas9 were picked and cultured in LB medium at 30°C for 1.0 h. L-arabinose was then added to a final concentration of 30 mM to induce λ-red system expression. When OD... 600 When the concentration reaches 0.6-0.8, prepare Escherichia coli BP6 / pCas9 competent cells.

[0056] (5) Electroporate 500 ng of the target plasmid pTargetF with poxB-specific target gRNA (20 bp) constructed in step (1) and 1000 ng of the homology repair template constructed in step (2) to the E. coli BP6 / pCas9 competent cells prepared in step (4), spread them on LB plates (kanamycin and spectinomycin), and incubate at 30℃ for 16-24 h. Perform colony PCR verification on the single colonies that grow on the plates, screen positive transformants and perform gene sequencing.

[0057] (6) For verified single colonies, the pTargetF-poxB and pCas9 plasmids were eliminated. Single colonies were inoculated into LB liquid medium (kanamycin resistant) and cultured at 30°C and 200 rpm until the logarithmic growth phase. IPTG was added to a final concentration of 0.5 mmol / L and cultured overnight to induce pTargetF-poxB plasmid inactivation. The bacterial suspension was streaked onto LB plates containing kanamycin and cultured at 30°C and 200 rpm for 12 h. Single colonies were spotted onto plates resistant to both kanamycin and spectinomycin. No colony growth indicated successful pTargetF-poxB plasmid elimination.

[0058] (7) The pCas9 plasmid is a temperature-sensitive plasmid. Single colonies of successfully eliminated pTargetF-poxB plasmid were transferred to LB broth without antibiotics and passaged at 42°C to eliminate the pCas9 plasmid. After streaking the bacterial solution onto antibiotic-free LB plates, the culture was kept at 37°C. Single colonies were spotted onto LB broth containing kanamycin. If no single colony grew, it indicated that the pCas9 plasmid was successfully eliminated. The constructed gene-deleted strains without pTargetF-poxB plasmid and pCas9 plasmid were stored at -80°C for later use.

[0059] (8) The knockout of genes poxB, pta-ackA, pflB, ldhA and lacI and the integration of the corresponding HpM32, manC-manB, gmd-wcaG and manA are performed according to the above steps. The construction steps of the gene-edited strains involved in other embodiments are all in accordance with Example 1.

[0060] Table 1. Primers for gene knockout and integration

[0061]

[0062]

[0063]

[0064] In recombinant engineered bacteria, the accumulation of glycolysis byproducts not only has a toxic effect on cell growth but also competes with GDP-L-fucose for carbon sources. To enhance the synthesis of the precursor GDP-L-fucose, we blocked the generation of byproducts lactic acid, formic acid, and acetic acid through gene knockout. We further achieved efficient FL production in engineered E. coli by regulating four enzymes that may participate in the competitive pathway. The engineered strains constructed in this example are shown in Table 2. The fucoidosyl lactose synthesis capacity, cell biomass, and byproduct accumulation of the engineered strains were tested. The results showed that after deleting poxB and introducing the HpfutC / HpM32 gene, a FL-producing strain was initially constructed. The 2'-FL and 3-FL titers of strains BP8-0 and BP9-0 were 1.39 and 0.98 g / L, respectively, with 2.97 and 3.32 g / L of lactose remaining in the fermentation supernatant, respectively. After eliminating all glycolysis byproducts, no accumulation of acetic acid or lactic acid was detected in the recombinant strains during fermentation. Strains BP8-4 and BP9-4 exhibited higher biomass and lactose production reaching 6.85 and 6.64 g / L, respectively. Furthermore, the extracellular export of 2'-FL and 3-FL increased from 1.89 and 1.24 g / L (initial strains BP8-0 and BP-9-0) to 3.34 and 2.78 g / L, respectively. Figure 2The above indicates that weakening the glycolysis pathway allows more carbon to flow to the FL and lactose synthesis pathways, which is beneficial for the efficient biosynthesis of FL.

[0065] Table 2. Detailed information on engineered bacteria that integrate the fucose-lactose metabolic pathway into their genome.

[0066]

[0067] Example 2: Upregulating key genomic enzymes to enhance the efficient production of fucoidosyllactose

[0068] Escherichia coli contains an endogenous synthetic pathway for GDP-L-fucose. GDP-L-fucose can be synthesized from Fru-6-P, the most basic sugar intermediate in the glycolysis pathway, through five consecutive biocatalytic processes: manA, manB, manC, gmd, and wcaG. Figure 1 GDP-L-fucose, as a precursor in the coralamic acid biosynthesis pathway, determines the total yield of fucosyl lactose (FL) biosynthesis due to its intracellular availability. However, the yield of GDP-L-fucose produced by wild-type *E. coli* is negligible, hindering further FL synthesis. To accelerate the carbon flux of the GDP-L-fucose synthesis module, a promoter engineering strategy was employed to improve the precursor supply of GDP-L-fucose and achieve efficient FL synthesis in antibiotic-free strains.

[0069] Promoter engineering is an effective method for moderately regulating protein expression. In this embodiment, the strong promoter T7 is used to replace the promoter of the "key enzyme" itself on the *E. coli* genome, upregulating the expression levels of the manA, manB, manC, and gmd genes in the GDP-L-fucose biosynthesis pathway, in order to achieve efficient production of fucoidan. The gene editing primers involved in this embodiment are shown in Table 3.

[0070] Table 3 Promoter Replacement Primers

[0071]

[0072]

[0073] Based on engineered strains BP8-4 and BP9-4, the original promoters of manA, manB, manC, and gmd in the GDP-L-fucose pathway on the genome were replaced with the strong promoter T7, resulting in 2'-FL producing strains (BP10-0, BP10-1, BP10-2, and BP10-3) and 3-FL producing strains (BP11-0, BP11-1, BP11-2, and BP11-3), respectively. The strains constructed in this example are shown in Table 4. Fermentation results showed that upregulation of the "key enzyme" in the GDP-L-fucose pathway on the genome increased the yield of all 2'-FL and 3-FL strains. Under the condition of integrating sugar efflux proteins, the extracellular export yields of 2'-fucosyllactose and 3-fucosyllactose were 4.36 and 3.23 g / L, respectively, which were 30.5% and 16.1% higher than those of strains BP8-4 and BP9-4 in Example 1. Figure 3 ).

[0074] Table 4. Detailed information on engineered bacteria with promoter substitution

[0075]

[0076] Example 3: Detection of intracellular GDP-L-fucose content and transcription level of related genes in engineered strains

[0077] To determine the effectiveness of the promoter engineering strategy, the FL-producing strain from Example 2 was cultured in shake flasks, and the cells after 24 hours of fermentation were used as samples to detect the intracellular GDP-L-fucose concentration. Simultaneously, real-time quantitative PCR analysis was performed on the transcriptional levels of the "key enzyme" genes in the GDP-L-fucose module. The results are shown in Table 5. The intracellular GDP-L-fucose concentration of BP10-3 was 547 mg / g DCW, an increase of 98% compared to the control BP8-4. The transcriptional levels of manA, manB, manC, and gmd genes were 2.1, 1.5, 2.5, and 1.8 times higher than those of BP8-4, respectively. Figure 4 Similarly, strain BP11-3 also showed significant enhancements in GDP-L-fucose titer and gene transcription levels. The promoter engineering strategy can increase lactose consumption, forcing carbon metabolism to the GDP-L-fucose biosynthesis pathway, improving carbon economy, and is of great significance for FL synthesis.

[0078] Table 5. Intracellular GDP-L-fucose concentration of different engineered bacteria

[0079]

[0080] Example 4: Production of fucoidan-based lactose in a 3L fermenter using a fed-batch process

[0081] To prepare high yields of 2'-FL and 3-FL, antibiotic-free strains BP10-3 and BP11-3 were used for high-density fed-batch fermentation in 3L fermenters.

[0082] Fermentation conditions: 50 mL of overnight cultured seed culture was inoculated into 1 L of fermentation medium. The culture temperature was 37℃. The initial concentrations of glycerol and glucose were 30 g / L and 10 g / L, respectively. NH4OH was used to maintain a constant pH of 6.80 throughout the fermentation process. To maintain cell growth and fucoidan synthesis, 800 g / L of glycerol (containing 20 g / L MgSO4·7H2O) was added after the initial glycerol was consumed to replenish the carbon source. pH feedback was used to maintain a low glycerol concentration in the fermentation system (glycerol is used for cell growth and metabolism, with a concentration of approximately 0 g / L) until the end of fermentation. After the initial glucose was consumed, 300 g / L of glucose was manually added to maintain a final concentration of approximately 10 ± 0.5 g / L in the fermentation system. If glucose concentrations decreased during fermentation, glucose was added again until the end of fermentation. The system was cascaded controlled during fermentation, and the dissolved oxygen in the tank was maintained at 30 ± 5% by adjusting the rotation speed, aeration rate, and oxygen supply.

[0083] Sampling was performed regularly throughout the fermentation process, and cell OD was measured. 600 1 mL of fermentation broth was boiled for 15 min to completely lyse the cells, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm membrane. During fermentation, the production of lactose, 2'-FL, and 3-FL, as well as the consumption of glucose and glycerol, were detected using HPLC. Figure 5 and Figure 6 The results showed that the lactose content of the product remained at 8-15 g / L during fermentation. After fermentation (a total of 100 hours), the concentration of extracellular 2'-FL reached 40.44 g / L, and the concentration of extracellular 3-FL reached 30.42 g / L.

[0084] 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 recombinant Escherichia coli, characterized in that, Knocking out the ubiquinone-dependent pyruvate dehydrogenase gene in a host cell poxB and integrating at the ubiquinone-dependent pyruvate dehydrogenase gene poxB site α -1,2-fucosyltransferase gene HpfutC and / or α -1,3-fucosyltransferase gene HpM32 Knocking out the phosphoacetyltransferase and acetate kinase gene cluster pta - ackA and integrating at pta - ackA the phosphomannomutase and mannose-1 -phosphate guanylyltransferase gene cluster manC-manB ​ Knocking out the formate cleavage enzyme gene pflB and integrating a GDP-mannose-6-dehydrogenase and GDP-fucose synthetase gene cluster at pflB gmd wcaG Knocking out the D-lactate dehydrogenase gene ldhA and integrating a mannose-6-phosphate isomerase gene at ldhA manA ;​​​ Knockout of lactose operon repressor protein lacI The host cell was a *E. coli* cell that had been knocked out. β -Galactosidase gene lacZ、 UDP-glucose lipid carrier transferase gene wcaJ、 GDP-mannose-mannosyl hydrolase gene nudD、 6-Phosphofructokinase-1 gene pfkA、 protease gene lon The glucose-specific transporter EⅡABC was knocked out. Glc Component-encoding genes crr and ptsG and in crr and ptsG The sites were integrated respectively SetA and Glf ; The UDP-glucose-6-dehydrogenase gene was knocked out ugd and in ugd The gene contains an integrated UDP-glucose-4-epimerase gene. GalE; Knockout of glucokinase gene Glk and in the glucokinase gene G lk Integration at the site originates from Neisseria meningitidis β -1,4-galactosyltransferase NmlgtB The α -1,2-fucosyltransferase gene HpfutC , α -1,3-fucosyltransferase gene HpM32 Gene clusters manC - manB Gene clusters gmd - wcaG and mannose-6-phosphate isomerase gene manA All expressions are started using the T7 promoter; Replace the E. coli genome with the strong promoter T7. manC , manB , gmd - wcaG and manA The self-promoter of the gene encoding, the α -1,2-fucosyltransferase gene HpfutC Derived from Helicobacter pylori ATCC 26695, α -1,3-fucosyltransferase gene HpM32 Derived from Helicobacter pylori NCTC11639, the α -1,2-fucosyltransferase gene HpfutC, α -1,3-fucosyltransferase gene HpM32 The above β -1,4-galactosyltransferase NmlgtB The nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

2. The use of the recombinant Escherichia coli according to claim 1 in the production of 2'-fucosylated lactose and / or 3-fucosylated lactose.

3. Use according to claim 2, characterized in that, Using the recombinant Escherichia coli as the fermentation strain, 2'-fucosylated lactose and / or 3-fucosylated lactose are produced in a fermentation system with glycerol and glucose as carbon sources.

4. Use according to claim 3, characterized in that, The recombinant Escherichia coli was inoculated into a shake-flask fermentation medium. At the beginning of fermentation, glucose with a final concentration of 8 g / L was added, and the mixture was cultured at 30-40℃ and 150-250 rpm for 72 h.

5. Use according to claim 3, characterized in that, Fermentation medium is added to a fermenter, and the recombinant Escherichia coli is inoculated for fermentation. The fermentation system contains 20-30 g / L glycerol, 5-10 g / L glucose, 10-15 g / L potassium dihydrogen phosphate, 1-2 g / L citric acid, 3-5 g / L diammonium hydrogen phosphate, 1-2 g / L magnesium sulfate heptahydrate, 8-10 g / L yeast extract, and 8-10 mL / L trace metal solution.

6. The application according to claim 4, characterized in that, The recombinant Escherichia coli was cultured at 20-40°C, maintaining dissolved oxygen at 30±5% and pH at 6.5-7.0 in the fermentation system. After the initial glucose was consumed, glucose was added to maintain the glucose concentration in the fermentation system at 10±0.5 g / L.

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