CN116064345A

CN116064345A - Non-antibiotic genetic engineering bacteria for efficiently producing fucosyllactose and application thereof

Info

Publication number: CN116064345A
Application number: CN202210980879.5A
Authority: CN
Inventors: 张涛; 李梦丽; 骆叶姣; 江波
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-08-16
Filing date: 2022-08-16
Publication date: 2023-05-05

Abstract

The invention relates to an antibiotic-free genetically engineered bacterium for efficiently producing fucosyllactose and application thereof, belonging to the fields of metabolic engineering and food fermentation. The invention adopts CRISPR/Cas9 gene editing technology to reconstruct chassis microorganism, and realizes construction of gene engineering bacteria without resistance and efficient synthesis of fucosyllactose by reconstructing 2' -FL and 3-FL synthesis paths in lactose synthesis strains, regulating central carbon metabolism, weakening byproduct paths, up-regulating key enzymes of the pathway of de-head synthesis, removing repressor inhibition, enhancing extracellular output of products and other strategies. The recombinant escherichia coli obtained by the invention can be used for efficiently synthesizing the fucosyl lactose by utilizing the glycerol and the glucose, no antibiotics are added in the fermentation process, the product is safe and harmless, the production cost is low, the production level of the product is high, the social and economic benefits are high, and the market development prospect is wide.

Description

Non-antibiotic genetic engineering bacteria for efficiently producing fucosyllactose and application thereof

Technical Field

The invention relates to an antibiotic-free genetically engineered bacterium for efficiently producing fucosyllactose and application thereof, belonging to the fields of metabolic engineering and food fermentation.

Background

The production of high value-added products by using inexpensive biomass through green biological processes is an important way to achieve carbon neutralization and environmentally friendly economy. 2 '-fucosyllactose (2' -FL) and 3-fucosyllactose (3-FL) are neutral Fucosyllactoses (FL) secreted most abundantly in breast milk, accounting for about 35% of the total amount of breast milk oligosaccharides (HMOs). Beneficial properties of FL (e.g., maintaining intestinal ecological balance, resisting adhesion of pathogenic bacteria, immunomodulation, and promoting development and repair of the nervous system) have attracted great attention for their potential use in nutraceutical and pharmaceutical applications.

The development of green, efficient and safe chassis microorganisms is a key for solving the mass production and application of HMOs. With the development of metabolic engineering and synthetic biology, many models of microorganisms have been explored as potential microbial cell factories for HMOs production. Microorganisms of the species Escherichia coli, saccharomyces cerevisiae, yarrowia lipolytica, and Bacillus subtilis have been successfully used for de novo biosynthesis of 2' -FL. Currently, 3-FL producing strains are commonly found in E.coli, with fewer reports and lower yields. It has been reported that under anaerobic and microaerobic conditions, glycerol or glucose based catabolism may lead to the production of different products (acetic acid, lactic acid, formic acid, etc.) at high concentrations. The presence of byproducts slows or stops the growth of the strain, resulting in a severe decrease or complete cessation of the production rate of the target, and therefore, elimination of these byproduct pathways is particularly important in the production of FL. In addition, the production of fucosyl lactose requires the supply of exogenous lactose, the lactose raw material cost is high, and the development of a novel lactose synthesis technology in microorganisms can solve the problem of high lactose cost in the FL efficient biosynthesis process.

From the selection of host bacteria, the FL production strain with industrial competitiveness can be constructed by utilizing strategies such as metabolic pathway reconstruction, weakening of competing pathways, cofactor and energy regeneration, metabolic flux optimization and the like. However, most FL strains were constructed based on overexpression of plasmid genes. The development of plasmid cell factories involves the risk of genetic instability and antibiotic addition during fermentation. Thus, the preparation of FL using E.coli without antibiotics and inducers has become apparent to be environmentally safe and commercially valuableProduction strategy. Before this time,

the de novo and salvage synthetic pathways for 2'-FL were introduced on the chromosome of E.coli JM109 strain band, yielding 20.28.+ -. 0.83g/L of 2' -FL in a fed-batch fermentation without antibiotics. Parschat et al developed a 2'-FL producing strain with sucrose alone as a substrate, which produced lactose intracellularly without the addition of antibiotics and was used for 2' -FL production. In addition, 2' -FL producing strains without antibiotics and inducers were not reported.

The invention aims to realize the construction of the non-antibiotic genetic engineering bacteria and the efficient synthesis of fucosyllactose by utilizing the synthetic biological means and by utilizing strategies such as reconstructing 2' -FL and 3-FL synthesis paths in lactose synthesis strains, regulating central carbon metabolism, weakening by-product paths, up-regulating key enzymes of the de-leader synthesis paths, removing repressor inhibition, enhancing extracellular output of products and the like. The research method provides strain safety and substrate flexibility for industrial use of FL, enriches and develops technical research of microbial metabolism regulation, provides new methods and cases for reconstructing a microbial metabolism network and improving carbon atom economy, provides new ideas for rational design and construction of a new generation of microbial cell factories, and has important values in theoretical and practical application.

Disclosure of Invention

[ technical problem ]

The prior art has low efficiency of synthesizing the fucosyl lactose, the plasmid strain expression has risks of genetic instability and antibiotic addition, the acceptor lactose and the inducer are expensive, the strain for safely and efficiently producing the fucosyl lactose cannot be provided, and the preparation method of the fucosyl lactose with low cost and environmental protection cannot be provided.

Technical scheme

The invention provides a genetic engineering bacterium for efficiently producing lactose and fucosyl lactose and a construction method thereof, which aim to solve the problems that lactose and inducer are expensive, plasmid strains have genetic instability, and the risk of adding antibiotics and the like in the synthesis process of fucosyl lactose.

The invention discloses a genetic engineering bacterium BP6 capable of self-producing lactose by utilizing low-cost carbon sources (glycerol and glucose), wherein the BP6 is prepared by taking a strain BZWNANAAL as an initial strain and knocking out glucose specific transporter EIIABC ^Glc The component codes the genes crr and ptsG, and integrates SetA and Glf at crr and ptsG sites respectively; knocking out UDP-glucose-6-dehydrogenase gene ugd, and integrating UDP-glucose-4-epimerase gene GalE at the ugd gene; the glucokinase gene Glk was knocked out and the beta-1, 4-galactosyltransferase NmlgtB from Neisseria meningitidis was integrated at the Glk locus of the glucokinase gene, respectively.

The strain BZWNANAAL is BL21 (DE 3) DeltalacZ DeltawcaJ DeltanudDeltapfkA Deltalon, and the construction method is disclosed in the patent document with the publication number CN 114480240A.

The genetic engineering bacteria provided by the invention knock out the ubiquinone dependent pyruvate dehydrogenase gene poxB on the basis of the strain BP6, and integrate alpha-1, 2-fucosyltransferase genes HpfutC and alpha-1, 3-fucosyltransferase genes HpM at the poxB locus of the ubiquinone dependent pyruvate dehydrogenase gene; knocking out phosphoacetyl transferase and acetate kinase gene cluster pta-ackA, and integrating phosphomannose mutase and mannose-1-phosphoguanyl transferase gene cluster manC-manB at pta-ackA; knocking out formate lyase gene pflB, and integrating GDP-mannose-6-dehydrogenase and GDP-fucose synthase gene cluster gmd-wcaG at pflB site; knocking out the D-lactate dehydrogenase gene ldhA, and integrating a mannose-6-phosphate isomerase gene manA at the ldhA site; the lactose operon repressor lacI was knocked out.

In one embodiment, the alpha-1, 2-fucosyltransferase gene HpfutC is derived from helicobacter pylori ATCC 26695, and the alpha-1, 3-fucosyltransferase gene HpM is derived from helicobacter pylori NCTC 11639, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

In one embodiment, the beta-galactosidase Gene lacZ has a Gene ID of 945006, the UDP-glucose lipid carrier transferase Gene wcaJ has a Gene ID of 946583, the GDP-mannosyl hydrolase Gene nudD has a Gene ID of 946559,6-phosphofructokinase-1 Gene pfkA of 948412, the protease Gene lon has a Gene ID of 945085, the glucose-specific transporter protease genes crr and ptsG have a Gene ID of 946880 and 945651, the UDP-glucose-6-dehydrogenase Gene ugd has a Gene ID of 946571, and the UDP-glucose-4-epimerase Gene GalE has a Gene ID of 945354; gene ID of the glucokinase Gene Glk was 946858; the nucleotide sequence of beta-1, 4-galactosyltransferase NmlgtB from neisseria meningitidis is shown in SEQ ID NO. 3.

In one embodiment, the α -1, 2-fucosyltransferase gene HpfutC, the α -1, 3-fucosyltransferase gene HpM, the gene cluster manC-manB, the gene cluster gmd-wcaG and the mannose-6-phosphate isomerase gene manA all utilize the promoter T7 to initiate expression.

In one embodiment, the self-promoter of the manC, manB, gmd-wcaG and manA encoding genes in the E.coli genome is replaced with a strong promoter T7.

In one embodiment, the ubiquinone-dependent pyruvate dehydrogenase Gene poxB has a Gene ID of 946132, the phosphoacetyl transferase 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 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 guanyl transferase 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.

It is a second object of the present invention to provide the use of said recombinant E.coli for the production of 2' -fucosyllactose and/or 3-fucosyllactose.

In one embodiment, the recombinant escherichia coli is used as a fermentation strain, and 2' -fucosyllactose and/or 3-fucosyllactose are produced in a fermentation system using glycerol and glucose as carbon sources.

In one embodiment, 2' -fucosyllactose and/or 3-fucosyllactose is produced by fermentation in shake flasks or fermentors.

In one embodiment, the recombinant escherichia coli is inoculated in a shake flask fermentation medium, glucose with the final concentration of 8g/L is added at the beginning of fermentation, and the recombinant escherichia coli is cultured for 72 hours at the temperature of 30-40 ℃ and the rpm of 150-250 rpm.

In one embodiment, fermentation medium is added to the fermentor and the recombinant E.coli described for BP10-3 and BP11-3 are inoculated for fermentation.

In one embodiment, the glycerol is present in the fermenter at a concentration of 10 to 40g/L.

In one embodiment, the fermentation system contains 20-30 g/L of glycerin, 5-10 g/L of glucose, 10-15 g/L of monopotassium phosphate, 1-2 g/L of citric acid, 3-5 g/L of diammonium phosphate, 1-2 g/L of magnesium sulfate heptahydrate, 8-10 g/L of yeast extract and 8-10 mL/L of trace metal solution.

In one embodiment, the trace metal solution contains 8-10 g/L of ferric 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.

In one embodiment, the genetically engineered bacterium is cultured at 20-40 ℃ and maintains dissolved oxygen in a fermentation system at 30+/-5% and pH at 6.5-7.0.

In one embodiment, glycerol is fed after initial glycerol consumption in the reaction system, so that glycerol concentration can maintain bacterial growth and metabolism.

In one embodiment, glucose is added after initial glucose consumption to maintain a concentration of 10.+ -. 0.5g/L.

In one embodiment, the fermentation time is not less than 70 hours.

Preferably, the fermentation time is 100 hours.

The invention has the beneficial effects that:

the invention introduces the biosynthesis path of fucosyl lactose into lactose producing bacteria, and realizes the construction of aseptic strains and the efficient synthesis of fucosyl lactose without exogenous lactose addition by weakening the byproduct path, regulating and controlling the central carbon metabolism, up-regulating key enzymes of the de-head synthesis path, releasing the repression and inhibition of the repressor protein, enhancing the extracellular output of the product and other strategies. Under the shake flask fermentation condition without antibiotics and inducers, the capacity of the genetically engineered bacteria constructed by the application for producing 2' -FL and 3-FL reaches 4.36 g/L and 3.23g/L respectively; under the culture condition of a 3L fermentation tank, the yields of 2' -FL and 3-F respectively reach 40.44g/L and 30.42g/L, thereby laying a foundation for the industrial production of fucosyl lactose.

Drawings

FIG. 1 is a schematic diagram of the metabolic process of producing fucosyllactose using glucose and glycerol as substrates.

FIG. 2 is a graph comparing yields of engineering bacteria that attenuate the byproduct pathway and introduce the fucosyllactose pathway.

FIG. 3 is a graph showing the effect of up-regulating the GDP-L-fucose pathway on fucosyllactose production in engineering bacteria.

FIG. 4 shows the relative transcript levels of GDP-L-fucose module genes in strains BP10-3 and BP 10-4.

FIG. 5 is a 3L fermenter fed-batch fermentation of strain BP 10-3.

FIG. 6 is a 3L fermenter fed-batch fermentation of strain BP 11-3.

Detailed Description

The present invention will be further described with reference to examples and drawings, wherein plasmids, PCR reagents, restriction enzymes, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercially available, and the specific operations are performed according to the kit instructions.

Embodiments of the invention are not limited thereto, and other unspecified experimental operations and process parameters are conducted in accordance with conventional techniques.

Vectors pCas9 and pTargetF are purchased from Addgene.

The sequencing of DNA products and plasmids was completed by Tianzhan biotechnology (tin-free) Limited.

Preparation of E.coli competence: kit for Shanghai engineering and biological engineering company.

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

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

Fermentation medium: 30g/L of glycerin, 13.5g/L of monopotassium phosphate, 1.7g/L of citric acid, 4.0g/L of diammonium phosphate, 1.4g/L of magnesium sulfate heptahydrate, 10g/L of yeast extract, 10mL/L of trace metal solution (10 g/L of ferric citrate, 2.25g/L of magnesium sulfate heptahydrate, 1.0g/L of cupric sulfate pentahydrate, 0.35g/L of manganese sulfate monohydrate, 0.23g/L of borax, 0.11g/L of ammonium molybdate, 2.0g/L of calcium chloride dihydrate) and pH 6.8.

Method for measuring 2' -FL,3-FL and GDP-L-fucose:

determination using HPLC: 1mL of the fermentation broth was boiled at 100℃for 10min, centrifuged at 12000r/min for 5min, and the supernatant was subjected to 0.22 μm membrane filtration, and the amount of fucosyllactose produced and the consumption of glucose and glycerol were measured by HPLC. HPLC detection conditions: a differential refractive detector; the chromatographic column is Rezex ROA-organic acid (Phenomenex, USA) with the column temperature of 50 ℃; h with mobile phase of 0.005mol/L ₂ SO ₄ The flow rate of the aqueous solution is 0.6mL/min; the sample loading was 10. Mu.L. HPLC detection conditions for GDP-L-fucose: an ultraviolet detector; a detection wavelength of 254nm; the chromatographic column is Inertsil ODS-SP (GL Sciences, kyoto, japan); mobile phase a was a 20mM aqueous triethylamine-glacial acetic acid solution (pH 6.0) and mobile phase B was an acetonitrile solution; gradient elution; the flow rate is 0.6mL/min; the sample loading was 10. Mu.L.

Shake flask fermentation conditions for the strains in the examples below: inoculating engineering bacteria single colony to LB liquid culture medium, shaking flask culture at 37deg.C and 200rpm for 12 hr to obtain seed liquid; inoculating the seed solution into 50mL fermentation medium with 3% (v/v) inoculum size, shaking flask culture at 37deg.C and 200rpm to OD ₆₀₀ Glucose was added to a final concentration of 8g/L at 25℃and incubated at 200rpm for 72h.

Example 1: derepression protein and glycolysis by-product inhibition are released, and an antibiotic-free fucosyllactose production strain is constructed

The synthesis of fucosyllactose requires the supply of exogenous lactose, and a genetic engineering bacterium capable of self-producing lactose by using low-cost carbon sources (glycerol and glucose) has been constructed before. On this basis, in order to prevent metabolic overflow of the glycolytic pathway, the byproduct pathway is weakened and the fucosyllactose synthesis pathway is introduced. Taking escherichia coli BP6 as an initial strain, knocking out poxB by using a CRISPR-Cas9 gene editing system, and integrating double-gene copy alpha-1, 2/3-fucosyltransferase gene HpfutC/HpM at the locus; knocking out pta-ackA and integrating manC-manB at this site; knocking out pflB and integrating gmd-wcaG at the site; knockout ldhA and integration of manA at this site; finally, the repressor gene lacI was knocked out. The metabolic pathways of fucosyllactose using glycerol and glucose as substrates in lactose producing strains are shown in fig. 1, and the specific steps of gene knockout and integration are as follows (the primer sequences involved are shown in table 1):

(1) Taking the α -1, 2-fucosyltransferase gene HpfutC gene with poxB knocked out and chromosome integrated in double tandem as an example, searching for a specific target gRNA (20 bp) of the poxB gene by http:// www.regenome.net/cas-offinder, performing PCR amplification by using the poxB-gRNA-F/gRNA-R upstream and downstream primers and pTargetF plasmid (Addgene: # 62226) as a template, and performing restriction enzyme Dpn I digestion on the amplified product to remove redundant circular plasmid pTargetF. The amplified product was then transformed into E.coli DH 5. Alpha. Competent cells, miniplasmids, and the successfully constructed knockdown plasmid was identified by sequencing and designated pTargetF-poxB.

(2) The genome of the escherichia coli BP6 strain is used as a template, an upstream homology arm primer poxB-US-F/poxB-US-R is utilized, a middle homology arm primer 2HpfutC-MS-F/2HpfutC-MS-R and a downstream homology arm primer poxB-DS-F/poxB-DS-R are respectively amplified to obtain three sequence fragments, and after the products are purified and recovered, the three fragments are connected by adopting a SOE-PCR method and using the primer poxB-US-F/poxB-DS-R to obtain the gene homology repair template.

(3) pCas9 plasmid (Addgene: # 62225) and E.coli BP6 electrotransduce competent cells, thaw competent cells after 5min on ice, add 10. Mu.L plasmid into 100. Mu.L competent cells, mix gently. Transferring the plasmid and the electrotransfer competent cells into a precooled electrotransfer cup, carrying out electric shock for 5ms at 2.5kV, rapidly adding precooled liquid LB after electric shock, slightly blowing and uniformly mixing, and transferring a culture medium mixed with the plasmid and the competent cells into a new centrifuge tube for expansion culture for 1.5h. Centrifuging at 6000r/min for 2min, discarding supernatant, coating thallus on LB plate containing kana resistance, and culturing at 30deg.C overnight.

(4) E.coli BP6/pCas9 single colony was picked up in LB medium, cultured at 30℃for 1.0h, and L-arabinose was added at a final concentration of 30mM to induce the expression of lambda-red system. When OD is ₆₀₀ When reaching 0.6-0.8, the bacillus coli BP6/pCas9 competence is prepared.

(5) And (3) electrotransferring 500ng of targeting plasmid pTargetF with poxB specific target gRNA (20 BP) constructed in the step (1) and 1000ng of homologous repair template constructed in the step (2) to competent cells of escherichia coli BP6/pCas9 prepared in the step (4), coating the competent cells on LB plates (kanamycin and spectinomycin), culturing at 30 ℃ for 16-24h, performing colony PCR verification on single colonies growing on the plates, screening positive transformants and performing gene sequencing.

(6) The elimination of pTargetF-poxB and pCas9 plasmids was performed on single colonies which were confirmed to be correct, and the single colonies were inoculated into LB liquid medium (kana resistance), cultured at 30℃for 200r/min to the logarithmic growth phase, and cultured overnight with the addition of IPTG at a final concentration of 0.5mmol/L to induce inactivation of pTargetF-poxB plasmids. The bacterial liquid is streaked on LB plate containing Kan, and cultured for 12h at 30 ℃ and 200 r/min. Single colonies were plated on dual resistance plates of kana and spectinomycin, indicating successful elimination of the pTargetF-poxB plasmid if colonies were aseptically grown.

(7) The pCas9 plasmid is a temperature sensitive plasmid, and a single colony of the successfully eliminated pTargetF-poxB plasmid is transferred into LB non-resistant liquid medium for subculturing at 42 ℃ to eliminate the pCas9 plasmid. And (3) after streaking a non-resistant LB plate by bacterial liquid, culturing at a constant temperature of 37 ℃, and if a single colony does not grow in a LB culture medium containing the kana resistance, indicating that the pCas9 plasmid is successfully eliminated, and preserving the constructed gene deletion strain without pTargetF-poxB plasmid and pCas9 plasmid at the temperature of-80 ℃ for later use.

(8) Knock-out of genes poxB, pta-ackA, pflB, ldhA and lacI and corresponding HpM, manC-manB, gmd-wcaG, manA integration procedures were referred to above, and the construction procedure of the gene editing strain referred to in other examples was referred to in example 1.

TABLE 1 Gene knockout and integration primers

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In recombinant engineering bacteria, accumulation of byproducts of the glycolytic pathway not only has deleterious effects on cell growth, but also competes with the synthesis of GDP-L-fucose for carbon source material. To enhance synthesis of the precursor GDP-L-fucose, we blocked the production of by-products lactate, formate and acetate by gene knockout. Efficient production of FL in engineered E.coli is further achieved by modulating four enzymes that may be involved in competing pathways. The engineering strain constructed in this example was tested for the ability to synthesize fucosyllactose, biomass of cells and accumulation of byproducts as shown in Table 2. As a result, it was revealed that after deletion of poxB and introduction of HpfutC/HpM32 gene, FL-producing strains were initially constructed, and 2' -FL and 3-FL titers of BP8-0 and BP9-0 strains were determined to be 1.39 and 0.98g/L by shake flask culture, with 2.97 and 3.32g/L lactose remaining in the fermentation supernatant. After all byproducts of the glycolytic pathway are released, the recombinant strain does not detect accumulation of acetic acid, lactic acid during fermentation. Strains BP8-4 and BP9-4 showed higher biomass, and the yield of synthetic lactose reached 6.85 and 6.64g/L. In addition, the extracellular output contents of 2' -FL and 3-FL were increased from 1.89 and 1.24g/L (initial strains BP8-0 and BP-9-0) to 3.34 and 2.78g/L (FIG. 2). The above shows that weakening the glycolytic pathway allows more carbon to flow to the FL and lactose synthesis pathway, favoring efficient biosynthesis of FL.

TABLE 2 details of engineering bacteria whose genome integrates fucosyllactose Metabolic pathway

Example 2: efficient production of fucosyllactose is enhanced by up-regulating genome "key enzyme

Coli contains an endogenous synthetic pathway for the production of GDP-L-fucose. GDP-L-fuse can be synthesized from the most basic sugar intermediate Fru-6-P in the glycolytic pathway, through manA, manB, manC, gmd, and wcaG, five consecutive biocatalytic processes (FIG. 1). The availability of GDP-L-fucose, as a precursor of the cola biosynthetic pathway, in cells determines the overall yield of fucosyl lactose biosynthesis. However, the production of GDP-L-fucose by wild E.coli is very little, which is disadvantageous for further synthesis of FL. In order to accelerate the carbon flux of the GDP-L-fucose synthesis module, promoter engineering strategies were employed to improve the precursor supply of GDP-L-fucose and achieve efficient synthesis of FL in the antibiotic-free strain.

The promoter engineering strategy is an effective method for moderately regulating the expression of proteins, and the embodiment replaces the promoter of the key enzyme on the genome of escherichia coli with a strong promoter T7 to up-regulate the expression levels of manA, manB, manC and gmd genes in the GDP-L-fucose biosynthesis pathway so as to realize the efficient production of fucosyl lactose. The gene editing primers involved in this example are shown in Table 3.

TABLE 3 promoter replacement primers

The original promoters of manA, manB, manC and gmd in the GDP-L-fucose pathway on the genome were replaced with strong promoters T7 on the basis of engineering bacteria BP8-4 and BP9-4 to obtain 2' -FL producing strains (BP 10-0, BP10-1, BP10-2 and BP 10-3) and 3-FL producing strains (BP 11-0, BP11-1, BP11-2 and BP 11-3), respectively. The strains constructed in this example are shown in Table 4. Fermentation results showed that up-regulation of the "key enzyme" of the GDP-L-fucose pathway on the genome, increased production of all 2'-FL and 3-FL strains, with extracellular output of 4.36 and 3.23g/L for 2' -fucosyllactose and 3-fucosyllactose, by 30.5% and 16.1% compared to BP8-4 and BP9-4 strains in example 1, with integration of the sugar efflux proteins (FIG. 3).

TABLE 4 detailed information on engineering bacteria with replaced promoters

Example 3: detecting intracellular GDP-L-fucose content and transcription level of related genes in engineering strain

To determine whether the promoter engineering strategy was effective, the FL producing strain in example 2 was subjected to shake flask fermentation culture, and intracellular GDP-L-fucose concentration was detected by taking 24h of fermented cells as a sample. Meanwhile, in the GDP-L-fucose module, the transcription level of the "key enzyme" gene is subjected to real-time fluorescence quantitative analysis. As shown in Table 5, the intracellular GDP-L-fucose concentration of BP10-3 was 547mg/g DCW, which was 98% increased over that of the control BP 8-4. The transcript levels of manA, manB, manC and gmd genes were 2.1, 1.5, 2.5 and 1.8-fold higher than BP8-4, respectively (FIG. 4). Likewise, the strain BP11-3 also showed a significant enhancement in GDP-L-fucose titer and gene transcription level. The promoter engineering strategy can improve lactose consumption, force carbon metabolism to flow to GDP-L-fucose biosynthesis pathway, improve economy of carbon atoms, and has important significance for FL synthesis.

TABLE 5 intracellular GDP-L-fucose concentrations of different engineering bacteria

Example 4:3L fermenter batch fed-batch production of fucosyllactose

To produce high yields of 2' -FL and 3-FL, high density fed-batch fermentations were performed in 3L fermentors using sterile strains BP10-3 and BP11-3, respectively.

Fermentation conditions: 50mL of overnight cultured seed solution is inoculated into 1L of fermentation medium, the culture temperature is 37 ℃, the initial concentration of glycerol and glucose is 30g/L and 10g/L, and NH is used in the whole fermentation process ₄ The OH controls the pH of the tank to be constant at 6.80. To maintain cell growth and fucosyllactose synthesis, 800g/L glycerol (20 g/L MgSO) was fed after initial glycerol consumption ₄ ·7H ₂ O) to supplement the carbon source, the concentration of the glycerol in the fermentation system is maintained at a lower concentration level (the glycerol is used for thallus growth and metabolism, the concentration is about 0 g/L) through pH feedback adjustment (the flow rate is set to be 20 mL/h) until the fermentation is finished, 300g/L of glucose is manually added after the initial glucose consumption is finished, the final concentration of the glucose in the fermentation system is maintained at about 10+/-0.5 g/L, and the glucose is continuously added until the fermentation is finished when the glucose consumption is reduced to the lower concentration in the fermentation process. In the fermentation process, the system is controlled in cascade, and the dissolved oxygen in the tank is 30+/-5% by adjusting the rotating speed, the ventilation and the oxygen.

Sampling and measuring the OD of the thallus in the whole fermentation process ₆₀₀ 1mL of the fermentation broth was boiled for 15min to completely break the cells, centrifuged for 10min at 12000r/min, and the supernatant was subjected to 0.22 μm membrane filtration, and the production amounts of lactose, 2' -FL and 3-FL and the consumption amounts of glucose and glycerol were measured by HPLC during fermentation (FIGS. 5 and 6). The result shows that the lactose product is maintained at 8-15g/L in the fermentation process, and after the fermentation is finished (100 hours of total fermentation), the concentration of extracellular 2' -FL can reach 40.44g/L, and the concentration of extracellular 3-FL can reach 30.42g/L.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A recombinant escherichia coli characterized in that a ubiquinone-dependent pyruvate dehydrogenase gene poxB is knocked out in a host cell, and an α -1, 2-fucosyltransferase gene HpfutC and/or an α -1, 3-fucosyltransferase gene HpM are integrated at the poxB site of the ubiquinone-dependent pyruvate dehydrogenase gene; knocking out phosphoacetyl transferase and acetate kinase gene cluster pta-ackA, and integrating phosphomannose mutase and mannose-1-phosphoguanyl transferase gene cluster manC-manB at pta-ackA; knocking out formate lyase gene pflB, and integrating GDP-mannose-6-dehydrogenase and GDP-fucose synthase gene cluster gmd-wcaG at pflB site; knocking out the D-lactate dehydrogenase gene ldhA, and integrating a mannose-6-phosphate isomerase gene manA at the ldhA site; the lactose operon repressor lacI was knocked out.

2. The recombinant E.coli according to claim 1, wherein the host cell is a gene for knocking out beta-galactosidase gene lacZ, UDP-glucose lipid carrier transferase gene wcaJ, GDP-mannosyl hydrolase gene nudD, 6-phosphofructokinase-1 gene pfkA, protease gene lon, glucose specific transporter enzyme EIIB ABC in E.coli ^Glc The component codes the genes crr and ptsG, and integrates SetA and Glf at crr and ptsG sites respectively; knocking out UDP-glucose-6-dehydrogenase gene ugd, and integrating UDP-glucose-4-epimerase gene GalE at the ugd gene; the glucokinase gene Glk was knocked out and the beta-1, 4-galactosyltransferase NmlgtB from Neisseria meningitidis was integrated at the position Glk of the glucokinase gene.

3. Recombinant E.coli according to claim 1 or 2, characterized in that the alpha-1, 2-fucosyltransferase gene HpfutC is derived from helicobacter pylori ATCC 26695 and the alpha-1, 3-fucosyltransferase gene HpM32 is derived from helicobacter pylori NCTC 11639.

4. A recombinant escherichia coli according to any one of claims 1 to 3, wherein the α -1, 2-fucosyltransferase gene HpfutC, α -1, 3-fucosyltransferase gene HpM, the gene cluster manC-manB, the gene cluster gmd-wcaG and the mannose-6-phosphate isomerase gene manA all utilize the promoter T7 to initiate expression.

5. The recombinant E.coli according to any one of claims 1 to 4, wherein the self-promoter of the manC, manB, gmd-wcaG and manA coding genes in the E.coli genome is replaced by a strong promoter T7.

6. Use of a recombinant escherichia coli according to any one of claims 1-5 for the production of 2' -fucosyllactose and/or 3-fucosyllactose.

7. The use according to claim 6, wherein the recombinant E.coli is used as a fermentation strain for producing 2' -fucosyllactose and/or 3-fucosyllactose in a fermentation system using glycerol and glucose as carbon sources.

8. The use according to claim 7, wherein the recombinant E.coli is inoculated in shake flask fermentation medium, glucose is added at a final concentration of 8g/L at the beginning of fermentation, and the culture is carried out at 30-40℃for 72h at 150-250 rpm.

9. The use according to claim 7, wherein a fermentation medium is added to a fermenter and the recombinant escherichia coli is inoculated for fermentation; the fermentation system contains 20-30 g/L of glycerin, 5-10 g/L of glucose, 10-15 g/L of monopotassium phosphate, 1-2 g/L of citric acid, 3-5 g/L of diammonium phosphate, 1-2 g/L of magnesium sulfate heptahydrate, 8-10 g/L of yeast extract and 8-10 mL/L of trace metal solution.

10. The use according to claim 7, wherein the genetically engineered bacterium is cultivated at 20-40 ℃ to maintain dissolved oxygen in the fermentation system at 30±5% and pH at 6.5-7.0; glucose is added after the initial glucose consumption is finished, so that the glucose concentration in the fermentation system is maintained to be 10+/-0.5 g/L.