CN116064345A - High-efficiency production of fucosyllactose without genetically engineered bacteria and its application - Google Patents

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

Antibiotic-free genetically engineered bacteria for efficient production of fucosyllactose and its application

Technical Field

The invention relates to an antibiotic-free genetic engineering bacterium capable of efficiently producing fucosyllactose and application thereof, and belongs to the field of metabolic engineering and food fermentation.

Background Art

Using cheap biomass to produce high value-added products 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 fucosylated lactose (FL) secreted in breast milk, accounting for about 35% of the total human milk oligosaccharides (HMOs). The beneficial properties of FL (such as maintaining intestinal ecological balance, resisting the adhesion of pathogens, immune regulation, and promoting the development and repair of the nervous system) have attracted great attention for their potential applications in nutrition, health care and pharmaceutical uses.

The development of green, efficient and safe chassis microorganisms is the key to solving the large-scale production and application of HMOs. With the development of metabolic engineering and synthetic biology, many model microorganisms have been discovered as potential microbial cell factories for the production of HMOs. Model microorganisms such as Escherichia coli, Saccharomyces cerevisiae, Yarrowia lipolytica and Bacillus subtilis have been successfully used for the de novo biosynthesis of 2'-FL. At present, 3-FL production strains are common in Escherichia coli, with few reports and low yields. It is 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 by-products slows down or stops the growth of the strain, resulting in a serious decrease or complete cessation of the production rate of the target product. Therefore, eliminating these by-product pathways is particularly important in the production of FL. In addition, the production of fucosyllactose requires the supply of exogenous lactose, and the cost of lactose raw materials is high. The development of new lactose synthesis technologies in microorganisms can solve the problem of high lactose cost in the efficient biosynthesis of FL.

等人在大肠杆菌JM109菌株带的染色体上引入了2’-FL的从头合成和补救合成途径,在无抗生素的补料分批发酵中产生了20.28±0.83g/L的2’-FL。Parschat等人开发了一种仅以蔗糖为底物的2’-FL生产菌株，该菌株在不添加无抗生素的情况下胞内自产乳糖并用于2’-FL的生产。除此之外，无抗生素和诱导剂的2’-FL生产菌株未见报道。Starting from the selection of host bacteria, industrially competitive FL production strains can be constructed by using strategies such as metabolic pathway reconstruction, weakening of competitive pathways, cofactor and energy regeneration, and metabolic flux optimization. However, the construction of most FL strains is based on the overexpression of plasmid genes. The development of plasmid-based cell factories has the risk of genetic instability and antibiotic addition during the fermentation process. Therefore, the use of antibiotic- and inducer-free Escherichia coli to prepare FL has clearly become a green, safe, and commercially valuable production strategy. Previously,

et al. introduced the de novo synthesis and salvage synthesis pathways of 2'-FL on the chromosome of the Escherichia coli JM109 strain, and produced 20.28±0.83 g/L of 2'-FL in antibiotic-free fed-batch fermentation. Parschat et al. developed a 2'-FL production strain that only uses sucrose as a substrate. The strain produces lactose intracellularly without the addition of antibiotics and is used for the production of 2'-FL. In addition, no 2'-FL production strains without antibiotics and inducers have been reported.

The present invention aims to use synthetic biology to achieve the construction of non-resistant genetically engineered bacteria and the efficient synthesis of fucosyllactose by reconstructing the 2'-FL and 3-FL synthesis pathways in lactose synthesis strains, regulating central carbon metabolism and weakening the byproduct pathway, upregulating the key enzymes of the de novo synthesis pathway, relieving the inhibition of repressor proteins, and enhancing the 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 atom economy, and provides new ideas for the rational design and construction of a new generation of microbial cell factories, which has important value in both theory and practical application.

Summary of the invention

[Technical issues]

The existing technology for synthesizing fucosyllactose is not efficient, and there are risks of genetic instability and antibiotic addition in the expression of plasmid strains. The acceptor lactose and inducer are expensive. It is impossible to provide a strain for safely and efficiently producing fucosyllactose, nor is it possible to provide a low-cost and environmentally friendly method for preparing fucosyllactose.

[Technical solution]

The present invention provides a genetically engineered bacterium for efficiently producing lactose and fucosyllactose and a construction method thereof in order to solve the problems of expensive lactose and inducers, genetic instability of plasmid strains and the risk of adding antibiotics in the synthesis process of fucosyllactose.

The invention discloses a genetically engineered bacterium BP6 which can utilize low-cost carbon sources (glycerol and glucose) to self-produce lactose. The BP6 is based on the strain BZWNAPAL as a starting strain, wherein the genes crr and ptsG encoding the glucose-specific transporter protease EⅡABC ^Glc component are knocked out, and SetA and Glf are respectively integrated at the crr and ptsG sites; the UDP-glucose-6-dehydrogenase gene ugd is knocked out, and the UDP-glucose-4-epimerase gene GalE is integrated at the ugd gene; the glucose kinase gene Glk is knocked out, and the beta-1,4-galactosyltransferase NmlgtB derived from Neisseria meningitidis is respectively integrated at the glucose kinase gene Glk site.

The strain BZWNAPAL is BL21 (DE3) ΔlacZ ΔwcaJ ΔnudD ΔpfkA Δlon, and the construction method has been disclosed in the patent document with publication number CN114480240A.

The genetically engineered bacteria provided by the present invention are based on strain BP6, wherein the ubiquinone-dependent pyruvate dehydrogenase gene poxB is knocked out, and the α-1,2-fucosyltransferase gene HpfutC and the α-1,3-fucosyltransferase gene HpM32 are integrated at the site of the ubiquinone-dependent pyruvate dehydrogenase gene poxB; the phosphate acetyltransferase and acetate kinase gene cluster pta-ackA is knocked out, and the phosphomannose mutase and mannose-1-phosphate guanyltransferase gene cluster manC-manB are integrated at the site of pta-ackA; the formate lyase gene pflB is knocked out, and the GDP-mannose-6-dehydrogenase and GDP-fucose synthase gene cluster gmd-wcaG are integrated at the pflB site; the D-lactate dehydrogenase gene ldhA is knocked out, and the mannose-6-phosphate isomerase gene manA is integrated at the ldhA site; and the lactose operon repressor protein lacI is knocked out.

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.

In one embodiment, the Gene ID of the β-galactosidase gene lacZ is 945006, the Gene ID of the UDP-glucose lipid carrier transferase gene wcaJ is 946583, the Gene ID of the GDP-mannose mannosyl hydrolase gene nudD is 946559, the Gene ID of the 6-phosphofructokinase-1 gene pfkA is 948412, the GeneID of the protease gene lon is 945085, the Gene IDs of the glucose-specific transporter protease genes crr and ptsG are 946880 and 945651, the Gene ID of the UDP-glucose-6-dehydrogenase gene ugd is 946571, the GeneID of the UDP-glucose-4-isomerase gene GalE is 945354; the Gene ID of the glucose kinase gene Glk is 946858; the nucleotide sequence of the β-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 HpM32, the gene cluster manC-manB, the gene cluster gmd-wcaG and the mannose-6-phosphate isomerase gene manA are all expressed using promoter T7.

In one embodiment, the strong promoter T7 is used to replace the native promoters of the genes encoding manC, manB, gmd-wcaG and manA in the E. coli genome.

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

The second object of the present invention is to provide the use of the recombinant Escherichia coli in producing 2'-fucosyllactose and/or 3-fucosyllactose.

In one embodiment, the recombinant Escherichia coli is used as a fermentation strain to produce 2'-fucosyllactose and/or 3-fucosyllactose in a fermentation system with 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 into a shake flask fermentation medium, glucose with a final concentration of 8 g/L is added at the beginning of the fermentation, and the culture is carried out at 30-40° C. and 150-250 rpm for 72 hours.

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

In one embodiment, the concentration of glycerol in the fermenter is 10-40 g/L.

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.

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.

In one embodiment, the genetically engineered bacteria are cultured at 20-40° C., and the dissolved oxygen in the fermentation system is maintained at 30±5% and the pH is maintained at 6.5-7.0.

In one embodiment, glycerol is added after the initial glycerol in the reaction system is consumed, so that the concentration of glycerol can maintain the growth and metabolism of the bacteria.

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

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

Preferably, the fermentation time is 100 h.

Beneficial effects of the present invention:

The present invention introduces the biosynthetic pathway of fucosyllactose into lactose-producing bacteria, and realizes the construction of non-resistant strains and the efficient synthesis of fucosyllactose without exogenous lactose addition by weakening the byproduct pathway, regulating central carbon metabolism, upregulating key enzymes of the de novo synthesis pathway, relieving the repression inhibition of repressor proteins, and enhancing the extracellular output of products. Under the conditions of shake flask fermentation without adding antibiotics and inducers, the ability of the genetically engineered bacteria constructed in this application to produce 2'-FL and 3-FL reached 4.36 and 3.23 g/L respectively; under the conditions of 3L fermenter culture, the output of 2'-FL and 3-F reached 40.44 and 30.42 g/L respectively, laying the foundation for the industrial production of fucosyllactose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the metabolic process of producing fucosyllactose using glucose and glycerol as substrates.

FIG2 is a comparison chart showing the yields of engineered bacteria that weakened the byproduct pathway and introduced the fucosyllactose pathway.

FIG3 shows the effect of up-regulating the GDP-L-fucose pathway in engineered bacteria on the production of fucosyllactose.

FIG4 shows the relative transcription levels of GDP-L-fucose module genes in strains BP10-3 and BP10-4.

FIG5 shows the fed-batch fermentation of strain BP10-3 in a 3 L fermenter.

FIG6 shows the fed-batch fermentation of strain BP11-3 in a 3 L fermenter.

DETAILED DESCRIPTION

The specific implementation of the present invention is further described below in conjunction with examples and drawings. 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 according to the kit instructions.

The embodiments of the present invention are not limited thereto, and other unspecified experimental operations and process parameters are performed according to conventional techniques.

Vectors pCas9 and pTargetF were purchased from Addgene.

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

Preparation of competent E. coli: kit provided by Shanghai Sangon Biotechnology Co., Ltd.

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

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

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.

Determination method of 2'-FL, 3-FL and GDP-L-fucose:

HPLC determination: 1 mL of fermentation broth was boiled at 100°C for 10 min, centrifuged at 12000 r/min for 5 min, the supernatant was filtered through a 0.22 μm membrane, and HPLC was used to detect the production of fucosyllactose and the consumption of glucose and glycerol. HPLC detection conditions: differential refractive index detector; chromatographic column: Rezex ROA-organic acid (Phenomenex, USA), column temperature: 50°C; mobile phase: 0.005 mol/L H ₂ SO ₄ aqueous solution, flow rate: 0.6 mL/min; injection volume: 10 μL. HPLC detection conditions of GDP-L-fucose: UV detector; detection wavelength 254 nm; chromatographic column Inertsil ODS-SP (GL Sciences, Kyoto, Japan); mobile phase A is 20 mM triethylamine-glacial acetic acid aqueous solution (pH 6.0), mobile phase B is acetonitrile solution; gradient elution; flow rate is 0.6 mL/min; injection volume is 10 μL.

The shake flask fermentation conditions of the strains in the following examples are as follows: a single colony of the engineered bacteria was inoculated into LB liquid medium, and the culture was shaken at 37°C, 200 rpm for 12 h to obtain a seed solution; the seed solution was inoculated into 50 mL of fermentation medium at an inoculum rate of 3% (v/v), and the culture was shaken at 37°C, 200 rpm until the _OD600 was 0.6, and glucose was added to a final concentration of 8 g/L, and the culture was induced at 25°C, 200 rpm for 72 h.

Example 1: Removing the inhibition of repressor proteins and glycolysis byproducts to construct a strain producing non-resistant fucosyllactose

The synthesis of fucosyllactose requires the supply of exogenous lactose. Previously, a genetically engineered bacterium that can produce lactose using low-cost carbon sources (glycerol and glucose) was constructed. On this basis, in order to prevent metabolic overflow of the glycolysis pathway, the byproduct pathway was weakened and the fucosyllactose synthesis pathway was introduced. Using Escherichia coli BP6 as the starting strain, the CRISPR-Cas9 gene editing system was used to knock out poxB, and a double gene copy of the 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 with glycerol and glucose as substrates in the lactose-producing strain is shown in Figure 1. The specific steps of gene knockout and integration are as follows (the primer sequences involved are shown in Table 1):

(1) Taking the knockout of poxB and the chromosome integration of the double tandem α-1,2-fucosyltransferase gene HpfutC as an example, the specific target gRNA (20 bp) of the poxB gene was searched through http://www.regenome.net/cas-offinder, and the upstream and downstream primers of poxB-gRNA-F/gRNA-R were used to perform PCR amplification with the 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, the plasmid was extracted, and the successfully constructed knockout plasmid was named pTargetF-poxB by sequencing.

(2) Using the genome of Escherichia coli strain BP6 as a template, three sequence fragments were amplified using the upstream homology arm primers poxB-US-F/poxB-US-R, the midstream homology arm primers 2HpfutC-MS-F/2HpfutC-MS-R and the downstream homology arm primers poxB-DS-F/poxB-DS-R respectively. After the products were purified and recovered, the three fragments were connected using the SOE-PCR method using primers poxB-US-F/poxB-DS-R to obtain the gene homology repair template.

(3) Take pCas9 plasmid (Addgene: #62225) and E. coli BP6 electroporated competent cells, place on ice for 5 minutes, thaw the competent cells, take 10μL plasmid and add 100μL competent cells, and mix gently. Transfer the plasmid and electroporated competent cells into a pre-cooled electroporation cup, electroporate at 2.5kV for 5ms, quickly add pre-cooled liquid LB after electroporation, gently blow and mix, and transfer the culture medium mixed with plasmid and competent cells to a new centrifuge tube for expansion culture for 1.5h. Centrifuge at 6000r/min for 2min, discard the supernatant, spread the bacteria on an LB plate containing kanamycin resistance, and place in a 30℃ incubator for overnight culture.

(4) Pick a single colony of E. coli BP6/pCas9 in LB medium, culture at 30°C for 1.0 h, and add L-arabinose with a final concentration of 30 mM to induce the expression of the λ-red system. When OD ₆₀₀ reaches 0.6-0.8, prepare the competent E. coli BP6/pCas9.

(5) 500 ng of the targeting plasmid pTargetF with poxB-specific target gRNA (20 bp) constructed in step (1) and 1000 ng of the homologous repair template constructed in step (2) were electroporated into the E. coli BP6/pCas9 competent cells prepared in step (4), spread on LB plates (kanamycin and spectinomycin), and cultured at 30°C for 16-24 h. Single colonies grown on the plates were verified by colony PCR, positive transformants were screened, and gene sequencing was performed.

(6) Eliminate the pTargetF-poxB and pCas9 plasmids from the verified single colonies. Inoculate the single colonies in LB liquid medium (kananamycin resistance), culture at 30°C and 200 r/min until the logarithmic growth phase, add IPTG with a final concentration of 0.5 mmol/L for overnight culture to induce inactivation of the pTargetF-poxB plasmid. Streak the bacterial liquid on an LB plate containing Kan and culture at 30°C and 200 r/min for 12 hours. Spot the single colony on a double-resistant plate of kanamycin and spectinomycin. If no colony grows, it indicates that the pTargetF-poxB plasmid has been successfully eliminated.

(7) The pCas9 plasmid is a thermosensitive plasmid. The single colony that successfully eliminated the pTargetF-poxB plasmid was transferred to LB liquid medium without resistance and subcultured at 42°C to eliminate the pCas9 plasmid. The bacterial liquid was streaked on an LB plate without resistance and cultured at 37°C. A single colony was spotted on an LB medium containing kanamycin resistance. If the single colony did not grow, it indicated that the pCas9 plasmid was successfully eliminated. The constructed gene-deficient strain without pTargetF-poxB plasmid and pCas9 plasmid was stored at -80°C for future use.

(8) The knockout of genes poxB, pta-ackA, pflB, ldhA and lacI and the corresponding integration of HpM32, manC-manB, gmd-wcaG and manA refer to the above steps. The construction steps of gene editing strains involved in other embodiments refer to Example 1.

Table 1 Gene knockout and integration primers

In recombinant engineered bacteria, the accumulation of byproducts of the glycolysis pathway is not only toxic to cell growth, but also competes with the synthesis of GDP-L-fucose for carbon sources. In order to strengthen the synthesis of the precursor GDP-L-fucose, we blocked the generation of byproducts lactic acid, formic acid and acetic acid by gene knockout. The efficient production of FL in engineered Escherichia coli was further achieved by regulating four enzymes that may participate in the competitive pathway. The engineered strains constructed in this embodiment are shown in Table 2, and the synthesis ability of fucosyllactose, bacterial biomass and byproduct accumulation of the engineered strains were detected. The results showed that after the deletion of poxB and the introduction of HpfutC/HpM32 genes, the FL production strain was initially constructed, and the 2'-FL and 3-FL titers of BP8-0 and BP9-0 strains were 1.39 and 0.98 g/L by shaking flask culture, and 2.97 and 3.32 g/L lactose remained in the fermentation supernatant. After all byproducts of the glycolysis pathway were removed, the accumulation of acetic acid and lactic acid was not detected in the recombinant strains during fermentation. Strains BP8-4 and BP9-4 showed higher biomass, and the production of synthesized lactose reached 6.85 and 6.64 g/L. In addition, the extracellular output content 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 (Figure 2). The above shows that weakening the glycolysis pathway allows more carbon to flow to the FL and lactose synthesis pathways, which is conducive to the efficient biosynthesis of FL.

Table 2. Detailed information of engineered bacteria with integrated fucosyllactose metabolic pathway in their genome

Example 2: Upregulating the genomic "key enzyme" to enhance the efficient production of fucosyllactose

Escherichia coli contains an endogenous synthetic pathway for the production of 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 of manA, manB, manC, gmd, and wcaG (Figure 1). As a precursor of the colanic acid biosynthetic pathway, the availability of GDP-L-fucose in the cell determines the total yield of fucosyllactose biosynthesis. However, the production of GDP-L-fucose by wild Escherichia coli is negligible, which is not conducive to the further synthesis of FL. In order to accelerate the carbon flux of the GDP-L-fucose synthesis module, a promoter engineering strategy was used to improve the precursor supply of GDP-L-fucose and achieve efficient synthesis of FL in an antibiotic-free strain.

The promoter engineering strategy is an effective method for moderately regulating protein expression. In this example, the strong promoter T7 replaces the promoter of the "key enzyme" itself on the E. coli genome, and upregulates the expression levels of manA, manB, manC and gmd genes in the GDP-L-fucose biosynthesis pathway, in order to achieve efficient production of fucosyllactose. The gene editing primers involved in this example are shown in Table 3.

Table 3 Promoter replacement primers

On the basis of engineered bacteria BP8-4 and BP9-4, the strong promoter T7 was used to replace the original promoters of manA, manB, manC and gmd in the GDP-L-fucose pathway on the genome, and 2'-FL production strains (BP10-0, BP10-1, BP10-2 and BP10-3) and 3-FL production strains (BP11-0, BP11-1, BP11-2 and BP11-3) were obtained respectively. The strains constructed in this embodiment are shown in Table 4. The fermentation results showed that the production of all 2'-FL and 3-FL strains was improved by upregulating the "key enzyme" of the GDP-L-fucose pathway on the genome. Under the condition of integrating sugar efflux protein, the extracellular output of 2'-fucosyllactose and 3-fucosyllactose was detected to be 4.36 and 3.23 g/L, which was 30.5% and 16.1% higher than that of BP8-4 and BP9-4 strains in Example 1 (Figure 3).

Table 4. Detailed information of the engineered bacteria with promoter replacement

Example 3: Detection of intracellular GDP-L-fucose content and transcription levels of related genes in engineered strains

In order to determine whether the promoter engineering strategy is effective, the FL producing strain in Example 2 was cultured by shaking flask fermentation, and the cells fermented for 24 hours were taken as samples for intracellular GDP-L-fucose concentration detection. At the same time, in the GDP-L-fucose module, the transcription level of the "key enzyme" gene was analyzed by real-time fluorescence quantitative analysis. The test results are shown in Table 5. The intracellular GDP-L-fucose concentration of BP10-3 was 547 mg/g DCW, which was 98% higher than that of the control BP8-4. The transcription 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 enhancement in GDP-L-fucose titer and gene transcription level. The promoter engineering strategy can improve lactose consumption, force carbon metabolism to flow to the GDP-L-fucose biosynthesis pathway, improve the economy of carbon atoms, and is of great significance to the synthesis of FL.

Table 5. Intracellular GDP-L-fucose concentrations of different engineered bacteria

Example 4: Production of fucosyllactose in a 3L fermenter in fed-batch format

In order to produce high-yield 2’-FL and 3-FL, high-density fed-batch fermentation was carried out in 3 L fermentors using the antibiotic-free strains BP10-3 and BP11-3, respectively.

Fermentation conditions: 50 mL of overnight cultured seed liquid was inoculated into 1 L of fermentation medium, the culture temperature was 37 °C, the initial glycerol and glucose concentrations were 30 and 10 g/L, and NH ₄ OH was used to control the pH of the tank to be constant at 6.80 during the fermentation process. In order to maintain bacterial growth and the synthesis of fucosyllactose, 800 g/L of glycerol (containing 20 g/L of MgSO ₄ ·7H ₂ O) was added to supplement the carbon source after the initial glycerol was consumed. The concentration of glycerol in the fermentation system was maintained at a low concentration level (glycerol was used for bacterial growth and metabolism, and the concentration was about 0 g/L) by pH feedback regulation (the flow rate was set to 20 mL/h) until the end of fermentation. After the initial glucose was consumed, 300 g/L of glucose was manually added to maintain its final concentration in the fermentation system at about 10±0.5 g/L. If glucose was consumed to a lower concentration during the fermentation process, glucose was continued to be added until the end of fermentation. During the fermentation process, the system is cascade controlled to adjust the speed, ventilation volume and oxygen to keep the dissolved oxygen in the tank at 30±5%.

During the whole fermentation process, samples were taken regularly and the bacterial OD ₆₀₀ was measured. 1 mL of fermentation liquid was boiled for 15 min to completely break the cells, centrifuged at 12000 r/min for 10 min, and the supernatant was filtered through a 0.22 μm membrane. During the fermentation process, HPLC was used to detect the production of lactose, 2'-FL and 3-FL, as well as the consumption of glucose and glycerol (Figures 5 and 6). The results showed that the product lactose was maintained at 8-15 g/L during the fermentation process. After the fermentation was completed (a total of 100 hours of fermentation), the concentration of extracellular 2'-FL could reach 40.44 g/L, and the concentration of extracellular 3-FL could reach 30.42 g/L.

Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (10)

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.

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