A method for efficiently synthesizing lactose and its application

Abstract

The present invention relates to a method for efficiently synthesizing lactose and its application, belonging to the fields of metabolic engineering and food fermentation. The present invention uses CRISPR / Cas9 gene editing technology to transform chassis microorganisms, and achieves the construction of antibiotic-free and antibiotic-resistant strains through strategies such as reconstructing glucose transport pathways, lactose synthesis pathways, regulating central carbon metabolism, relieving repressive inhibition of repressor proteins, and optimizing modular pathways. The recombinant engineered bacteria obtained by the present invention can achieve lactose production and can also use glycerol and glucose to achieve efficient lactose synthesis. The product production level is high, with strong social and economic benefits and broad market development prospects.

Description

Technical Field

[0001] The invention relates to a method for efficiently synthesizing lactose and application thereof, and belongs to the fields of metabolic engineering and food fermentation. Background Art

[0002] Lactose is a carbohydrate unique to mammalian milk. It is a disaccharide composed of glucose and galactose linked by a 1,4-glycosidic bond. Lactose synthesis occurs within the Golgi apparatus within mammary epithelial cells, a process catalyzed by multiple enzymatic reactions. Lactose is readily broken down into glucose and galactose by β-galactosidase in the human intestine. Glucose primarily provides energy, while galactose glycosidically binds to ceramide to form galactocerebroside, which plays a role in brain development. Furthermore, lactose can promote the production of certain lactic acid bacteria in the human intestine, inhibit the growth of putrefactive bacteria, and promote intestinal motility. Therefore, lactose is often used in infant foods and pharmaceutical products.

[0003] Traditionally, lactose is extracted from milk whey through concentration, crystallization, refining, recrystallization, and drying, a cumbersome and costly process. In recent years, the development of high-value-added products such as human milk oligosaccharides (HMOs) using lactose as a receptor has increased. The synthesis of these functional products requires a continuous supply of exogenous lactose, significantly increasing production costs. Therefore, by mimicking the mammalian lactose synthesis pathway and designing and developing new lactose synthesis technologies using microbial cell factories, the cost of lactose biosynthesis can be reduced.

[0004] The development of green, efficient, and safe microbial platforms is key to scalable lactose production and application. With the advancement of metabolic engineering and synthetic biology, numerous model microorganisms have been explored as potential microbial cell factories for chemicals and natural products. Model microorganisms such as Escherichia coli, Bacillus, Lactobacillus, Corynebacterium glutamicum, and Saccharomyces cerevisiae have been successfully used for the biosynthesis of functional sugars. Given the rapid growth, simple cultivation, stable recombinants, and comprehensive vector-receptor systems of these model organisms, combined with the efficient CRISPR-Cas9 gene editing system, the production of lactose products using recombinant engineered bacteria has become a green, safe, and commercially viable production strategy. This paper aims to utilize synthetic biology to construct antibiotic-free and antibiotic-resistant strains and achieve efficient lactose synthesis by reconstructing glucose transport and lactose synthesis pathways, regulating central carbon metabolism, relieving repressor inhibition, and optimizing plasmid-based pathways. This research approach will enrich and advance the research on microbial metabolic regulation, provide new methods and examples for reconstructing microbial metabolic networks and improving carbon economy, and offer new insights into the rational design and construction of a new generation of microbial cell factories, possessing significant theoretical and practical value. Summary of the Invention

[0005] [Technical Issues]

[0006] Existing lactose production methods are derived from animal milk and are limited by dairy separation, requiring multiple purification steps and cumbersome procedures. Existing technologies cannot provide microbial strains that can safely and efficiently produce lactose, nor can they provide a low-cost and environmentally friendly method for preparing lactose.

[0007] [Technical solution]

[0008] In order to solve the defects of limited lactose sources, high costs, and complex purification processes, the present invention provides a recombinant microorganism that can efficiently produce lactose using glycerol and glucose as substrates. It has the advantages of high conversion efficiency, high environmental friendliness, and low production costs, and is suitable for industrial production of lactose.

[0009] The first object of the present invention is to provide a genetically engineered bacterium, wherein the genetically engineered bacterium is a host bacterium BL21 (DE3) in which the β-galactosidase gene lacZ and the glucose-specific transporter protease EⅡABC are knocked out. Glc The component encoding gene was integrated into the sugar efflux transporter gene SetA and the glucose transporter gene Glf at this site, or the glucose-specific transporter protease EⅡABC was knocked out in the host bacteria BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlon. Glc The component encodes genes, and the sugar efflux transporter gene SetA and the glucose transporter gene Glf are integrated at this site; the genes wcaJ, nudD, pfkA, and lon have no effect on the synthesis of lactose.

[0010] Preferably, the glucose-specific transporter EIIABC Glc Component encoding genes include crr and ptsG.

[0011] Preferably, the genetically engineered bacteria is based on the host bacteria in which crr is knocked out and the sugar efflux transporter gene SetA is integrated at the site.

[0012] Preferably, the genetically engineered bacteria is based on the host bacteria, with crr and ptsG knocked out, and the sugar efflux transporter gene SetA is integrated into the crr site, and the glucose transporter gene Glf is integrated into the ptsG site.

[0013] In one embodiment, 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 β-1,4-galactosyltransferase is integrated at the glucose kinase gene Glk site.

[0014] In one embodiment, the β-1,4-galactosyltransferase is derived from AaGalT of Actinobacillus actinomycetemcomitans, NmlgtB from Neisseria meningitidis, PmGalT from Pasteurella multocida, NLlgtB from Neisseria lactis, KpGalT from Klebsiella pneumoniae, HsGalT from Histophilus somnus, PawaaX from Pseudomonas aeruginosa, SdwaaX from Shigella dysenteriae or EcwaaX from Escherichia coli, etc.

[0015] In one embodiment, the sugar efflux transporter gene SetA, the glucose transporter gene Glf, the UDP-glucose-4-epimerase gene GalE, and the β-1,4-galactosyltransferase gene are all expressed using promoter T7.

[0016] In one embodiment, the ubiquinone-dependent pyruvate dehydrogenase gene poxB is knocked out, the phosphate acetyltransferase and acetate kinase gene cluster pta-ackA is knocked out, the formate lyase gene pflB is knocked out, and the D-lactate dehydrogenase gene ldhA is knocked out.

[0017] In one embodiment, the genes pgi, pgm, GalU, GalE, and NmlgtB are overexpressed using pRSFDuet-1, pETDuet-1, or pCDFDuet-1 vectors.

[0018] In one embodiment, the genetically engineered bacteria overexpress genes pgi, pgm, GalU, GalE, and NmlgtB using pRSFDuet-1, pETDuet-1, or pCDFDuet-1 vectors.

[0019] In one embodiment, pRSFDuet-1 is used to overexpress genes pgi, pgm, and GalU, and pETDuet-1 is used to overexpress genes GalE and NmlgtB.

[0020] In one embodiment, pRSFDuet-1 is used to overexpress genes pgi, pgm, and GalU, and pCDFDuet-1 is used to overexpress genes GalE and NmlgtB.

[0021] In one embodiment, pETDuet-1 is used to overexpress genes pgi, pgm, and GalU, and pRSFDuet-1 is used to overexpress genes GalE and NmlgtB.

[0022] In one embodiment, pETDuet-1 is used to overexpress genes pgi, pgm, and GalU, and pCDFDuet-1 is used to overexpress genes GalE and NmlgtB.

[0023] In one embodiment, pCDFDuet-1 is used to overexpress genes pgi, pgm, and GalU, and pRSFDuet-1 is used to overexpress genes GalE and NmlgtB.

[0024] In one embodiment, pCDFDuet-1 is used to overexpress genes pgi, pgm, and GalU, and pETDuet-1 is used to overexpress genes GalE and NmlgtB.

[0025] In one embodiment, the Gene ID of the β-galactosidase gene lacZ is 945006, 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 Gene ID of the UDP-glucose-4-epimerase gene GalE is 945354, and the Gene ID of the glucokinase gene Glk is 946858; 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 IdhA is 946315, the Gene ID of the formate lyase gene pflB is 945514, and the Gene ID of the lactose operon repressor protein lacI is 946400. The gene ID is 945007, and the gene ID of the D-lactate dehydrogenase gene ldhA is 946315.

[0026] In one embodiment, the glucose transporter gene Glf is derived from Zymomonas mobilis, the sugar efflux transporter gene SetA is derived from Yersinia burnetii ATCC 43970, and their nucleotide sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively; the β-1,4-galactosyltransferase is derived from AaGalT of Actinobacillus actinomycetemcomitans, NmlgtB from Neisseria meningitidis, PmGalT from Pasteurella multocida, NLlgtB from Neisseria lactis, KpGalT from Klebsiella pneumoniae, HsGalT from Histophilus somnus, PawaaX from Pseudomonas aeruginosa, SdwaaX from Shigella dysenteriae or EcwaaX from Escherichia coli, and their nucleotide sequences are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31 NO.11.

[0027] In one embodiment, the host is Escherichia coli, Bacillus, lactic acid bacteria, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus oryzae or Aspergillus niger.

[0028] The second object of the present invention is to provide the use of the genetically engineered bacteria in synthesizing lactose and its derivative products.

[0029] In one embodiment, the lactose-derived products include human milk oligosaccharides, galacto-oligosaccharides, lactofructo-oligosaccharides, lactulose, lactulose, lactulose, lactitol, lactobionic acid, tagatose, and the like.

[0030] In one embodiment, the genetically engineered bacteria are inoculated into a fermentation medium, a single colony of the genetically engineered bacteria is picked and placed in LB liquid medium, and cultured in a shake flask at 37°C, 200 rpm for 12 hours, the seed liquid is inoculated into the fermentation medium at an amount of 3% (v / v), glucose with a final concentration of 8 g / L is added at the beginning of fermentation, and culture is carried out at 30-40°C, 150-250 rpm for 48 hours.

[0031] Preferably, the recombinant Escherichia coli is BP20.

[0032] 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.

[0033] Beneficial effects of the present invention:

[0034] The genetically engineered bacteria provided by the present invention inactivate PTS Glc Transfer system and enhanced NPTS Glc The research team developed a novel transport system that enhanced glucose internalization efficiency and prevented glucose phosphorylation. They screened for the optimal source of β-1,4-galactosyltransferase and constructed a lactose synthesis pathway. They also weakened the byproduct pathway and relieved the repressor protein's inhibition. Through strategies such as plasmid-based pathway optimization, they achieved the construction of both antibiotic-resistant and antibiotic-resistant strains and the efficient synthesis of lactose. Furthermore, the system can use glycerol and glucose as substrates, making them inexpensive and widely available, and the conversion efficiency to lactose is high, reducing production costs and environmental pollution. This lays the foundation for the industrial production of lactose and the microbial production of lactose-based chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the metabolic process for producing lactose using glucose and glycerol as substrates.

[0036] Figure 2 For PTS Glc Inactivation and NPTS Glc Activation pathway design in engineered Escherichia coli.

[0037] Figure 3 For PTS Glc Comparison of glucose consumption rate and biomass of modified strains.

[0038] Figure 4 To compare the lactose production performance of engineered strains of β-1,4-GalT from different sources.

[0039] Figure 5 To weaken the impact of the by-product pathway on lactose production. DETAILED DESCRIPTION

[0040] 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 carried out according to the kit instructions.

[0041] The embodiments of the present invention are not limited thereto, and other unspecified experimental operations and process parameters are performed according to conventional techniques.

[0042] The construction method of strain BZWNAPAL has been disclosed in patent document with publication number CN114480240A.

[0043] Vectors pCas9 and pTargetF were purchased from Addgene.

[0044] The sequencing of DNA products and plasmids was completed by Tianlin Biotechnology (Wuxi) Co., Ltd.

[0045] Preparation of competent E. coli: kit provided by Shanghai Sangon Biotechnology Co., Ltd.

[0046] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0047] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 18 g / L agar powder.

[0048] 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.

[0049] Lactose determination method:

[0050] HPLC determination was performed: 1 mL of fermentation broth was boiled at 100°C for 10 minutes, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm membrane. Lactose production and glucose and glycerol consumption were determined by HPLC. HPLC conditions included a differential refractive index detector (RID) on a Rezex ROA-organic acid column (Phenomenex, USA) at a column temperature of 50°C; the mobile phase consisted of 0.005 mol / L aqueous H₂SO₄ at a flow rate of 0.6 mL / min; and the injection volume was 10 μL.

[0051] The shake flask fermentation conditions for the strains in the following examples were as follows: a single colony of the engineered bacteria was inoculated into LB liquid medium and cultured at 37°C, 200 rpm, for 12 hours to obtain a seed solution. This seed solution was then inoculated into 50 mL of fermentation medium at a 3% (v / v) inoculum. Glucose was added at a final concentration of 8 g / L at the beginning of the fermentation, and the culture was incubated at 37°C, 200 rpm, for 48 hours. If the strain is resistant, the corresponding antibiotic should be added to the medium before inoculation.

[0052] Example 1: Inactivation of PTS Glc system and strengthen NPTS Glc System to enhance glucose internalization efficiency

[0053] Taking Escherichia coli as an example, there are two glucose internalization modes in E. coli, namely PTS Glc and NPTS Glc Transport system. When the lactose synthesis pathway is reconstructed using glucose as a substrate, the PTS of glucose Glc The system converts the phosphate of phosphoenolpyruvate (PEP) into Glc -EIICB Glc Transferred to glucose, Glu-6-P is generated and enters the intracellular metabolism. In the process of endoplasmic glucose and glycolysis, a large amount of pyruvate is produced, which can easily lead to the overflow of central carbon metabolism and generate by-products such as acetate, formate, and lactate. In addition, NPTS composed of glucose / hydrogen ion co-transporter (encoded by gene GalP) and glucose kinase (encoded by gene Glk) Glc Transfer system in PTS Glc After inactivation, it is activated to restore glucose metabolism ( Figure 2 Using Escherichia coli BL21 (DE3) ΔlacZΔwcaJΔnudDΔpfkAΔlon (BZWNDPAL) as the starting strain, the glucose-specific transporter EⅡABC was knocked out using the CRISPR-Cas9 gene editing system. Glc The component encodes genes crr and ptsG, and the sugar efflux transporter gene SetA and glucose transporter gene Glf are integrated at this site. Figure 1 As shown, the specific steps are as follows (the primer sequences involved are shown in Table 1):

[0054] (1) Taking the knockout of ptsG and chromosomal integration of the Glf gene as an example, the specific target gRNA (20 bp) of the ptsG gene was searched through http: / / www.regenome.net / cas-offinder. PCR amplification was performed using the upstream and downstream primers ptsG-gRNA-F / gRNA-R and the pTargetF plasmid (Addgene: #62226) as a template. The amplified product was digested with the restriction endonuclease Dpn I to remove the excess circular plasmid pTargetF. The amplified product was then transformed into E. coli DH5α competent cells, the plasmid was extracted, and sequencing was performed using the primers gRNA-PF / gRNA-PR. The successfully constructed knockout plasmid was named pTargetF-ptsG.

[0055] (2) Using the Escherichia coli BZWNDPAL genome as a template, three sequence fragments were amplified using the upstream homology arm primers ptsG-US-F / ptsG-US-R, the midstream homology arm primers Glf-MS-F / Glf-MS-R, and the downstream homology arm primers ptsG-DS-F / ptsG-DS-R. After the products were purified and recovered, the three fragments were connected using the SOE-PCR method using primers ptsG-US-F / ptsG-DS-R to obtain the gene homology repair template.

[0056] (3) Take pCas9 plasmid (Addgene: #62225) and Escherichia coli BZWNDPAL electroporated competent cells, place on ice for 5 minutes to thaw the competent cells, take 10 μL of plasmid and add 100 μL of competent cells, and mix gently. Transfer the plasmid and electroporated competent cells into a pre-cooled electroporation cup, electroporate at 2.5 kV for 5 ms, 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 1.5 hours. Centrifuge at 6000 r / min for 2 minutes, discard the supernatant, spread the bacteria on an LB plate containing kanamycin resistance, and place in a 30°C incubator for overnight culture.

[0057] (4) Pick a single colony of E. coli BZWNDPAL / pCas9 and place it in LB medium, culture it at 30℃ for 1.0h, and add L-arabinose with a final concentration of 30mM to induce the expression of the λ-red system. 600 When the concentration reaches 0.6-0.8, prepare the competent E. coli BZWNDPAL / pCas9.

[0058] (5) 500 ng of the targeting plasmid pTargetF with ptsG-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 Escherichia coli BZWNDPAL / 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.

[0059] (6) Eliminate the pTargetF-ptsG and pCas9 plasmids from the verified single colony. Inoculate the single colony into LB liquid medium (kanama resistant) and culture at 30°C and 200 r / min until the logarithmic growth phase. Add IPTG with a final concentration of 0.5 mmol / L and culture overnight to induce inactivation of the pTargetF-nudD plasmid. Streak the bacterial liquid onto an LB plate containing Kan and culture at 30°C and 200 r / min for 12 hours. Spot the single colony onto a double-resistant plate containing kanamycin and spectinomycin. If no colony grows, it indicates that the pTargetF-ptsG plasmid has been successfully eliminated.

[0060] (7) The pCas9 plasmid is a thermosensitive plasmid. The single colony that has successfully eliminated the pTargetF-ptsG plasmid is transferred to LB liquid medium without resistance and subcultured at 42°C to eliminate the pCas9 plasmid. The bacterial liquid is streaked onto an LB plate without resistance and cultured at 37°C. A single colony is plated onto an LB plate containing kanamycin resistance. If the single colony does not grow, it indicates that the pCas9 plasmid has been successfully eliminated. The constructed gene deletion strain without the pTargetF-ptsG plasmid and the pCas9 plasmid is stored at -80°C for future use.

[0061] (8) The knockout of the crr gene and the integration of the sugar efflux transporter gene SetA were performed with reference to the above steps. The construction steps of the gene-edited strains involved in other embodiments were all performed with reference to Example 1.

[0062] Table 1 Gene knockout and integration primers

[0063]

[0064] The engineered strains constructed in this embodiment are shown in Table 2. The glucose consumption rate and bacterial biomass of the engineered strains were detected. The fermentation results showed that when ptsG was missing, the function of the PTS system was reduced, glucose consumption and cell growth were significantly reduced, and the rate of glucose transport was reduced to 65%-70% of the control group (BZWNDPAL). In order to achieve glucose transport and prevent glucose phosphorylation, the ptsG gene was replaced by the Glf gene. The results showed that the glucose transport capacity and biomass of the strain were significantly improved. The improvement of glucose transport rate and cell biomass is the key to enhancing NPTS. Glc As a result, the dual effects of GalP and Glf promoted glucose internalization. In addition, the PTS was further deleted in strain BP3. Glc IIA in the system Glc (encoded by gene ptsG) and introduced sugar efflux transporter SetA, which significantly inhibited glucose transport and cell growth compared to BZWNDPAL. During the fermentation process, we found that some glucose was still phosphorylated and entered the glycolysis pathway and a large amount of by-products (acetic acid) accumulated in the fermentation broth. Phosphorylation is also a PTS - The main limiting factor of the glucose utilization efficiency of the strain was the knockout and replacement of the glucokinase gene Glk in Example 2.

[0065] Table 2. Inactivated PTS Glc Detailed information on the engineered bacteria of the transport system

[0066]

[0067] Example 2: Genomic screening of β-1,4-galactosyltransferase and construction of lactose-resistant strains

[0068] Lactose synthase from mammalian mammary epithelial cells is involved in the biosynthesis of lactose. Lactose synthase is composed of two subunits: β-1,4 galactosyltransferase (β-1,4-GalT1) and α-lactalbumin. In the absence of α-lactalbumin, the ability of this enzyme to catalyze UDP-galactose and glucose to form lactose is extremely low. Generally, overexpression of lactose synthase in Escherichia coli is unsuccessful. In order to get rid of the problem of limited traditional lactose sources, the strain is engineered to produce its own inherent lactose supply pathway. Lactose synthesis involves the regulation of several metabolic pathways ( Figure 1), such as the uptake of extracellular glucose, the conversion of intracellular glycerol to UDP-galactose, and the synthesis of lactose from UDP-galactose and glucose catalyzed by heterologous β-1,4-galactosyltransferases. Genes involved in the lactose synthesis pathway include glucose uptake (Glf, GalP, etc.), the conversion of glycerol to UDP-galactose (pgi, pgm, GalU, GalE, etc.), and the synthesis of lactose from UDP-galactose and glucose (GalT). Efficient expression of β-1,4-galactosyltransferase is crucial for lactose synthesis. For comparison, three β-1,4-galactosyltransferases from different sources were selected: the Glk gene was replaced with AaGalT from Actinobacillus actinomycetemcomitans, NmlgtB from Neisseria meningitidis, and PmGalT from Pasteurella multocida. To inhibit glucose phosphorylation and prevent UDP-galactose degradation, the GalE gene, which converts UDP-glucose to UDP-galactose, was integrated into the ugd locus of the chromosome. The gene editing primers involved in the lactose synthesis pathway are shown in Table 3.

[0069] Table 3 Gene knockout and integration primers

[0070]

[0071]

[0072] The engineered strains constructed in this embodiment are shown in Table 4. The lactose synthesis ability and bacterial biomass of the engineered strains were detected. The fermentation results showed that when the recombinant bacteria BP4 only contained the GalU and GalE genes for strengthening the precursor UDP-galactose and lacked the β-1,4-GalT gene, the fermentation results showed that no lactose was produced. The β-1,4-galactosyltransferase (NmlgtB) derived from Neisseria meningitidis has a strong lactose synthesis ability. When the strain was fermented for 48h, the lactose content reached 3.25g / L and the yield reached 0.62g / g glucose. In contrast, the ability of PmGalT derived from Pasteurella multocida and AaGalT derived from Actinobacillus actinomycetemcomitans to synthesize lactose is weaker. During the entire fermentation process, there is still accumulation of some by-products acetic acid and lactic acid. It is speculated that the addition of glucose may cause the strain to consume more glycerol to enter the glycolysis pathway, resulting in overflow of central carbon metabolism. To solve this problem, the inhibition of glycolysis by-products was further removed by gene editing means. For details, see Example 3.

[0073] Table 4. Detailed information of lactose self-synthesizing engineered bacteria

[0074]

[0075] Example 3: Removing the inhibition of repressor proteins and glycolysis byproducts to construct a lactose-free production strain

[0076] 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 lactose for carbon sources. In order to enhance the synthesis of the precursor UDP-galactose, this example knocked out the byproduct acetic acid synthesis gene poxB and pta-ackA gene cluster, the formic acid synthesis gene pflB, and the lactic acid synthesis gene ldhA based on the BP6 strain. In order to achieve the construction of an inducer-free strain, this example deleted lacI on the genome to relieve the inhibition of the lactose operon repressor protein. The gene editing primers involved in the lactose synthesis pathway are shown in the following table.

[0077] Table 5 Gene knockout and integration primers

[0078]

[0079]

[0080] The engineered strains constructed in this embodiment are shown in Table 6, and the lactose synthesis ability, bacterial biomass and by-product accumulation of the engineered strains were tested. After the by-products of the glycolysis pathway were eliminated, no accumulation of by-products such as formic acid, acetic acid and lactic acid was detected in the fermentation broth of the recombinant strains. The recombinant strains BP11 and BP12 showed stronger growth, with the maximum bacterial mass in shake flask fermentation reaching 6.64 and 6.44 g / L, respectively, which were 1.39 and 1.35 times that of the initial strain BP6 (4.76 g / L). Moreover, the production of lactose synthesized by the two strains reached 7.15 and 6.87 g / L at 48 h, which was 120% and 111% higher than that of strain BP6, indicating that weakening the glycolysis pathway allows more carbon to flow to the downstream lactose synthesis pathway, which is beneficial to the microbial synthesis of lactose.

[0081] Table 6. Detailed information of engineered bacteria with genome-integrated fucosyllactose metabolic pathway

[0082]

[0083] Example 4: Modular pathway engineering to optimize de novo lactose synthesis

[0084] Gene assembly was performed using a plasmid expression system, and expression vectors with varying copy numbers were constructed to increase the expression levels of key upstream and downstream genes. Three expression vectors of varying strengths (pCDFDuet-1, pETDuet-1, and pRSFDuet-1) were optimized. Key genes, including pgi, pgm, and GalU from the de novo synthesis pathway, were divided into upstream modules, while GalE and NmlgtB were divided into downstream modules. Ultimately, a multi-copy plasmid system encompassing the complete lactose metabolic pathway was generated. Expression vectors with different module combinations were screened, and the lactose production performance of these combinations was compared.

[0085] Table 7. Detailed information of engineered bacteria with different plasmid combinations

[0086]

[0087] Fermentation experiments revealed that the optimal plasmid for lactose production using BP12 as the host strain was pET-GalE-NmlgtB. Compared to the antibiotic-free strain, the engineered strain containing the plasmid did not significantly increase yield. On the contrary, the addition of the plasmid reduced the strain's biomass and increased its metabolic burden. The yield of the host strain expressing a single plasmid was slightly higher than that of the dual-plasmid strain. The optimal strain, BP20, achieved a maximum lactose yield of 7.89 g / L after 48 hours of fermentation, a 14.8% increase compared to the antibiotic-free strain.

[0088] Although the present invention has been disclosed above in terms of preferred embodiments, 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

1. A genetically engineered bacterium, characterized in that: The engineered bacteria are β -galactosidase gene lacZ, UDP-glucose lipid transporter transferase gene wcaJ, GDP-mannose mannosyl hydrolase gene nudD, 6-phosphofructokinase-1 gene pfkA, Protease gene lon The host strain was the one that knocked out the glucose-specific transporter EⅡABC. Glc EⅡABC Glc Sugar efflux transporter gene integrated into the component encoding gene locus SetA and glucose transporter genes Glf ; Knockout of the glucokinase gene Glk , and in G lk Integrated at the site β -1,4-galactosyltransferase gene; UDP-glucose-6-dehydrogenase gene was knocked out ugd , and in ugd UDP-glucose-4-epimerase gene was integrated into the GalE ; The EⅡABC Glc The component encoding gene is crr and ptsG ; The glucose transporter gene Glf The nucleotide sequence of the sugar efflux transporter gene is shown in SEQ ID NO.

1. SetA The nucleotide sequence is shown in SEQ ID NO.2; described β -1,4-galactosyltransferase is a nucleotide sequence as shown in SEQ ID NO.4 NmlgtB , the nucleotide sequence is shown in SEQ ID NO.3 AaGalT Or the nucleotide sequence is shown in SEQ ID NO.5 PmGalT ; The UDP-glucose-4-epimerase gene GalE The Gene ID is 945354; The host is Escherichia coli.

2. The genetically engineered bacterium according to claim 1, characterized in that Knockout of the ubiquinone-dependent pyruvate dehydrogenase gene poxB , knocked out the phosphate acetyltransferase and acetate kinase gene clusters pta - ackA, Knockout of the formate lyase gene pfL , knocked out the D-lactate dehydrogenase gene ldhA and the repressor gene responsible for lactose manipulation lacI .

3. The genetically engineered bacterium according to claim 2, characterized in that Overexpression of genes using pRSFDuet-1, pETDuet-1, or pCDFDuet-1 vectors pgi 、 pgm 、 GalU 、 GalE as well as NmlgtB; The gene GalE The Gene ID is 945354; The gene NmlgtB The nucleotide sequence is shown in SEQ ID NO.

4.

4. Use of the genetically engineered bacteria according to any one of claims 1 to 3 in synthesizing lactose and its derivative products; the derivative products are human milk oligosaccharides, galacto-oligosaccharides, lactofructo-oligosaccharides, lactulose, lactulose, lactulose, lactitol, lactobionic acid, and tagatose.

5. The use according to claim 4, characterized in that The seed liquid of the genetically engineered bacteria is added to the fermentation medium, and glucose with a final concentration of 8 g / L is added at the beginning of the fermentation. The culture is carried out at 35-40° C. and 150-250 rpm for 45-55 hours.

6. The use according to claim 5, characterized in that The seed liquid of the engineered bacteria is obtained by picking a single colony in LB liquid culture medium and culturing in a shake flask at 35-40° C. and 150-250 rpm for 12-14 hours.

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