Recombinant strain for efficiently producing fucosylated lactose and application thereof

Abstract

The application discloses a recombinant strain for efficiently producing fucosyllactose and application thereof, and belongs to the field of synthetic biology and metabolic engineering. The recombinant Escherichia coli BGM-18 is successfully constructed by replacing the promoters of manC-manB and gmd-wcaG on the chromosome, combining with transforming plasmids pET-M32(S98R / D340E)-H9-H9-(R T72 )mdfa(C139A / R190L) and pRSF-CBGW, with BZWNDLMEI as a starting strain. Under the condition of a shaking flask, the yield of 3-FL produced by the recombinant strain is significantly increased from 3.72 g / L to 7.65 g / L, with an increase of 105.6%. Under the condition of a 3L fermenter, the yield of 3-FL produced by the strain BGM-18 reaches 65.19 g / L, which shows a significant industrial production potential.

Description

Technical Field

[0001] This invention relates to a recombinant strain that efficiently produces fucoidan and its applications, belonging to the fields of synthetic biology and metabolic engineering. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk. They play a vital role in helping infants maintain healthy gastrointestinal function by preventing pathogens from adhering to the intestinal epithelium and promoting intestinal maturation. Analysis of infant fecal composition has shown that breast milk oligosaccharides are not only absorbed by infants but also act as prebiotics, promoting the healthy development of the gut microbiota. Among the oligosaccharides in breast milk, seven are recognized as Generally Recognized As Safe (GRAS) by the U.S. Food and Drug Administration (FDA), including 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2′-FL), demonstrating their safety and potential health benefits. Studies have also shown that fucoidan can effectively prevent colitis and help maintain gut health by enhancing intestinal barrier function and stimulating anti-inflammatory responses. Furthermore, research on the gut microbiota has found a close symbiotic relationship between fucoidan and beneficial bacteria in the human gut, indicating its important role in cultivating a healthy, balanced gut microbiota, which is crucial for health and disease prevention.

[0003] With the rapid development of metabolic engineering and synthetic biology, microbial systems, especially genetically modified *E. coli*, have become ideal platforms for fucosyllactose (FL) production. Modifying chassis strains, optimizing metabolic pathways to enhance substrate supply, and increasing the expression level and catalytic activity of rate-limiting enzymes such as fucosyltransferases (FucTs) are currently the core strategies for improving FL yield. However, existing technologies still face several bottlenecks. One prominent issue is the intracellular accumulation of products and intermediate metabolites. Studies have shown that some human milk oligosaccharides (FL) are difficult to expel from cells via passive diffusion. FL accumulation in cells easily triggers metabolic feedback inhibition, increased osmotic pressure, and intensified energy consumption, thereby inhibiting cell growth and reducing fermentation efficiency. Therefore, constructing an efficient product efflux system is considered an important way to improve FL yield. In recent years, research has begun to explore introducing efflux engineering into *E. coli* to improve FL secretion efficiency, for example, by overexpressing natural efflux protein genes to promote product export. However, most existing studies focus on the direct overexpression of natural efflux proteins, while systematic research on the regulation of efflux protein expression levels, substrate recognition characteristics, and engineering modification is relatively limited. Furthermore, multidrug efflux transporters typically possess broad-spectrum substrate characteristics, and their wild-type forms may not be suitable for the efficient and stable efflux of oligosaccharides such as FL under high-load fermentation conditions. On the other hand, GDP-L-fucose, as a key donor for fucosyl lactose synthesis, is considered a crucial factor affecting FL yield in terms of its synthesis throughput. *E. coli* BL21(DE3) naturally possesses an endogenous metabolic pathway for de novo GDP-L-fucose synthesis, but key genes such as manB, manC, gmd, and wcaG are regulated by natural promoters, resulting in limited transcriptional levels and difficulty in continuously providing sufficient precursors for high-throughput synthesis reactions. Although constitutive promoter substitution (such as P...) J23119 This has been successfully applied to enhance the expression of manC-manB and gmd-wcaG to improve 2′-FL production, but existing studies mainly rely on single P. J23119 Promoter enhancement did not consider the differential transcriptional requirements of individual gene cluster modules such as manC-manB and gmd-wcaG. Furthermore, in the FL biosynthesis system, a systematic framework integrating the transcriptional regulation of the GDP-L-fucose biosynthesis module with downstream output processes has not yet been established. Specifically, how engineered promoters with different transcriptional drive strengths affect precursor supply, flux allocation, and their synergistic relationship with efflux processes remains to be systematically explored.

[0004] Therefore, a comprehensive strategy is urgently needed that can enhance the supply capacity of GDP-L-fucose on the one hand, and improve the efflux efficiency of cells for fucoidosyl lactose, especially 3-FL and other products, thereby alleviating the inhibitory effect of product accumulation and providing a new technical approach for constructing microbial cell factories with high fucoidosyl lactose production. Summary of the Invention

[0005] Current fucoidan biosynthesis technologies still face several limitations. The primary issue is the insufficient supply of GDP-L-fucose, a precursor required for fucoidan synthesis. In particular, key genes in the synthesis pathway, such as gmd-wcaG and manC-manB, are regulated by the host's natural promoters, making it impossible to meet the demands of high-throughput synthesis. Secondly, the accumulation of intracellular fucoidan, especially 3-FL, is difficult to efflux in a timely manner, leading to increased cellular metabolic burden and even feedback inhibition, further reducing yield. To overcome the aforementioned technical shortcomings, this invention provides a recombinant *Escherichia coli* strain capable of efficiently synthesizing fucoidan and its construction method. This recombinant strain achieves efficient fucoidan synthesis through multi-strategy combination optimization.

[0006] The first technical solution provided by this invention is a multidrug efflux pump protein (MdfA) mutant, which is based on the multidrug efflux pump protein parent with the amino acid sequence shown in SEQ ID NO.3, and subjected to any of the following mutations: (1) The serine (S) at position 184 is mutated to alanine (A); (2) The isoleucine (I) at position 244 is mutated to valine (V); (3) The asparagine (N) at position 387 is mutated to alanine (A); (4) Cysteine ​​(C) at position 139 is mutated to alanine (A); (5) Arginine (R) at position 190 is mutated to leucine (L); (6) The serine (S) at position 184 is mutated to alanine (A), and the asparagine (N) at position 387 is mutated to alanine (A); (7) The isoleucine at position 244 (I) is mutated to valine (V), and the asparagine at position 387 (N) is mutated to alanine (A); (8) The isoleucine at position 244 (I) is mutated to valine (V), and the arginine at position 190 (R) is mutated to leucine (L); (9) The asparagine (N) at position 387 is mutated to alanine (A), and the arginine (R) at position 190 is mutated to leucine (L); (10) Cysteine ​​(C) at position 139 is mutated to alanine (A), and arginine (R) at position 190 is mutated to leucine (L).

[0007] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0008] The third technical solution provided by the present invention is to express the mutant described in the first technical solution, or to use a host cell containing the gene described in the second technical solution.

[0009] The fourth technical solution provided by the present invention is a genetically engineered bacterium that produces fucoidosyl lactose, wherein the genetically engineered bacterium is based on Escherichia coli BZWNDLMEI (B20) as the starting strain and overexpresses the efflux pump gene mdfa.

[0010] In some embodiments, the efflux pump gene mdfa is (a) or (b): (a) The gene encoding the parent of the multidrug efflux pump protein with the amino acid sequence shown in SEQ ID NO.3; (b) The gene described in the second technical solution.

[0011] In some embodiments, the efflux pump gene mdfa is expressed by an expression regulatory element containing a ribosome binding site (RBS), wherein the RBS is selected from any one of the commonly used RBS sequences and their variants T71 and T72 in the BBa_B0034, BBa_B0029, BBa_B0031, BBa_B0035, or T7 expression systems.

[0012] In some embodiments, the nucleotide sequences of BBa_B0034, BBa_B0029, BBa_B0031, BBa_B0035, T71, and T72 are shown in SEQ ID NO.13~18, respectively.

[0013] In some embodiments, Escherichia coli BZWNDLMEI (B20) is an Escherichia coli BZWNDLMEI chassis cell that overexpresses phosphogmannose mutase manB, mannose-1-phosphogguanine transferase manC, GDP-mannose-6-dehydrogenase gmd, GDP-fucose synthase wcaG, and α-1,3-fucosyltransferase mutants.

[0014] In some embodiments, the amino acid sequence of the α-1,3-fucosyltransferase mutant is as shown in SEQ ID NO.2, M32(S98R / D340E)-H9.

[0015] In some embodiments, in the aforementioned chassis cells, manB, manC, gmd, and wcaG are overexpressed using the pRSFDuet-1 vector, and one copy of wild-type α-1,3-fucosyltransferase is expressed in the MCSⅠ region using the pETDuet-1 vector, or two copies of the α-1,3-fucosyltransferase mutant are expressed in both the MCSⅠ and MCSⅡ regions.

[0016] In some implementations, strong promoters are used to replace the promoters of the manC-manB and gmd-wcaG encoding genes in the E. coli genome.

[0017] In some embodiments, T7, constitutive promoters J23100, J23105, and J23119 are used alone or in combination to replace the promoters encoding the manC-manB and gmd-wcaG genes in the Escherichia coli genome, wherein the nucleotide sequences of promoters T7, J23100, J23105, and J23119 are SEQ ID NO. 19~22.

[0018] In some embodiments, the *E. coli* BZWNDLMEI is *E. coli* BL21(D3) β-galactosidase gene lacZ, UDP-glucose lipotransferase gene wcaJ, GDP-mannose-mannosyl hydrolase gene nudD, protease gene lon, mannitol-1-phosphate dehydrogenase gene mtlD, collamylate biosynthesis glycosyltransferase gene WcaE, and collamylate biosynthesis fucosyltransferase gene WcaI.

[0019] The fifth technical solution provided by the present invention is a method for producing 3-fucosyl lactose, using glycerol as a carbon source and inducing fermentation to produce 3-fucosyl lactose by inoculating the genetically engineered bacteria described in the third technical solution into a fermentation system.

[0020] In some embodiments, the fermentation system contains glycerol at a concentration of 10–30 g / L.

[0021] In some embodiments, the fermentation system contains 20-30 g / L glycerol, 1-2 g / L casein amino acids, 3-5 g / L potassium dihydrogen phosphate, 5-10 g / L disodium hydrogen phosphate, 0.5-1 g / L ammonium chloride, 0.1-1 g / L sodium chloride, 1-2 g / L magnesium sulfate heptahydrate, 5-10 g / L yeast extract, 3-5 g / L betaine, 5-10 mg / L thiamine hydrochloride, and 1-2 mL / L trace metal solution.

[0022] In some embodiments, the trace metal solution contains 20-30 g / L ferric chloride hexahydrate, 2-3 g / L calcium chloride dihydrate, 2-3 g / L zinc chloride, 2-3 g / L sodium molybdate dihydrate, 2-3 g / L copper sulfate pentahydrate, 2-3 g / L manganese sulfate monohydrate, and 0.5-1 g / L boric acid.

[0023] In some embodiments, the trace metal solution in the fermenter contains 10-15 g / L ferrous sulfate heptahydrate, 2-3 g / L calcium chloride dihydrate, 2-3 g / L zinc sulfate heptahydrate, 2-3 g / L copper sulfate pentahydrate, 0.5-1 g / L manganese sulfate monohydrate, 0.5-1 g / L sodium molybdate dihydrate, and 0.5-1 g / L boric acid.

[0024] In some embodiments, the genetically engineered bacteria are cultured in a fermentation system to OD0.05. 600 The concentration of the solution was 0.6 ± 0.1. IPTG was added to a final concentration of 0.2–0.5 mM and lactose to a final concentration of 5–10 g / L. The mixture was then incubated at 20–30 °C for at least 70 h.

[0025] In some embodiments, the genetically engineered bacteria are cultured in a fermentation system to OD0.05. 600 Add IPTG to a final concentration of 0.1–0.5 mM and lactose to a final concentration of 5–15 g / L at a concentration of 20±3. Induce culture at 20–30 °C, maintain dissolved oxygen in the fermentation system at 20–50%, pH at 6.5–7.0, and culture at 20–30 °C for no less than 80 h.

[0026] In some implementations, glycerol is added after the initial glycerol in the reaction system is consumed, so that the concentration of glycerol can maintain the cell growth and metabolism.

[0027] In some implementations, lactose is added after the initial lactose is consumed to maintain its concentration at 10 g / L.

[0028] The sixth technical solution provided by the present invention is the application of the mutant described in the first technical solution, or the gene described in the second technical solution, or the genetically engineered bacteria described in the fourth technical solution in the production of 3-fucosylated lactose and / or 2'-fucosylated lactose.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, based on promoter substitution of key chromosomal genes and engineering modification of efflux pump genes, successfully improved the production efficiency of 3-FL and significantly optimized extracellular secretion. Specific beneficial effects are as follows: (1) This invention uses BZWNDLMEI as the starting strain and replaces the promoters of the "key enzymes" manC-manB and gmd-wcaG on the chromosome, combined with the transformation plasmid pET-M32(S98R / D340E)-H9-H9-(R T72 Recombinant Escherichia coli BGM-18 was successfully constructed using mdfa (C139A / R190L) and pRSF-CBGW. Under shake-flask conditions, the 3-FL yield of the recombinant strain significantly increased from the initial 3.72 g / L to 7.65 g / L, an increase of 105.6%. Under 3L fermenter conditions, the 3-FL yield of strain BGM-18 reached 60.57 g / L, demonstrating significant potential for industrial production.

[0030] (2) By optimizing the induction time in a 3L fermenter, the present invention further improved the yield of 3-FL. Finally, when induction was performed 11 h after inoculation, the yield of 3-FL was increased to 65.19 g / L.

[0031] This invention provides a new technical solution for the efficient microbial synthesis of fucoidan lactose. Attached Figure Description

[0032] Figure 1 This is a metabolic pathway diagram of fucoidan.

[0033] Figures 2-4 To investigate fed-batch fermentation of BGM-18 in a 3L fermenter at different induction times (6h, 11h and 21h after inoculation). Detailed Implementation

[0034] refer to Figures 1-4 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] 1. The plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, and DNA gel recovery kits used in the following examples are all commercially available products. Specific experimental procedures were performed according to the instructions for each kit. The embodiments of this invention are not limited to those described herein; other experimental operations and process parameters not specifically mentioned should be performed using conventional technical methods.

[0036] 2. Carrier pRSFDuet 1 and pETDuet 1. Purchased from Addgene.

[0037] 3. The sequencing of DNA products and plasmids was carried out by Tianlin Biotechnology Co., Ltd. (Wuxi) and Suzhou Genewise Biotechnology Co., Ltd.

[0038] 4. Preparation of competent Escherichia coli cells: Shanghai Sangon Biotech Co., Ltd. reagent kit.

[0039] 5. LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0040] 6. LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0041] 7. Fermentation medium: Glycerol 30.0 g / L, casein amino acids 2.0 g / L, yeast extract 10.0 g / L, potassium dihydrogen phosphate 3.0 g / L, disodium hydrogen phosphate 6.8 g / L, ammonium chloride 1.0 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 1.4 g / L, thiamine hydrochloride 10.0 mg / L, trace metal solution 1.0 mL / L (ferric chloride hexahydrate 25.0 g / L, calcium chloride dihydrate 2.3 g / L, zinc chloride 2.6 g / L, sodium molybdate dihydrate 2.6 g / L, copper sulfate pentahydrate 2.0 g / L, manganese sulfate monohydrate 2.5 g / L, boric acid 0.7 g / L), pH 6.8.

[0042] Fermentation medium: glycerol 20.0 g / L, casein amino acids 2.0 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 3.0 g / L, disodium hydrogen phosphate 6.8 g / L, ammonium chloride 1.0 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 1.4 g / L, thiamine hydrochloride 10.0 mg / L, trace metal solution 10 mL / L (ferrous sulfate heptahydrate 12 g / L, calcium chloride dihydrate 2.3 g / L, zinc sulfate heptahydrate 2.6 g / L, copper sulfate pentahydrate 2.5 g / L, manganese sulfate monohydrate 0.5 g / L, sodium molybdate dihydrate 0.5 g / L, boric acid 0.5 g / L), pH 6.8.

[0043] 8. Shake-flask fermentation conditions for the strains in the following examples: A single colony of the engineered strain was inoculated into LB liquid medium and cultured in a shake flask at 37 °C and 200 rpm for 12 h to obtain a seed culture; the seed culture was then inoculated into 25 mL of fermentation medium at an inoculation rate of 3% (v / v) and cultured in a shake flask at 37 °C and 200 rpm until OD was reached. 600 The concentration was 0.6; IPTG was added to a final concentration of 0.4 mM, and lactose was added to a final concentration of 6 g / L. The mixture was induced and cultured at 28 °C and 200 rpm for 72 h. The concentration was determined by HPLC. FL content.

[0044] 9. Determination method of 3-FL HPLC determination: The total 3-FL titer was determined by boiling 1 mL of fermentation broth at 100 °C for 10 min, centrifuging at 10625 × g for 10 min, and filtering the supernatant through a 0.22 μm membrane. The amount of 3-FL produced and the consumption of lactose and glycerol were detected by HPLC. For the determination of extracellular 3-FL titer, the fermentation broth was centrifuged at 10625 × g for 10 min, and the supernatant was filtered through a 0.22 μm membrane for HPLC detection. HPLC detection conditions: differential refractive index detector; Sugar-Pak I column, 6.5 mm × 300 mm (Waters Corporation, USA); column temperature: 85 °C; mobile phase: 50 mg / L EDTA Ca. 2+ The aqueous solution was injected at a flow rate of 0.4 mL / min, with an injection volume of 10 μL. Example 1: Screening of efflux pump genes (1) Construction of starting plasmid The plasmid pET-M32(S98R / D340E)-H9-H9 was constructed according to the method in patent CN120536328A, and the plasmid pRSF-CBGW was constructed according to the method in patent CN114480240A, with pET-M32 in patent CN114480240A used as the initial control. Furthermore, plasmid pET-M32(S98R / D340E)-H9-H9 was constructed by linking the nucleotide sequences shown in SEQ ID NO.1 to the MCSⅠ and MCSⅡ regions of the vector pETDuet-1, respectively; plasmid pRSF-CBGW was constructed by linking the manC, manB, gmd, and wcaG genes to the vector pRSFDuet-1. The plasmids pET-M32 and pET-M32(S98R / D340E)-H9-H9 were transformed with plasmid pRSF-CBGW into strain BZWNDLMEI (BL21(DE3)ΔlacZΔwcaJΔnudDΔlonΔmtlDΔwcaEΔwcaI, the construction method of which is referred to patent CN120536328A), respectively, to obtain strains B1 and B2 (B2 is strain B20 in patent CN120536328A), as shown in Table 3. After 72 h of shake-flask fermentation, the 3-FL yield of strain B1 was 3.72 g / L, and the 3-FL yield of B2 was 6.16 g / L.

[0045] (2) Construction and screening of efflux pump genes: To achieve efficient expression of the efflux pump genes mdfa, AcrD, MdtK, and EmrE (encoding protein amino acid sequences SEQ ID NO. 3, 5, 7, and 9) in *E. coli*, we cloned nucleotide fragments of these genes into the MCSII region of pETDuet-1 and expressed them under the same T7 promoter. The nucleotide sequences of the efflux pumps are shown in SEQ ID NO. 4, 6, 8, and 10. According to literature reports, the constitutive RBS of BBa_B0034 (nucleotide sequence shown in SEQ ID NO. 13) showed good expression performance; therefore, this RBS was selected for preliminary screening to obtain the plasmid pET-M32(S98R / D340E)-H9-H9-(R BBa_B0034 )AcrD, pET-M32(S98R / D340E)-H9-H9-(R BBa_B0034 )MdtK、pET-M32(S98R / D340E)-H9-H9-(R BBa_B0034 )EmrE, pET-M32(S98R / D340E)-H9-H9-(R BBa_B0034 )mdfa. pET-M32(S98R / D340E)-H9-H9-( R BBa_B0034 Taking MDFA as an example, upstream and downstream primers (R) are used. BBa_B0034 The target gene was amplified using mdfa_F / R to obtain the target gene fragment. The vector, pET-M32(S98R / D340E)-H9-H9, was successfully constructed as a template, and the backbone sequence was amplified using primers Mdfa_V_F / R. Primer sequences are shown in Table 1. According to In... Fusion cloning technology was used, and a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) was employed to insert the target gene fragment into the fucosyltransferase gene in the MCSII region. Positive clones were then screened and sequenced. The above plasmid and plasmid pRSF-CBGW were used to transform strain BZWNDLMEI to obtain strains B3-B6. As shown in Table 3, after 72 h of shake-flask fermentation, mdfa was the efflux pump gene that optimally improved 3-FL secretion efficiency, reaching 6.39 g / L, a 71.8% increase compared to B1. Furthermore, the extracellular fraction increased to 90%.

[0046] (3) Optimization of expression regulatory elements (RBS) of the mdfa gene In addition to BBa_B0034, this invention selects five other RBS sequences of different intensities, including BBa_B0029, BBa_B0031, BBa_B0035, or any one of the commonly used RBS sequences and their variants T71 and T72 in the T7 expression system (different RBS sequences are shown in Table 2) to regulate the protein translation intensity of the target gene. Taking the replacement of the original RBS T72 in pETDuet-1 as an example, primers (R... T72 ) mdfa_F / R with pET-M32(S98R / D340E)-H9-H9-(R BBa_B0034 PCR amplification was performed using the mdfa plasmid as a template. The product was digested with the restriction endonuclease Dpn I to remove excess circular template. The linear fragment was then recombined using the Clon Express® II One Step Cloning Kit (Vazyme) and subsequently transformed into [a specific enzyme / technology]. E. coli DH5α was cultured overnight, and single clones from the plates were picked and transferred to 5 mL of LB medium for plasmid extraction and sequencing. The successfully constructed plasmid was named pET-M32(S98R / D340E)-H9-H9-(R T72 mdfa. Other RBS replacement methods are the same as R. T72 The above plasmids and plasmid pRSF-CBGW were used to transform strain BZWNDLMEI to obtain strains B7-B11, as shown in Table 3. After 72 h of shake-flask fermentation, strain B8, expressed by T72, could further enhance the extracellular secretion efficiency of 3-FL, with a titer of 6.61 g / L, which was 77.7% higher than that of strain B1, and the extracellular proportion increased to 97%.

[0047] (4) Effect of co-expression of OmpF and mdfa on 3-FL secretion To verify whether outer membrane permeability can synergistically promote 3-FL efflux with mdfa, the ompF coding sequence (SEQ ID NO. 12) was cloned into the MCS I region of the pET-duet vector, located after the fucosyltransferase gene. Using different RBSs (SEQ ID NO. 13, 15) on the same plasmid as the transferase to improve translation initiation efficiency, the plasmid pET-M32(S98R / D340E)-H9-H9-(R T72 ) mdfa-(R T72 ) ompF、pET-M32(S98R / D340E)-H9-H9-(R T72 ) mdfa-(R BBa_B0034) ompF. pET-M32(S98R / D340E)-H9-H9-(R T72 ) mdfa-(R T72 Taking ompF as an example, upstream and downstream primers (R) are used. T72 The target gene was amplified using ompF_F / R to obtain the target gene fragment. The vector was then successfully constructed using PET-M32(S98R / D340E)-H9-H9-(R T72 Using MDFA as a template, the backbone sequence was amplified using primers ompF_V_F / R. Primer sequences are shown in Table 1. According to In... Fusion cloning technology was used, and a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) was employed to insert the target gene fragment into the fucosyltransferase gene in the MCSⅠ region. Positive clones were then screened and sequenced. The above plasmid and plasmid pRSF-CBGW were used to transform strain BZWNDLMEI to obtain strains B12-B13. As shown in Table 3, after 72 hours of shake-flask fermentation, although the extracellular partition ratio did not decrease significantly, the overall 3-FL titer decreased significantly. This may be attributed to the overexpression of outer membrane channel proteins disrupting cell membrane homeostasis, increasing non-specific permeability, thereby leading to impaired cell growth and increased metabolic burden, ultimately reducing the overall 3-FL synthesis capacity.

[0048] Table 1. Primer sequences for constructing the efflux pump gene

[0049] Table 2. RBS sequence

[0050] Table 3. Plasmids and genotypes contained in the host

[0051] Example 2: Mutation of the efflux pump gene (1) Point mutations were performed on the nucleotide sequence of mdfa. Single or multiple site-directed saturation mutations were performed on residues 12, 14, 33, 39, 47, 65, 123, 135, 138, 139, 164, 181, 184, 187, 190, 198, 224, 241, 244, 246, 248, 251, 274, 290, 292, 297, 322, 333, 352, 374, 378, 386, 387, and 389. Taking S184A as an example, the upstream and downstream primers of S184A_F / R (Table 4) were used, with pET-M32(S98R / D340E)-H9-H9-(R T72 PCR amplification was performed using the mdfa plasmid as a template, and the amplification product was subjected to restriction endonuclease... DpnThe PCR product was digested with enzyme I to remove excess circular template. The PCR product was then transformed. E. coli DH5α competent cells were plated with ampicillin and cultured overnight. Single colonies on the plates were transferred to 5 mL of LB medium and cultured for 12 h. Plasmids were extracted and sequenced. The successfully constructed mutant plasmid was named M32(S98R / D340E)-H9-H9-mdfa(S184A). Other mutation sites (Y47F, I244V, T248S, L274V, M290L, I297L, I322L, N374L, V386L, N387A, I389L, G12S, Q14A, G123S, E135D, V138D, C139A, W164L, R190A, R198S, Q241L, I246L, Q251G, N33L, G39L, G65L, A181L, G187L, G224V, Q241I, G292L, G333L, A352F, G378W) and the construction method of combined mutations are the same as S184A.

[0052] Table 4. Primer sequences for mutation sites

[0053] (2) pRSF-CBGW and M32(S98R / D340E)-H9-H9-mdfa combinations with different mutation sites were transformed into strain BZWNDLMEI. Finally, single or combined mutation sites (S184A, I244V, N387A, C139A, R190L, S184A / N387A, I244V / N387A, I244V / R190L, N387A / R190L, C139A / R190L) with significantly increased yields were obtained. Under shake-flask conditions, the highest yield of 3-FL produced by strain B23 expressing mutant C139A / R190L reached 7.31 g / L after 72 h of fermentation, which was 97% higher than that of strain B1 (3.72 g / L) (Table 5).

[0054] Table 5. Plasmids and genotypes contained in the host

[0055] Example 3: Promoter substitution of "key enzymes" on chromosomes To enhance the synthesis of the precursor GDP-L-fucose, this embodiment employs a promoter engineering strategy to moderately adjust the expression levels of key enzymes in the GDP-L-fucose module. Specifically, the strong promoter T7 and constitutive promoters J23100, J23105, and J23119 replace the promoters of manB-manC and gmd-wcaG on the *E. coli* genome. The specific steps are as follows (primer sequences are shown in Table 6): (1) Taking the replacement of the original promoter of the manC gene with the T7 promoter as an example, the specific target crRNA (23bp) of the target gene was found by searching http: / / chopchop.cbu.uib.no / and manC-crRNA1 was used. F / manC-crRNA1 R and manC crRNA2 F / manC-crRNA2 Using upstream and downstream primers R, PCR amplification was performed with pcrEG plasmid as a template. The amplification product was digested with restriction endonuclease Dpn I to remove excess circular pcrEG plasmid. The amplification product was then transformed... E. coli DH5α competent cells were used to extract plasmids, which were then identified using primers and crRNA-PF sequencing. The successfully constructed knockout plasmid was named pcrEG. manC.

[0056] (2) Using the genome of Escherichia coli BL21(DE3)ΔlacZΔwcaJΔnudDΔlonΔmtldΔwcaEΔwcaI as a template, the upstream homologous arm primer T7-manC was used. US F / T7-manC US R and downstream homologous arm primer T7-manC DS F / T7-manC DS R amplifies homologous arm sequence fragments separately, and the products are purified and recovered using SOE. The PCR method uses primers T7-manC US F / T7-manC DS R connects the two fragments to obtain a gene homology repair template.

[0057] (3) Take the helper plasmid pEcCpf1 (100-200 ng) and transfer it to the Escherichia coli strain BL21(DE3)ΔlacZΔwcaJΔnudDΔlonΔmtldΔwcaEΔwcaI to be edited into chemocompetent cells. After placing them on ice for 5 min, thaw the competent cells. Take 10 μL of plasmid and add it to 100 μL of competent cells. Mix gently and place on ice for 30 min. Heat shock in a 42 °C water bath for 90 s and immediately place on ice to cool for 5 min. Add 900 μL of LB medium (without antibiotics) and shake at 37 °C for 1 h (200-250 rpm). Finally, centrifuge the bacterial solution at 5000 rpm for 5 min and discard 900 μL of supernatant. Spread the bacterial cells on LB plates containing kanamycin and incubate overnight at 37 °C.

[0058] (4) Pick Escherichia coli BL21(DE3)ΔlacZΔwcaJΔnudDΔlonΔmtldΔwcaEΔwcaI / pEcCpf1 and single colonies of E. coli and incubate them in LB medium at 37 °C. Kanamycin should be added to maintain the stability of pEcCpf1, and it should be added at the beginning or OD. 600 When the concentration reaches 0.3, 10 mM arabinose is added to induce the expression of λ-red recombinase. The prepared electrocompetent cells can be temporarily stored at -80 °C.

[0059] (5) Electroporate 500 ng of the target plasmid pcrEG-manC with manC-specific target crRNA (23 bp) constructed in step (1) and 1000 ng of the homology repair template constructed in step (2) to the E. coli BL21(DE3)ΔlacZΔwcaJΔnudDΔlonΔmtldΔwcaEΔwcaI prepared in step (4) into competent cells, plate them on LB plates (kanamycin and spectinomycin), and incubate at 37 ℃ for 16 days. After 24 hours, colony PCR was performed on the single colonies that grew on the plate to verify the colonies, screen for positive transformants, and perform gene sequencing.

[0060] (6) The pcrEG-manC and pEcCpf1 plasmids were eliminated from the verified single colonies. One confirmed colony was selected and incubated overnight at 37 °C in 2 mL of LB medium containing 50 μg / mL kanamycin and L-rhamnose (10 mM). The next day, the culture was streaked on a plate containing 50 μg / mL kanamycin and incubated overnight at 37 °C. The culture was then harvested and screened on LB plates containing 50 µg / mL kanamycin and 100 μg / mL spectinomycin. It was confirmed that the single colony sensitive to spectinomycin but insensitive to kanamycin eliminated the crRNA expression plasmid.

[0061] (7) To eliminate pEcCpf1, single colonies of the successfully eliminated pcrEG-manC plasmid were further cultured in 2 mL of LB medium containing 5 g / L glucose at 37 °C for 12 hours. 16 hours. Then, the bacterial solution was streaked onto plates containing 5 g / L glucose and 10 g / L sucrose and incubated overnight at 37 °C. Single colonies were spotted onto LB medium containing kanamycin. If no single colony grew, it indicated that the pEcCpf1 plasmid was successfully eliminated. The constructed gene-deleted strains without pcrEG-manC and pEcCpf1 plasmids were then... Store at 80 °C for later use.

[0062] (8) The operation of replacing the original promoter of gene manC-manB with constitutive promoters J23100, J23105, J23119 and replacing the original promoter of gene gmd-wcaG with T7, constitutive promoters J23100, J23105, J23119 is the same as the above experiment of replacing manC with T7 promoter.

[0063] Table 6. Gene knockout primers

[0064] (9) After replacing the key enzyme promoters in the first and second rounds, strains such as BG1 were finally obtained from BZWNDLMEI (BL21(DE3)ΔlacZΔwcaJΔnudDΔlonΔmtlDΔwcaEΔwcaI) strains (see Table 7).

[0065] Table 7. Detailed information on strains

[0066] Example 4: Fed-batch fermentation of promoter-replaced strains The pET-M32(S98R / D340E)-H9-H9-(R from Example 1) T72 ) mdfa and pET-M32(S98R / D340E)-H9-H9-(R in Example 2 T72 ) mdfa (I244V / R190L), pET-M32(S98R / D340E)-H9-H9-(R T72 )mdfa (C139A / R190L) is converted to a form containing pRSF. Recombinant strains were obtained from the promoter replacement strains in Table 7 of CBGW (see Table 8).

[0067] (1) Inoculate single colonies of the above recombinant strains into 5 mL LB medium and culture overnight at 37 °C and 200 r / min on a shaker.

[0068] (2) The recombinant strain seed culture was inoculated into 25 mL of fermentation medium at an inoculation rate of 3.0% (v / v) and cultured at 37 °C and 200 r / min. 600 Up to 0.6 Add IPTG to a final concentration of 0.4 mM, along with 6 g / L lactose, and induce culture for 72 h.

[0069] (3) The fermentation products were detected by HPLC after 72 h of fermentation. The fermentation results are shown in Table 7. Under shake-flask conditions, the optimal strain BGM-18 produced product 3 after 72 h of fermentation. The yield of FL reached a maximum of 7.65 g / L, which was twice that of the original strain B1.

[0070] Table 8. Detailed information on strains

[0071] Example 5: Feed-in batch fermentation of strain BGM-18 in a 3L fermenter to produce 3-fucosyl lactose In order to prepare high-yield 3 FL uses recombinant strain BGM-18 for high-density fed-batch fermentation in a 3L fermenter.

[0072] Fermentation conditions: fermentation medium, inoculum size 10% (v / v), pre-induction culture temperature 37 °C, waiting for OD... 600At 20 (6 h post-inoculation), IPTG was added to induce protein expression, resulting in a concentration of 0.4 mmol / L in the fermentation system. The initial lactose concentration was 10 g / L, and the induction fermentation temperature was 25 °C. Glycerol was used as the primary carbon source for cell growth, and lactose was intermittently added as a substrate throughout the fermentation process, achieving continuous biotransformation of 3-FL. Ammonia was used to maintain a constant pH of 6.80 in the fermentation tank throughout the process. To maintain cell growth and 3-FL synthesis, 800 g / L of glycerol (containing 10 g / L of MgSO4·7H2O) was added after the initial glycerol was consumed to replenish the carbon source. pH feedback was used to maintain the glycerol concentration in the fermentation system at a low level (0–5 g / L). After the initial lactose was consumed, 250 g / L of lactose was manually added to maintain a final concentration of approximately 10 ± 2 g / L in the fermentation system. If the lactose concentration decreased during fermentation, lactose was added again until the end of fermentation. Here, the concentration of 3-FL was monitored in real time using HPLC during fermentation. When the 3-FL concentration showed no significant change between two consecutive samples, and the cell growth tended to stabilize (i.e., OD...), the 3-FL concentration was considered stable. 600 When the value stops rising, the fermentation process is considered to be nearing its end, and fermentation is terminated at this point. During fermentation, the system is cascaded and controlled to maintain the dissolved oxygen level in the tank at 30% ± 5% by adjusting the rotation speed, aeration rate, and oxygen supply.

[0073] Sampling was performed regularly throughout the fermentation process, and cell OD was measured. 600 1 mL of fermentation broth was boiled for 10 min to completely lyse the cells, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm membrane. HPLC was used to detect the 3... The amount of FL produced and the amount of lactose and glycerol consumed ( Figure 2 The results showed that BGM-18 fermented for a total of 119 hours after the fermentation was completed, producing 3 products. The concentration of FL can reach 60.57 g / L ( Figure 2 The lactose conversion rate and productivity were 0.94 mol3-FL / mol and 0.51 g / L / h, respectively.

[0074] Example 6: Optimization of induction conditions for the production of 3-fucosylated lactose in a fed-batch fermentation tank in a 3L fermenter To optimize the fermentation yield of 3-FL, recombinant strain BGM-18 was used for scale-up culture in a 3 L fermenter. The effects of induction at different times after fermentation inoculation (11 h and 21 h after inoculation) on the fermentation process were investigated. Figure 3-4Figure 3 shows the fermentation process and 3-FL production during induction at 11 h post-inoculation, and Figure 4 shows the fermentation process and 3-FL production during induction at 21 h post-inoculation. The results showed that when induction was initiated at 11 h post-inoculation, the 3-FL production further increased to 65.19 g / L after 102 h of fermentation (Figure 3), with lactose conversion rate and productivity of 0.90 mol 3-FL / mol and 0.64 g / L / h, respectively. In contrast, when induction was initiated at 21 h post-inoculation, although the cell biomass (OD200) decreased, the 3-FL production still increased significantly. 600 The yield of 3-FL was relatively high, but only reached 34.42 g / L after 119 h of fermentation (Figure 4). These results indicate that induction time has a significant impact on the synthesis efficiency of 3-FL. When induction is performed later, cellular metabolic resources may be more inclined to support cell growth, leading to a mismatch between target protein expression and metabolic flux establishment. This makes it difficult for precursor supply, energy allocation, and product synthesis to form an effective synergy in the early stages of fermentation, thus limiting the continuous accumulation of 3-FL.

[0075] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A mutant of a multidrug efflux pump protein, characterized in that, The mutant is defined by an amino acid sequence such as SEQ ID. Based on the parental multidrug efflux pump protein shown in NO.3, perform any of the following mutations: (1) mutate serine at position 184 to alanine; (2) mutate isoleucine at position 244 to valine; (3) mutate asparagine at position 387 to alanine; (4) mutate cysteine ​​at position 139 to alanine; (5) mutate arginine at position 190 to leucine; (6) mutate serine at position 184 to alanine and asparagine at position 387 to alanine; (7) mutate isoleucine at position 244 to valine and asparagine at position 387 to alanine; (8) mutate isoleucine at position 244 to valine and arginine at position 190 to leucine; (9) mutate asparagine at position 387 to alanine and arginine at position 190 to leucine; (10) mutate cysteine ​​at position 139 to alanine and arginine at position 190 to leucine.

2. The gene encoding the mutant of claim 1.

3. A host cell expressing the mutant of claim 1, or containing the gene of claim 2.

4. A genetically engineered bacterium that produces fucoidosyllactose, characterized in that, The genetically engineered bacteria used *Escherichia coli* BZWNDLMEI (B20) as the starting strain and overexpressed the efflux pump gene mdfa, wherein the efflux pump gene mdfa is (a) or (b): (a) The gene encoding the parent of the multidrug efflux pump protein with the amino acid sequence shown in SEQ ID NO.3; (b) The gene as described in claim 2.

5. The genetically engineered bacterium according to claim 4, characterized in that, The efflux pump gene mdfa is expressed by an expression regulatory element containing a ribosome binding site (RBS), wherein the RBS is selected from any one of BBa_B0034, BBa_B0029, BBa_B0031, BBa_B0035, T71, and T72, and the nucleotide sequences of BBa_B0034, BBa_B0029, BBa_B0031, BBa_B0035, T71, and T72 are shown in SEQ ID NO. 13-18, respectively.

6. The genetically engineered bacterium according to claim 4, characterized in that, Escherichia coli BZWNDLMEI (B20) is a mutant of Escherichia coli BZWNDLMEI using a chassis cell to overexpress phosphomannose mutase manB, mannose-1-phosguanine succinate transferase manC, GDP-mannose-6-dehydrogenase gmd, GDP-fucose synthase wcaG, and α-1,3-fucosyltransferase. The amino acid sequence of the α-1,3-fucosyltransferase mutant is shown in SEQ ID NO.

2.

7. The genetically engineered bacterium according to claim 6, characterized in that promoters T7, J23100, J23105, and J23119 are used alone or in combination to replace the promoters encoding the manC-manB and gmd-wcaG genes in the Escherichia coli genome, wherein the nucleotide sequences of promoters T7, J23100, J23105, and J23119 are SEQ ID NO.19~22.

8. A method for producing 3-fucosyllactose, characterized in that, Using glycerol as a carbon source, 3-fucosylated lactose is produced by inducing fermentation in a fermentation system using the genetically engineered bacteria described in any one of claims 4 to 7.

9. The method according to claim 8, characterized in that, The genetically engineered bacteria were cultured in the fermentation system until OD reached. 600 The concentration was 0.6 ± 0.

1. IPTG was added to a final concentration of 0.2–0.5 mM and lactose to a final concentration of 5–10 g / L. The mixture was incubated at 20–30 °C for at least 70 h. Alternatively, the genetically engineered bacteria can be cultured in a fermentation system until OD200. 600 Add IPTG to a final concentration of 0.1–0.5 mM and lactose to a final concentration of 5–15 g / L at a concentration of 20±3. Induce culture at 20–30 °C, maintain dissolved oxygen in the fermentation system at 20–50%, pH at 6.5–7.0, and culture at 20–30 °C for no less than 80 h.

10. The use of the mutant of claim 1, or the gene of claim 2, or the genetically engineered bacteria of any one of claims 4 to 7 in the production of 3-fucosylated lactose and / or 2'-fucosylated lactose.

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