An engineered strain of Bacillus licheniformis that efficiently produces 2′-fucosylated lactose using mannan.

Abstract

This invention discloses an engineered strain of *Bacillus licheniformis* capable of efficiently producing 2′-fucosylated lactose using mannan, belonging to the fields of synthetic biology and microbial metabolic engineering. This invention utilizes the food-safe microorganism *Bacillus licheniformis* as the host cell, expressing mannose-specific phosphotransferase (E. coli), phosphomannose mutase (E. coli), mannose-1-phosphate-guanylate transferase (E. coli), GDP-mannose-4,6-dehydratase (E. coli), GDP-L-fucose synthase (E. coli), and fucosylated transferase (Helicobacterpylori) using a promoter with quorum sensing effects. The recombinant strain possesses the ability to synthesize 2′-FL using inexpensive mannans, such as konjac flour or corn flour. This novel synthetic route offers significant advantages in production efficiency and cost compared to synthetic routes based on refined carbon sources such as glucose and glycerol.

Description

Technical Field

[0001] This invention provides an engineered strain of Bacillus licheniformis that efficiently produces 2′-fucosylated lactose using mannan, belonging to the fields of synthetic biology and microbial metabolic engineering. Background Technology

[0002] The composition of the gut microbiome influences human health. Breast milk is the most important food for infants, and its unique components, such as oligosaccharides, antibodies, and vitamins, affect health by shaping the composition of the infant's gut microbiome. Human milk oligosaccharides (HMOs) are the main soluble solids in breast milk, with 2'-fucosylated lactose (2'-FL) being the most abundant HMO, accounting for 30% of the total. It is composed of L-fucose, D-galactose, and D-glucose. The bioactivity of 2'-FL has received considerable attention in recent years, and researchers generally believe it is an important factor in ensuring healthy infant development. In-depth research has found that although infants have difficulty digesting 2'-FL, it plays a crucial role in the development of the immune system, such as regulating the gut and inhibiting pathogen infection. Currently, 2'-FL has been approved as a prebiotic for use in infant formula in the United States, Europe, and other countries.

[0003] In recent years, researchers have begun to widely use microbial fermentation to produce 2'-FL, primarily using model microorganisms such as *E. coli* and yeast. *E. coli* itself contains multiple genes involved in the 2'-FL synthesis pathway, such as mannose mutase and mannose-1-phosphate-guanylate transferase, making it easy to construct a complete synthetic pathway, hence its widespread research. However, *E. coli* is not a food safety microorganism, and its products pose problems when used in food. Since *Bacillus subtilis* is a food safety-grade strain, researchers have utilized this host to construct and ferment the 2'-FL synthesis pathway. The main problems currently are: low expression levels of heterologous genes in *Bacillus subtilis*, resulting in low synthesis efficiency; and, in existing 2'-FL fermentation methods, *Bacillus subtilis* primarily uses glucose and glycerol as culture media, while using more widely available and inexpensive corn flour as a raw material yields poor results. Furthermore, both *E. coli* and *Bacillus subtilis* face significant risks of bacteriophage infection in large-scale production.

[0004] Bacillus licheniformis is a widely used host for the production of food enzymes and important nutritional chemicals, and its products are certified by the FDA as "generally regarded as safe" (GRAS). In industrial fermentation, Bacillus licheniformis can ferment a wider variety of raw materials and exhibits stronger resistance to bacteriophages. Existing 2'-FL synthesis pathways in Bacillus licheniformis strains all use glucose, fructose, or glycerol as starting substrates. Bacillus licheniformis can secrete mannanase, thereby breaking down extracellular mannans for growth and metabolism. Since mannose is an important intermediate compound in the 2'-FL synthesis pathway, directly breaking down extracellular mannans and transporting mannose into the cell to participate in 2'-FL synthesis offers significant advantages in terms of production efficiency and cost compared to existing synthetic routes. Summary of the Invention

[0005] The purpose of this invention is to provide a promoter and recombinant plasmid for efficient expression of target genes in Bacillus licheniformis, and to construct and apply them.

[0006] This invention utilizes metabolic engineering to enable *Bacillus licheniformis* to efficiently transport mannose into cells using konjac flour, an inexpensive raw material, and synthesize GDP-fucose using GDP-mannose as a precursor. A high-yield 2'-FL *Bacillus licheniformis* strain was then obtained through heterologous expression of fucosyltransferase. The artificial pathway for *Bacillus licheniformis* to synthesize 2'-FL uses mannan substrates (konjac flour, guar gum, etc.) and corn flour as carbon sources. Mannanose and corn flour enzymes are used to hydrolyze the mannose and glucose. Glucose serves as the cell's energy source. After phosphorylation via the phosphotransferase system, mannose is converted to mannose hexaphosphate using heterologous expression of phosphomannose mutase. This is then converted to GDP-L-fucose using heterologous expression of mannose-1-phosphate-guanylate transferase, GDP-mannose-4,6-dehydratase, and GDP-L-fucose synthase, respectively. Finally, fucosyltransferase is used to produce 2'-FL. Figure 1 ).

[0007] This invention provides a high-yield 2'-FL Bacillus licheniformis engineered strain, wherein the engineered Bacillus licheniformis strain overexpresses mannose-specific phosphotransferase (N1), phosphomannose mutase (N2), mannose-1-phosphate-guanylate transferase (N3), GDP-mannose-4,6-dehydratase (N4), GDP-L-fucose synthase (N5), and fucosyltransferase (N6).

[0008] In one embodiment, the Bacillus licheniformis engineered strain overexpresses an expression cassette containing a mannose-specific phosphotransferase, an expression cassette encoding a phosphomannose mutase gene, an expression cassette encoding a mannose-1-phosphate-guanylate transferase gene, an expression cassette encoding a GDP-mannose-4,6-dehydratase gene, an expression cassette encoding a GDP-L-fucose synthase gene, and an expression cassette encoding a fucosyltransferase gene.

[0009] In one embodiment, the mannose-specific phosphotransferase is derived from *Escherichia coli*, the phosphomannose mutase is derived from *Escherichia coli*, the mannose-1-phosphate-guanylate transferase is derived from *Escherichia coli*, the GDP-mannose-4,6-dehydratase is derived from *Escherichia coli*, the GDP-L-fucose synthase is derived from *Escherichia coli*, and the fucosyltransferase is derived from *Helicobacter pylori*.

[0010] In one implementation, promoter P is used. lan Gene overexpression is performed, and the gene expression is terminated using the terminator ter.

[0011] In one embodiment, the amino acid sequence of the mannose-specific phosphotransferase is shown in SEQ ID NO. 3; the amino acid sequence of the phosphomannose mutase is shown in SEQ ID NO. 4; the amino acid sequence of the mannose-1-phosphate-guanylate transferase is shown in SEQ ID NO. 5; the amino acid sequence of the GDP-mannose-4,6-dehydratase is shown in SEQ ID NO. 6; the amino acid sequence of the GDP-L-fucose synthase is shown in SEQ ID NO. 7; and the amino acid sequence of the fucosyltransferase is shown in SEQ ID NO. 8.

[0012] In one embodiment, the starting strain of the Bacillus licheniformis engineered strain is Bacillus licheniformis ATCC9945A.

[0013] In one embodiment, the above-mentioned Bacillus licheniformis engineered bacteria are expressed using plasmids pHY and pHT43 as expression vectors.

[0014] In one embodiment, plasmid pHY is used to express mannose-specific phosphotransferase, phosphomannose mutase, and mannose-1-phosphate-guanylate transferase, and plasmid pHT43 is used to express GDP-mannose-4,6-dehydrase, GDP-L-fucose synthase, and fucosyltransferase.

[0015] In one embodiment, the nucleotide sequence of the gene encoding a mannose-specific phosphate transfer transporter is as shown in SEQ ID.

[0016] As shown in NO.9, the nucleotide sequence encoding the phosphomannose mutase gene is shown in SEQ ID NO.10, encoding mannose.

[0017] The nucleotide sequence of the -1-phosphate-guanylate transferase gene is shown in SEQ ID NO.11, the nucleotide sequence of the GDP-mannose-4,6-dehydratase gene is shown in SEQ ID NO.12, the nucleotide sequence of the GDP-L-fucose synthase gene is shown in SEQ ID NO.13, and the nucleotide sequence of the fucosyltransferase gene is shown in SEQ ID NO.14.

[0018] In one implementation, promoter P lan The nucleotide sequence of the terminator is shown in SEQ ID NO.1, and the nucleotide sequence of the terminator is shown in SEQ ID NO.2.

[0019] This invention also provides a method for constructing a Bacillus licheniformis strain for efficient synthesis of 2'-fucosylated lactose, wherein the Bacillus licheniformis contains plasmids pHY and pHT43; the pHY plasmid contains genes encoding a mannose-specific phosphotransferase, a phosphomannose mutase, and a mannose-1-phosphate-guanylate transferase; the pHT43 plasmid contains genes encoding GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase, and a fucosylated transferase.

[0020] In one implementation, promoter P is used. lan Gene overexpression is performed, and the gene expression is terminated using the terminator ter.

[0021] In one embodiment, the nucleotide sequence of the gene encoding a mannose-specific phosphate transfer transporter is as shown in SEQ ID.

[0022] As shown in NO.9, the nucleotide sequence encoding the phosphomannose mutase gene is shown in SEQ ID NO.10, encoding mannose.

[0023] The nucleotide sequence of the -1-phosphate-guanylate transferase gene is shown in SEQ ID NO.11, the nucleotide sequence of the GDP-mannose-4,6-dehydratase gene is shown in SEQ ID NO.12, the nucleotide sequence of the GDP-L-fucose synthase gene is shown in SEQ ID NO.13, and the nucleotide sequence of the fucosyltransferase gene is shown in SEQ ID NO.14.

[0024] In one implementation, promoter P lan The nucleotide sequence of the terminator is shown in SEQ ID NO.1, and the nucleotide sequence of the terminator is shown in SEQ ID NO.2.

[0025] In one implementation, the construction method includes the following steps:

[0026] (1) Using pHY as the starting plasmid, the promoter P, which has a quorum sensing effect, is utilized. lan The mannose-specific phosphate transfer transport protein gene was overexpressed on plasmid pHY, and the recombinant plasmid constructed was transformed into Escherichia coli to obtain plasmid pHY-N1;

[0027] (2) Using pHY-N1 as the starting plasmid, insert a plasmid containing the promoter P. lan The expression cassette of the phosphoglucono-mannose mutase gene and the terminator ter was used to construct a recombinant plasmid, which was then transformed into Escherichia coli to obtain plasmid pHY-N12.

[0028] (3) Using pHY-N12 as the starting plasmid, insert a plasmid containing the promoter P. lan The expression cassette of the mannose-1-phosphate-guanylate transferase gene and the terminator ter was used to construct a recombinant plasmid, which was then transformed into Escherichia coli to obtain plasmid pHY-N123.

[0029] (4) Using pHT43 as the starting plasmid, insert a plasmid containing the promoter P. lan The expression cassette of the GDP-mannose-4,6-dehydrase encoding gene and the terminator ter was used to construct a recombinant plasmid, which was then transformed into Escherichia coli to obtain plasmid pHT-N4.

[0030] (5) Using pHT43-N4 as the starting plasmid, insert a plasmid containing the promoter P. lan The expression cassette of the GDP-L-fucose synthase encoding gene and the terminator ter was used to construct a recombinant plasmid, which was then transformed into Escherichia coli to obtain plasmid pHT-N45.

[0031] (6) Using pHT43-N45 as the starting plasmid, insert a plasmid containing the promoter P. lanThe expression cassette of the fucosyltransferase encoding gene and the terminator ter was constructed, and the recombinant plasmid constructed was transformed into Escherichia coli to obtain plasmid pHT-N456.

[0032] (7) The plasmid pHY-N123 was transformed into Bacillus licheniformis ATCC 9945A to obtain recombinant strain BLH5.

[0033] (8) The plasmid pHT-N456 was transformed into Bacillus licheniformis BLH5 to obtain recombinant bacteria BLH6.

[0034] This invention provides a method for producing 2'-fucosylated lactose, wherein the method utilizes the above-mentioned engineered strain of Bacillus licheniformis to ferment and produce 2'-fucosylated lactose in a fermentation system with mannan as the carbon source.

[0035] In one embodiment, the mannan includes konjac flour and guar gum.

[0036] In one embodiment, the fermentation system also contains corn flour and lactose.

[0037] In one embodiment, the seed culture of the above-mentioned Bacillus licheniformis engineered bacteria is inoculated into the fermentation system, the aeration rate is controlled at 0.5 vvm, the stirring and DO are coupled to control DO at 25-35%, the rotation speed is set to 200-800 rpm, and after fermentation for 10-14 hours, 60-80 g / L of corn flour and 60-80 g / L of konjac flour are added at once.

[0038] In one embodiment, the seed culture is prepared by streaking the above-mentioned Bacillus licheniformis engineered bacteria on a plate, picking a single colony and inoculating it into LB medium, culturing it at 37°C and 250 rpm / min for 18-24 h, taking 2 ml of bacterial culture and transferring it into 100 ml of LB liquid medium, and culturing it at 37°C and 250 rpm for 18-24 h.

[0039] The present invention also provides the application of the above-mentioned Bacillus licheniformis in the synthesis of 2'-fucosylated lactose or products containing 2'-fucosylated lactose.

[0040] Compared with the prior art, the positive and progressive effects of the present invention are as follows:

[0041] This invention uses *Bacillus licheniformis* as the starting strain and leverages its ability to secrete mannanase to enhance mannose transport capabilities, enabling the strain to efficiently acquire mannose directly from the extracellular environment and participate in the 2'-FL synthesis pathway. Furthermore, it employs the promoter P, which exhibits quorum sensing effects. lanIt expresses mannose-specific phosphotransferase, phosphomannose mutase, mannose-1-phosphate-guanylate transferase, GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase, and fucosyltransferase, coordinating cell growth and product synthesis, and improving the yield and production rate of 2'-FL. The yield of 2'-FL can reach 98 g / L, with a maximum OD of 600 The value is 63. Bacillus licheniformis is a food-safe industrial microorganism that uses inexpensive mannan-based raw materials, such as konjac flour, as a carbon source to ferment and produce 2'-FL. Compared with Escherichia coli and Bacillus subtilis, which use refined carbon sources such as glucose or glycerol, the technical solution of this invention has significant advantages in terms of production cost and efficiency. Attached Figure Description

[0042] Figure 1 Design of the 2'-FL synthetic pathway in Bacillus licheniformis

[0043] Figure 2 : Recombinant plasmid structure diagram; A, pHYN123 expression vector, B, pHTN456 expression vector;

[0044] Figure 3 Physical map of recombinant plasmid;

[0045] Figure 4 : chromatographic quantitative analysis of 2'-FL in fermentation supernatant;

[0046] Figure 5 Changes in 2'-FL content in shake flasks over time under different culture temperatures;

[0047] Figure 6 Process curve for the production of 2'-FL by fed-batch fermentation of recombinant bacteria. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0049] (I) Culture medium

[0050] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride; add 20 g / L agar powder to prepare LB solid medium.

[0051] Shake-flask fermentation medium: sucrose 75 g / L, lactose 40 g / L, cottonseed protein 30 g / L, K2HPO4·3H2O 9.12 g / L, KH2PO4 1.36 g / L, (NH4)2HPO4 10 g / L; initial pH 7.5.

[0052] Fermentation medium: cottonseed protein 30 g / L, konjac flour 75 g / L, corn flour 75 g / L, lactose 80 g / L, K2HPO4·3H2O 9.12 g / L, KH2PO4 1.36 g / L, FeCl3 0.5 g / L, (NH4)2HPO4 10 g / L (pH 7.5).

[0053] (II) Extraction and detection of 2'-FL

[0054] The fermentation broth was centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm membrane and analyzed by HPLC. HPLC detection conditions: differential refractive index detector; chromatographic column: Polyamino HILIC 5 μm 250*4.6 mm (Dima Technology, China); column temperature: 40℃; mobile phase: 75% acetonitrile aqueous solution; flow rate: 0.8 mL / min; injection volume: 10 μL.

[0055] (iii) The strains and primers involved in the following examples.

[0056] Table 1. Strains involved in this invention

[0057]

[0058] Table 2 Primer sequences

[0059]

[0060]

[0061] Example 1: Construction of Recombinant Bacillus licheniformis BLH6

[0062] P was generated by overlap extension PCR. lan The promoter (nucleotide sequence shown in SEQ ID NO.1) and the terminator ter (nucleotide sequence shown in SEQ ID NO.2) of the xylose isomerase gene were fused with gene fragments of mannose-specific phosphotransferase (N1), phosphomannose mutase (N2), mannose-1-phosphate-guanylate transferase (N3), GDP-mannose-4,6-dehydratase (N4), GDP-L-fucose synthase (N5), and fucose-syltransferase (N6) to obtain gene expression fragments SEQ ID NO.9-14.

[0063] The gene expression fragment SEQ ID NO.9-14 was amplified using primer pairs P1-P6, thereby introducing restriction enzyme sites at both ends of the gene fragment. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min followed by the next cycle of 94℃ denaturation for 30 s, 54℃ annealing for 30 s, and 72℃ extension for 40 s, for 30 cycles; 72℃ extension for 10 min, followed by incubation at 4℃.

[0064] Expression cassettes containing N1, N2, and N3 were sequentially inserted into the plasmid via homologous recombination using restriction enzyme sites HindIII, BglII, BglII, and SalI (for ligation of the pHY plasmid), resulting in pHY-N123. Figure 2 Expression cassettes containing N4, N5, and N6 were inserted into the plasmid via homologous recombination using the restriction enzyme sites EcoRI, BamHI, SmaI, and SacI (for ligating the pHT43 plasmid), resulting in pHT-N456. Figure 2 One gene was inserted between every two adjacent restriction enzyme sites. The plasmid construction was further verified using 1% agarose gel electrophoresis. Figure 3 ).

[0065] The successfully constructed recombinant plasmid pHY-N123 was introduced into Bacillus licheniformis ATCC 9945A according to the method described in the literature Li, Y.; Jin, K.; Zhang, L.; Ding, Z.; Gu, Z.; Shi, G. Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon. Journal of Agricultural and Food Chemistry 2018, 66, 9456-9464, to obtain recombinant Bacillus licheniformis BLH5.

[0066] The successfully constructed recombinant plasmid pHT-N456 was then introduced into BLH5 using the same method described above to obtain recombinant Bacillus licheniformis BLH6.

[0067] Example 2: Shake-flask fermentation of 2'-FL

[0068] The recombinant BLH6 strain constructed in Example 1 was streaked onto LB agar plates for activation. After incubation at 37°C for 16 h, a single colony was picked and inoculated into 15 mL of LB medium at 37°C and 250 rpm. -1Culture for 16-18 hours to obtain the seed culture. Take 1 mL of the seed culture and transfer it to a 30 mL shake flask containing fermentation medium. Control the initial OD to be 0.5-1 and incubate at 37℃ or 42℃ and 250 rpm. -1 Culture. Samples were taken every 12 hours, and the fermentation broth was incubated at 4℃ and 12000 rpm. -1 Centrifuge for 10 minutes under the specified conditions, and use the supernatant for product detection. Figure 4 The result is as follows: Figure 5 The results show that 1.82 g / L of 2'-FL can be accumulated in the fermentation broth after 36 hours of cultivation at 42℃, and 1.52 g / L of 2'-FL is produced in the fermentation broth after 36 hours at 37℃.

[0069] Example 3: Effects of carbon source type and concentration on recombinant bacterial expression

[0070] The recombinant BLH6 strain constructed in Example 1 was streaked onto LB agar plates for activation. After incubation at 37°C for 16 h, a single colony was picked and inoculated into 15 mL of LB medium at 37°C and 250 rpm. -1 Culture for 16-18 hours to obtain the seed culture. Take 1 mL of the seed culture and transfer it to a 30 mL shake flask containing fermentation medium. Control the initial OD to be 0.5-1 and incubate at 37℃ or 42℃ and 250 rpm. -1 Cultivation. Fermentation for 36 hours, followed by incubation at 4℃ and 12000 rpm. -1 Centrifuge for 10 minutes under the specified conditions, and use the supernatant for product detection.

[0071] The carbon source in the shake-flask fermentation medium was replaced with different concentrations of konjac flour or guar gum (30 g / L, 45 g / L, 60 g / L, 75 g / L, and 90 g / L, respectively) to investigate the effect of different carbon source types and concentrations on the fermentation production of 2'-FL by recombinant Bacillus licheniformis. The results are shown in Table 3. Using 75 g / L konjac flour as the carbon source, the maximum yield of 2'-FL was 11.32 g / L; while using 75 g / L guar gum as the carbon source yielded 5.74 g / L of 2'-FL. This indicates that recombinant Bacillus licheniformis can grow and synthesize the product well in media containing different inexpensive mannan-based raw materials as the main carbon source.

[0072] Table 3. Effects of different carbon source types and concentrations on the fermentation of recombinant bacteria.

[0073]

[0074] Example 4: Production of 2'-FL in a 20L fermenter

[0075] The recombinant BLH6 strain constructed in Example 1 was streaked onto LB agar plates for activation. After incubation at 37°C for 16 h, a single colony was picked and inoculated into 15 mL of LB medium at 37°C and 250 rpm. -1 Cultured for 18-24 hours to obtain primary seed culture, 2 mL of primary seed culture was transferred to 100 mL LB medium and incubated at 37℃ and 250 rpm. -1 After 18-24 hours of cultivation, the culture was used as a secondary seed culture. Three bottles of the secondary seed culture were then inoculated into a 30L fermenter containing 15L of fermentation medium for fermentation.

[0076] Fermentation temperature was 42℃, initial pH 7.5. When the pH dropped to 7.0 during fermentation, 50% ammonia was added to maintain the pH at around 7.0. Aeration was controlled at 0.5 vvm, and stirring was coupled with dissolved oxygen (DO) to maintain DO at around 30%. The maximum stirring speed was set to 800 rpm. After 12 hours of fermentation, 75 g / L corn flour and 75 g / L konjac flour were added in a single batch.

[0077] Results of fed-batch fermentation at the fermenter scale: Figure 6 As shown, after 46 hours of fermentation, the yield of 2'-FL reached 98 g / L, with a maximum OD value of [missing information]. 600 It is 63.

[0078] Table 4 Fermentation Tank Process Parameters

[0079]

[0080] 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. An engineered strain of Bacillus licheniformis that produces 2'-FL, characterized in that, The engineered strain of *Bacillus licheniformis* uses mannan as a carbon source and overexpresses mannose-specific phosphotransferase, phosphomannose mutase, mannose-1-phosphate-guanylate transferase, GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase, and fucosyltransferase. The amino acid sequence of the mannose-specific phosphotransferase is shown in SEQ ID NO. 3; the amino acid sequence of the phosphomannose mutase is shown in SEQ ID NO. 4; the amino acid sequence of the mannose-1-phosphate-guanylate transferase is shown in SEQ ID NO. 5; the nucleotide sequence of the gene encoding the GDP-mannose-4,6-dehydratase is shown in SEQ ID NO. 12; the amino acid sequence of the GDP-L-fucose synthase is shown in SEQ ID NO. 7; and the amino acid sequence of the fucosyltransferase is shown in SEQ ID NO.

8. Using the promoter P with the nucleotide sequence shown in SEQ ID NO.1 lan The genes of the above 6 proteins were overexpressed, and the expression of the genes of the above 6 proteins was terminated using the terminator ter. The starting strain of the Bacillus licheniformis engineered strain is Bacillus licheniformis ATCC 9945A, using plasmids pHY and pHT43 as expression vectors. The pHY plasmid is used to express mannose-specific phosphotransferase, phosphomannose mutase and mannose-1-phosphate-guanylate transferase, and the pHT43 plasmid is used to express GDP-mannose-4,6-dehydrase, GDP-L-fucose synthase and fucosyltransferase.

2. The engineered strain of Bacillus licheniformis according to claim 1, characterized in that, The mannose-specific phosphate transfer transporter protein is derived from Escherichia coli (E. coli) Escherichia coli ), mannose phosphate mutase is derived from Escherichia coli ( Escherichia coli ), Mannose-1-phosphate-guanylate transferase is derived from Escherichia coli ( Escherichia coli GDP-mannose-4,6-dehydrase is derived from Escherichia coli ( Escherichia coli GDP-L-fucose synthase is derived from Escherichia coli ( Escherichia coli ) and fucosyltransferase derived from Helicobacter pylori ( Helicobacter pylori ).

3. A method for constructing Bacillus licheniformis that synthesizes 2'-fucosylated lactose, characterized in that, Starting with Bacillus licheniformis ATCC 9945A, the strain contains plasmids pHY and pHT43. Plasmid pHY contains genes encoding a mannose-specific phosphotransferase, a phosphomannose mutase, and a mannose-1-phosphate-guanylate transferase. Plasmid pHT43 contains genes encoding GDP-mannose-4,6-dehydrase, GDP-L-fucose synthase, and fucosyltransferase. The promoter P, with the nucleotide sequence shown in SEQ ID NO.1, was used. lan The above six genes were overexpressed, and the expression of the above six genes was terminated using the terminator ter. The amino acid sequence of the mannose-specific phosphotransferase is shown in SEQ ID NO.3; the amino acid sequence of the phosphomannose mutase is shown in SEQ ID NO.4; the amino acid sequence of the mannose-1-phosphate-guanylate transferase is shown in SEQ ID NO.5; the nucleotide sequence of the gene encoding GDP-mannose-4,6-dehydratase is shown in SEQ ID NO.12; the amino acid sequence of the GDP-L-fucose synthase is shown in SEQ ID NO.7; and the amino acid sequence of the fucosyltransferase is shown in SEQ ID NO.

8.

4. A method for producing 2'-fucosylated lactose, characterized in that, The method involves using the Bacillus licheniformis engineered strain described in claim 1 or 2 as the fermentation strain to produce 2'-fucosylated lactose in a fermentation system with mannan as the carbon source.

5. The method according to claim 4, characterized in that, The mannan includes konjac powder and guar gum.

6. The method according to claim 4, characterized in that, The fermentation system also contains corn flour and lactose.

7. The use of Bacillus licheniformis as described in claim 1 or 2 in the synthesis of 2'-fucosylated lactose or products containing 2'-fucosylated lactose.

Images