Recombinant bacterium for high-yield production of lactose-N-fucopentasaccharide 1 and application of recombinant bacterium

Abstract

The invention relates to a recombinant bacterium for high yield of lactose-N-fucopentasaccharide 1 and application of the recombinant bacterium, and belongs to the technical field of fermentation. According to the method disclosed by the invention, UTP regenerated protein is recruited by utilizing the specificity of the phase separation protein PodJ, and a highly-ordered micro-region structure is formed in a cell, so that efficient in-situ regeneration of UTP is realized. According to the strategy, the substrate supply of key enzyme in the synthetic route of the lactosyl-N-fucopentasaccharide 1 is obviously enhanced, and the synthetic efficiency of the saccharide is effectively improved. On the basis, a synthetic route of the lactose-N-fucopentasaccharide 1 is further optimized, a recombinant strain for high yield of the lactose-N-fucopentasaccharide 1 is successfully constructed, the strain is subjected to system optimization in genetic background and metabolic pathways, efficient concentration and cyclic utilization of cofactors can be realized in a subcellular level micro interval, and the yield of the lactose-N-fucopentasaccharide 1 is increased. Dependence of exogenous cofactor addition is greatly reduced, the overall economical efficiency and stability of the system are improved, and the method has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a recombinant strain that produces high levels of lactose-N-fucopentose 1 and its applications. Background Technology

[0002] Lactose-N-fucopentose 1 is one of the core functional components of oligosaccharides in human breast milk, hailed as "the first prebiotic of life." It is not only a crucial guardian of infant gut health but also a highly promising raw material in the research and development of modern functional foods, high-end infant formula, and immunomodulatory drugs. Compared to the drawbacks of chemical methods, such as high equipment requirements, low product yield, and significant pollution, the microbial fermentation method for synthesizing lactose-N-fucopentose 1 is environmentally friendly and holds significant strategic importance for optimizing my country's food ingredient industry.

[0003] Currently, in recent years, metabolic engineering techniques have enabled the synthesis of lactose-N-fucopentose 1 using renewable resources as substrates through microbial cell factories. Related research mainly focuses on three aspects: metabolic pathway design and construction, expression optimization, and protein engineering modification. Efficient synthesis of lactose-N-fucopentose 1 can be achieved by knocking out and expressing related enzymes in the lactose-N-fucopentose 1 metabolic pathway; yield can be further increased by controlling copy number and screening promoters; and the yield of lactose-N-fucopentose 1 can be increased by mutating related enzymes to enhance their activity. However, these methods suffer from low modification efficiency and require the addition of large amounts of exogenous cofactors, resulting in low synthesis efficiency of lactose-N-fucopentose 1. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of an efficient method for synthesizing lactose-N-fucopentose 1 in the prior art.

[0005] To address the aforementioned technical challenges, this invention utilizes the phase-separating protein PodJ to specifically recruit UTP regeneration proteins, forming highly ordered microdomain structures within the cell, thereby achieving efficient in-situ regeneration of UTPs. This strategy significantly enhances the substrate supply for key enzymes in the lactose-N-fucopentose 1 synthesis pathway, effectively improving the synthesis efficiency of this sugar. Building upon this, the invention further optimizes the lactose-N-fucopentose 1 synthesis pathway, successfully constructing a high-yield recombinant strain of lactose-N-fucopentose 1. This strain has undergone systematic optimization in both its genetic background and metabolic pathways, enabling efficient concentration and recycling of cofactors within subcellular microdomains. This greatly reduces dependence on exogenous cofactor addition, improves the overall economy and stability of the system, and demonstrates promising application prospects.

[0006] The first objective of this invention is to provide a recombinant bacterium that produces high levels of lactose-N-fucopentose 1, wherein the recombinant bacterium is *Escherichia coli* (…). Escherichia coli The starting strain was heterologously expressed, and the following proteins were isolated: PodJ, UTP regeneration protein, β-1,3-N-acetylglucosamine transferase LgtA, β-1,3-galactosyltransferase WbgO, phosphogannatase ManB, mannose-1-phosphogguanosyltransferase ManC, GDP-mannose 4,6-dehydratase Gmd, GDP-L-fucose synthase WcaG, and fucosyltransferase FutC.

[0007] Furthermore, the phase-separating protein PodJ is expressed under the regulation of RBS as shown in SEQ ID NO.1-3.

[0008] Furthermore, the gene encoding the phase-separating protein PodJ is fused with a gene encoding a first interacting short peptide, and the gene encoding the UTP regeneration protein is fused with a gene encoding a first interacting short peptide, and the first and second interacting short peptides can specifically bind.

[0009] Furthermore, the combination of the first interacting short peptide and the second interacting short peptide is selected from one or more of the following: short peptide RIAD and short peptide RIDD, short peptide CC-Di-A and short peptide CC-Di-B, short peptide SZ1 and short peptide SZ2.

[0010] Furthermore, the nucleotide sequence of the UTP regenerating protein is as shown in PrS (SEQ ID NO.4) or NdK (SEQ ID NO.5).

[0011] Further, the nucleotide sequence of the β-1,3-N-acetylglucosamine transferase LgtA is shown in SEQ ID NO.6, the nucleotide sequence of the β-1,3-galactosyltransferase WbgO is shown in SEQ ID NO.7, the nucleotide sequence of the phosphogmannose mutase ManB is shown in SEQ ID NO.8, the nucleotide sequence of the mannose-1-phosphogguanosyltransferase ManC is shown in SEQ ID NO.9, the nucleotide sequence of the GDP-mannose 4,6-dehydratase Gmd is shown in SEQ ID NO.10, the nucleotide sequence of the GDP-L-fucose synthase WcaG is shown in SEQ ID NO.11, the nucleotide sequence of the fucosyltransferase FutC is shown in SEQ ID NO.12, and the nucleotide sequence of the phase-separating protein PodJ is shown in SEQ ID NO.15.

[0012] Furthermore, the sequence of RIAD is shown in SEQ ID NO.13, and the sequence of RIDD is shown in SEQ ID NO.14.

[0013] Furthermore, the recombinant bacteria also had the UDP-acetylglucosamine epiisomerase wecB gene, β-galactosidase lacZ gene, and glucosamine-6-phosphate deaminase nagB gene knocked out.

[0014] A second objective of this invention is to provide a microbial agent comprising the aforementioned recombinant bacteria.

[0015] A third objective of this invention is to provide the application of the above-mentioned recombinant bacteria or the above-mentioned microbial agents in the production of lactose-N-fucopentose 1.

[0016] A fourth objective of the present invention is to provide a method for producing lactose-N-fucopentose 1, wherein the above-mentioned recombinant bacteria or the above-mentioned microbial agent are inoculated into a fermentation medium for fermentation culture.

[0017] Furthermore, the fermentation culture temperature is 30-38℃.

[0018] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0019] This invention utilizes the phase-separating protein PodJ to recruit UTP regeneration proteins, thereby enhancing the synthesis efficiency of lactose-N-fucopentose 1 and successfully constructing a high-yield recombinant strain of lactose-N-fucopentose 1. The method is simple in design and can concentrate cofactor regeneration within micro-intervals. The recombinant strain of this invention achieves a yield of 2.15 g / L of lactose-N-fucopentose 1 at the shake-flask level, with a production intensity of 0.03 g / L / h, demonstrating good potential for industrial application. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0021] Figure 1 This is a diagram showing the construction and characterization of the phase-separating protein PodJ;

[0022] Figure 2 This is a diagram illustrating the construction and validation of a UTP regeneration system mediated by the phase-separating protein PodJ.

[0023] Figure 3 This is a diagram illustrating the synthetic pathway of lactose-N-fucopentose 1;

[0024] Figure 4 This is a graph showing the change in lactose-N-fucopentose 1 content in the shake flask;

[0025] Figure 5 This is a graph showing the changes in lactose-N-fucopentose 1 content among different strains. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0027] The culture medium formulations involved in the following examples are as follows:

[0028] Seed culture medium: LB medium, containing 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

[0029] Fermentation medium: The composition includes standard fermentation medium (DM medium, 1 L): 4.0 g / L ammonium phosphate, 1.4 g / L magnesium sulfate heptahydrate, 13.5 g / L potassium dihydrogen phosphate, 4.5 mg / L vitamin B1, 1.7 g / L citric acid, 10 mL / L trace metal element solution (10 g / L ferric chloride, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate, pH 6.8); with the appropriate concentration of glucose added (30 g / L glycerol for shake-flask fermentation). Glycerol was autoclaved separately, and before being added to the fermentation medium, the pH was adjusted to 7 with KOH and sterilized through a 0.22 µm membrane.

[0030] The methods for determining the content of lactose-N-fucopentose 1 involved in the following examples are as follows:

[0031] Fermentation sample preparation: Take the fermentation broth sample, centrifuge at 12000 rpm for 5 min, take the supernatant or dilute the supernatant, filter it through a 0.22 μm aqueous membrane to remove impurities, and then perform quantitative analysis by HPLC.

[0032] Determination of lactose-N-fucopentose 1 content: The sample was analyzed using a Shimadzu high performance liquid chromatograph and differential detector with a FinoM Carbohydrate Analysis (Rezex ROA-organic acid H+ (8%)) column. The mobile phase was 0.005M H2SO4, filtered through a 0.22 μm filter membrane, degassed by sonication for 30 min, with a flow rate of 0.6 mL / min and a column temperature of 65℃. The injection volume was 10 μL.

[0033] The strain information involved in the following examples is shown in Table 1.

[0034] Table 1. Strain information and yield of lactose-N-fucopentose 1

[0035]

[0036] Example 1: Construction and characterization of the phase-separating protein PodJ

[0037] Using the pRSF engineered plasmid as a template, green fluorescent protein (GFP) was fused with the phase-separating protein PodJ synthesized by Anhui General Biotechnology Co., Ltd. The GFP fragment was amplified using primers KZ-GFP-POPZ-S1 and KZ-GFP(POPZ)-A, and the PodJ fragment was amplified using primers KZ-POPZ-S and KZ-GFP-POPZ-A1. The two fragments were then fused via homologous arms to obtain the GFP-PodJ fusion fragment. The GFP-PodJ fusion protein was further digested with Nde1 enzyme in the pRSF engineered plasmid. Using Novovitek-mediated recombinant synthesis, the GFP-PodJ fusion protein was inserted into the digested pRSF plasmid to obtain the pRSF-GFP-PodJ plasmid. This plasmid was then transformed into *E. coli* BL21 competent cells to obtain the GFP-PodJ strain. Fluorescence changes were observed under a fluorescence microscope after inoculation and transformation. Figure 1 The results showed that after induction at 30℃, distinct micro-regions could be formed intracellularly after 12 hours, mainly concentrated at the cellular poles. This indicates that the plasmid pRSF-GFP-PodJ has a phase separation function, forming effective droplet micro-regions intracellularly.

[0038] Table 2. Some primers and their sequences used in Example 1

[0039]

[0040] Example 2: Construction and validation of a phase-separation protein PodJ-mediated UTP regeneration system

[0041] The GFP protein was removed from plasmid pRSF-GFP-PodJ by whole plasmid PCR, and the RIDD fragment was added based on KZ-RIDD-S and KZ-RIDD-A primers to obtain plasmid pRSF-RIDD-PodJ. Then, the UTP regeneration proteins PrS and NdK from E. coli K12 were fused with RIAD. Based on primers KZ-PrS-RIAD-S and KZ-PrS-RIAD-A, KZ-NdK-RIAD-S and KZ-NdK-RIAD-A, the RIAD fragments were fused to the C-terminus of the UTP regeneration proteins PrS and NdK, respectively, to obtain the PrS-RIAD fragment and the NdK-RIAD fragment. These were then combined with the pRSF-RIDD-PodJ engineered plasmid digested with Xho1 enzyme. Through Norvitic recombinant recombination, the PrS-RIAD fragment and the NdK-RIAD fragment were inserted into the pRSF-RIDD-PodJ engineered plasmid to obtain the pRSF-RIDD-PodJ-PrS-RIAD and pRSF-RIDD-PodJ-NdK-RIAD plasmids.

[0042] Following the same method, the UTP regenerated proteins PrS and NdK were inserted into the pRSF engineered plasmid via homologous recombination. The NdK fragment was amplified using KZ-ndk-s and KZ-ndk-a primers, and the PrS fragment was amplified using KZ-prs-s and KZ-prs-a primers. Homologous recombination was then performed to obtain pRSF-PrS and pRSF-NdK plasmids, which were then introduced into E. coli BL21 to obtain PrS and NdK bacteria, respectively.

[0043] PrS and NdK bacteria were inoculated into LB medium and induced at 30℃ for 24 h at 200 rpm. UTP concentration was measured, and the results showed that the UTP concentrations of PrS and NdK bacteria were significantly higher than those of the control strain (wild type). E. coli The expression of the UTP regeneration protein was 1.5 times and 1.3 times that of the control strain (wild-type Escherichia coli BL21), indicating that the expression of the UTP regeneration protein is beneficial to improving UTP regeneration. Furthermore, the pRSF-RIDD-PodJ-PrS-RIAD and pRSF-RIDD-PodJ-NdK-RIAD plasmids were transformed into E. coli BL21, respectively, to obtain PrS-PodJ and NdK-PodJ bacteria. These strains were inoculated into LB medium, induced at 30℃, and cultured at 200 rpm for 24 h. The UTP concentration was detected, and the results showed that the UTP concentration of PrS-PodJ and NdK-PodJ bacteria was 1.9 times and 1.6 times that of the control strain (wild-type Escherichia coli BL21), respectively, indicating that phase separation recruits the UTP regeneration protein, which is beneficial to improving UTP regeneration efficiency.

[0044] Table 3 Primers and their sequences used in Example 2

[0045]

[0046] Example 3: Determination of lactose-N-fucopentose 1 content in shake flasks

[0047] According to such Figure 3 The de novo synthesis pathway of lactose-N-fucopentose 1 is shown. Using the pACYC engineered plasmid as a template, the vector was linearized by NdeI digestion and gel recovery. The LgtA fragment was amplified using KZ-lgtA-S and KZ-lgtA-A primers, and the WbgO fragment was amplified using KZ-wbgO-S and KZ-wbgO-A primers. The LgtA protein derived from Neisseria meningitidis and the WbgO protein derived from Escherichia coli O55:H7 were fused to obtain the LgtA-WbgO protein. This LgtA-WbgO protein was then inserted into the pACYC engineered plasmid via homologous recombination, yielding the pACYC-LgtA-WbgO plasmid. Furthermore, using a similar approach, the FutC protein from Helicobacter pylori was inserted into the pACYC-LgtA-WbgO plasmid to obtain the pACYC-LgtA-WbgO-FutC plasmid. Additionally, using the pETduet engineered plasmid as a template, the vector was linearized by Nde1 digestion and gel recovery. Based on KZ-manCB-S and KZ-manCB-A primers, the ManC-ManB gene cluster from Escherichia coli K12 was amplified and homologously recombinated with the Nde1-digested pETduet engineered plasmid, integrating the ManC-ManB gene cluster into the pETduet engineered plasmid to obtain the pETduet-ManC-ManB plasmid. Following the same method, the GmD-WcaG gene cluster derived from Escherichia coli K12 was amplified using primers KZ-gmD-wcaG-S and KZ-gmD-wcaG-A, and homologously recombined with the pETduet-ManC-ManB plasmid digested with Xho1 to obtain the pETduet-ManC-ManB-GmD-WcaG plasmid.

[0048] The obtained recombinant plasmids pRSF-RIDD-PodJ-PrS-RIAD and pRSF-RIDD-PodJ-NdK-RIAD were introduced into competent Escherichia coli BL(21)ΔwecBΔlacZΔnagB cells (from a previously constructed Escherichia coli BL(21)ΔwecBΔlacZΔnagB cell (derived from Pathway Reinforcement and Chassis Optimization of Escherichia coli BL21 for Enhancing Lacto-N-tetraose Production, Appl BiochemBiotechnol, 2025, 197: 7450–7463.. DOI: 10.1007 / s12010-025-05397-9)) to obtain LP and LN bacteria. In addition, the control bacteria were *Escherichia coli* BL(21)ΔwecBΔlacZΔnagB containing the plasmids pACYC-LgtA-WbgO-FutC and pETduet-ManC-ManB-GmD-WcaG. The LP, LN, and control bacteria were cultured in shake flasks in DM medium containing glycerol, and the content of lactose-N-fucopentose 1 in the fermentation broth was identified. The results are as follows: Figure 4 As shown, with prolonged culture time, the yields of lactose-N-fucopentose 1 from recombinant strains LP and LN were 1.8 g / L and 1.51 g / L, respectively, while the control strain only reached 1.1 g / L. The recombinant strain LP showed a 19.2% and 63.6% increase in lactose-N-fucopentose 1 yield compared to LN and the control strain, respectively, indicating that the recombinant strain LP can effectively increase the yield of lactose-N-fucopentose 1.

[0049] To increase the yield of lactose-N-fucopentose 1, based on strain LP, the expression of PodJ and PrS proteins was enhanced by RBS1-3 sequences (RBS1 relative intensity 1.0, RBS2 relative intensity 0.65, RBS3 relative intensity 0.25). Based on primers KZ-RBS1-S and KZ-RBS1-A, KZ-RBS2-S and KZ-RBS2-A, and KZ-RBS3-S and KZ-RBS3-A, plasmids pRSF-RBS1-RIDD-PodJ-PrS-RIAD, pRSF-RBS2-RIDD-PodJ-PrS-RIAD, and pRSF-RBS3-RIDD-PodJ-PrS-RIAD were constructed. The pRSF-RIDD-PodJ-PrS-RIAD plasmid of strain LP was replaced to obtain strains LP-1, LP-2, and LP-3. Fermentation experiments showed that the lactose-N-fucopentose 1 yields of strains LP-1, LP-2, and LP-3 reached 1.58 g / L, 2.15 g / L, and 1.95 g / L, respectively. Among them, the lactose-N-fucopentose 1 yield of strain LP-2 was 95.5% higher than that of the control strain, indicating that the optimized cofactor regulation system is feasible. Figure 5 ).

[0050] Table 4 RBS sequence

[0051]

[0052] Table 5 Primers involved in Example 3

[0053]

[0054] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant bacterium that produces high levels of lactose-N-fucopentose 1, characterized in that, The recombinant bacteria is Escherichia coli (Escherichia coli) Escherichia coli The starting strain was heterologously expressed, and the following proteins were isolated: PodJ, UTP regeneration protein, β-1,3-N-acetylglucosamine transferase LgtA, β-1,3-galactosyltransferase WbgO, phosphogannatase ManB, mannose-1-phosphogguanosyltransferase ManC, GDP-mannose 4,6-dehydratase Gmd, GDP-L-fucose synthase WcaG, and fucosyltransferase FutC.

2. The recombinant bacteria according to claim 1, characterized in that, The phase-separating protein PodJ is expressed under the regulation of RBS as shown in SEQ ID NO.1-3.

3. The recombinant bacteria according to claim 1, characterized in that, The gene encoding the phase-separating protein PodJ is fused with a first interaction short peptide gene, and the gene encoding the UTP regeneration protein is fused with a second interaction short peptide gene, and the first and second interaction short peptides can specifically bind.

4. The recombinant bacteria according to claim 3, characterized in that, The combination of the first interacting short peptide and the second interacting short peptide is selected from one or more of the following: short peptide RIAD and short peptide RIDD, short peptide CC-Di-A and short peptide CC-Di-B, short peptide SZ1 and short peptide SZ2.

5. The recombinant bacteria according to claim 1, characterized in that, The nucleotide sequence of the UTP regenerated protein is shown in SEQ ID NO.4 or 5.

6. The recombinant bacteria according to claim 1, characterized in that, The nucleotide sequence of the β-1,3-N-acetylglucosamine transferase LgtA is shown in SEQ ID NO.6, the nucleotide sequence of the β-1,3-galactosyltransferase WbgO is shown in SEQ ID NO.7, the nucleotide sequence of the phosphogmannose mutase ManB is shown in SEQ ID NO.8, the nucleotide sequence of the mannose-1-phosphogguanosyltransferase ManC is shown in SEQ ID NO.9, the nucleotide sequence of the GDP-mannose 4,6-dehydratase Gmd is shown in SEQ ID NO.10, the nucleotide sequence of the GDP-L-fucose synthase WcaG is shown in SEQ ID NO.11, the nucleotide sequence of the fucosyltransferase FutC is shown in SEQ ID NO.12, and the nucleotide sequence of the phase-separating protein PodJ is shown in SEQ ID NO.

15.

7. A microbial agent comprising the recombinant bacteria according to any one of claims 1-6.

8. The use of the recombinant bacteria according to any one of claims 1-6 or the microbial agent according to claim 7 in the production of lactose-N-fucopentose 1.

9. A method for producing lactose-N-fucopentose 1, characterized in that, The recombinant bacteria according to any one of claims 1-6 or the microbial agent according to claim 7 are inoculated into a fermentation medium for fermentation culture.

10. The method according to claim 9, characterized in that, The fermentation culture temperature is 30-38℃.

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