A dynamically regulated strain for high-efficiency production of 2'-fucosyllactose from glucose and application thereof

Abstract

The application discloses a kind of dynamic regulation and control strain for efficiently producing 2'-fucosyllactose by glucose and application thereof, and relates to the technical field of biology.The application provides a kind of dynamic regulation and control strain for efficiently producing 2'-fucosyllactose by glucose, and the dynamic regulation and control strain is obtained by replacing the initial promoter of the gene of host strain with σ²⁸-dependent promoter P gltA fliC The application discloses a kind of dynamic regulation and control strain for efficiently producing 2'-fucosyllactose by glucose and application thereof, and relates to the technical field of biology.The application provides a kind of dynamic regulation and control strain for efficiently producing 2'-fucosyllactose by glucose, and the dynamic regulation and control strain is obtained by replacing the initial promoter of the gene of host strain with σ²⁸-dependent promoter P gltA fliC The application discloses a kind of dynamic regulation and control strain for efficiently producing 2'-fucosyllactose by glucose and application thereof, and relates to the technical field of biology.The application provides a kind of dynamic regulation and control strain for efficiently producing 2'-fucosyllactose by glucose.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a dynamically regulated strain that efficiently produces 2′-fucosylated lactose from glucose and its applications. Background Technology

[0002] Human milk oligosaccharides (HMOs) are unique oligosaccharide components derived from breast milk, crucial for regulating the immune system and providing nutrition to newborns. Based on monosaccharide type and spatial configuration, HMOs are classified into three main categories: neutral fucoidan, neutral non-fucosylanalose, and acidic sialyl lactose. Fucosylanalose, primarily 2′-fucosylanalose (2′-FL), constitutes the largest proportion and exhibits particularly significant bioactivity. Multiple studies have shown that 2′-FL significantly demonstrates positive effects on infant gut function and flora regulation, and therefore has been approved for use as a nutritional additive in infant formula, with its prebiotic benefits widely recognized.

[0003] Most known 2′-FL microbial cell factories use glycerol as a carbon source, achieving 2′-FL biosynthesis through the introduction of exogenous GDP-L-fucose synthesis pathway. As a commonly used fermentation substrate, glycerol has a clear metabolic pathway and bypasses the lactose uptake module, making it the preferred carbon source. However, its large quantity and high cost, coupled with the problem of later-stage production stagnation, make large-scale industrial production difficult. A small number of cell factories choose to add glucose proportionally as a shared carbon source or use glucose as the sole carbon source, but this results in low conversion efficiency, high modification difficulty, and long fermentation cycles, failing to achieve breakthroughs in yield. Furthermore, because the metabolic flow of 2′-FL as the end product and its relationship with the central metabolic modules within the microorganism are still unclear, the construction of cell factories remains focused on static control and module assembly, and its production potential has not been fully realized. Dynamic regulation strategies are an effective means to achieve fermentation process control and improve bio-fermentation capacity, but currently, no effective dynamic regulation methods regarding the relationship between the 2′-fucosylated lactose synthesis module and the main metabolic pathways of microorganisms have been reported. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a dynamically regulated strain for the efficient production of 2′-fucosylated lactose using glucose and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a dynamically regulated strain for the efficient production of 2′-fucosylated lactose from glucose, wherein the dynamically regulated strain regulates the host strain's... gltA The initial promoter of the gene is replaced with σ² 8 Dependency promoter P fliC The result.

[0006] This invention addresses the problems of existing 2′-fucosylated lactose-producing strains, primarily *E. coli*, which lack efficient precursor metabolite (GDP-L-fucose) synthesis capabilities, have unclear metabolic flux distribution, low carbon source conversion efficiency, and struggle to effectively control industrial production costs. It provides a dynamically regulated strain for the efficient production of 2′-fucosylated lactose from glucose. This is achieved by adjusting the host strain's... gltA The initial promoter of the gene is replaced with σ² 8 Dependency promoter P fliC This enables the efficient production of 2′-fucosylated lactose using glucose as a carbon source.

[0007] In a preferred embodiment of the dynamically regulated strain described in this invention, the host strain includes Kosakonia sp. Strains, Escherichia coli, or yeast.

[0008] As a preferred embodiment of the dynamically regulated strain described in this invention, the σ² 8 Dependency promoter P fliC The nucleotide sequence is shown in SEQ ID NO. 1.

[0009] The present invention also provides the application of the aforementioned dynamically regulated strain in the preparation of 2′-fucosylated lactose or products containing 2′-fucosylated lactose.

[0010] The present invention also provides a method for efficiently producing 2′-fucosylated lactose, wherein the method uses glucose as a substrate and the dynamically regulated strain as a fermentation strain to produce 2′-fucosylated lactose.

[0011] In a preferred embodiment of the method described in this invention, the dynamically regulated strain is inoculated into a fermentation system and fermented at 30°C, pH 6.9, rotation speed 500 rpm, and aeration value 5 for 96 h.

[0012] In a preferred embodiment of the method described in this invention, the fermentation system contains 40 g / L glucose, 3.6 g / L tryptone, 7.2 g / L yeast extract, 0.7 g / L KH2PO4, 0.4 g / L (NH4)2HPO4, 1 g / L MgSO4·7H2O, 0.5 g / L NaCl, 0.8 g / L citric acid monohydrate, 3 mL Tween 80, 1 mL defoamer, and 30 g / L lactose.

[0013] In a preferred embodiment of the method described in this invention, when glucose is consumed to 10 g / L during fermentation, glucose is added to maintain a glucose concentration of not less than 10 g / L.

[0014] This invention also provides a method for improving the production of 2′-fucosylated lactose by improving the host strain's... gltA The initial promoter of the gene is replaced with σ² 8 Dependency promoter P fliC .

[0015] In a preferred embodiment of the method described in this invention, the host strain includes Kosakonia sp. Strains, Escherichia coli, or yeast.

[0016] The beneficial effects of this invention: Guided by a genome-scale metabolic flux analysis model, this invention modifies 2′-fucosylated lactose-producing strains with a GDP-L-fucose production preference and glucose as a carbon source, and modifies key metabolic node genes. gltA The original promoter is replaced with a σ² that is associated with growth. 8 Dependency promoter P fliC This method increases the yield of 2′-fucosylated lactose by 45% in shake flasks, achieving a final yield of 119.21 g / L after 96 hours of fermentation in a 5L fermenter, with a glucose conversion rate of 85%. This method enables the efficient production of 2′-fucosylated lactose using glucose as a carbon source, demonstrating potential for large-scale production, commercial application value, and promising industrialization prospects. Attached Figure Description

[0017] Picture 1 for Kosakonia sp. Analysis of the expression intensity of the endogenous GDP-L-fucose synthesis pathway in engineered strains.

[0018] Picture 2 The graph shows the FBA calculation results with GDP-L-fucose as the final product.

[0019] Picture 3 for gltA Gene knockout humidity experiment verification results.

[0020] Picture 4 This is a schematic diagram of a dynamic metabolic regulation strategy.

[0021] Picture 5 This diagram illustrates the verification of dynamic control of strains during shake-flask fermentation.

[0022] Picture 6 To dynamically regulate the fermentation results of the strain in a 5L fermenter fed with feed. Detailed Implementation

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0024] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0025] The culture medium formulation involved in this invention is as follows: LB (Luria-Bertani) liquid medium: Weigh 5 g yeast powder, 10 g peptone, and 10 g sodium chloride, add 1 L of water and dissolve thoroughly. Dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 min, and cool before use.

[0026] TB medium: Weigh 12 g yeast powder, 24 g peptone, and 60 g glucose, add 1 L of water to dissolve them completely, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 min, and cool before use.

[0027] Seed culture medium: glucose 20 g / L, tryptone 3.6 g / L, yeast extract 7.2 g / L.

[0028] 5L fermentation medium: glucose (40 g / L), tryptone (3.6 g / L), yeast extract (7.2 g / L), KH2PO4 (0.7 g / L), (NH4)2HPO4 (0.4 g / L), MgSO4·7H2O (1 g / L), NaCl (0.5 g / L), citric acid monohydrate (0.8 g / L), Tween 80 (3 mL), defoamer (1 mL), lactose (30 g / L).

[0029] Kosakonia sp. The engineered recombinant strain was obtained through independent screening by the research group, and its accession number is: CCTCCM2018092; The RNA extraction kit was purchased from Omega Bio-Tek. The reverse transcription kit was purchased from Novizan. Example 1 Kosakonia sp. The culture of the engineered recombinant strain (K1 strain) and the analysis of gene expression intensity in the GDP-L-fucose synthesis pathway were conducted using the following experimental methods: 1) Shake flask fermentation 100 µL of the engineered recombinant Kosakonia sp. strain was inoculated into LB liquid medium and cultured overnight at 30°C on a shaker at 220 rpm. The next day, 1% was inoculated into TB liquid medium supplemented with lactose and fermented at 30°C on a shaker for 72 h at 220 rpm.

[0030] 2) Analysis of gene expression intensity in the GDP-L-fucose synthesis pathway based on RT-qPCR collect Kosakonia sp. Total RNA was extracted from the fermentation broth of the recombinant strain using an RNA kit. cDNA was prepared from the total RNA using a reverse transcription kit. Primers for the pathway genes (manB, manC, gmd, and fcl) were designed based on their gene sequences (as shown in Table 1), maintaining the detection sequence at 100-200 bp, and the annealing temperature was 60℃. The reaction system was prepared, and expression intensity was analyzed using real-time quantitative PCR. Results are as follows: Picture 1 As shown, Kosakonia sp. The engineered recombinant strain possesses two GDP-L-fucose synthesis gene clusters. Compared to the gene cluster located in the colacid synthesis pathway, the GDP-L-fucose synthesis gene cluster located in the rhamnose synthesis pathway exhibits higher expression intensity.

[0031] Table 1 Example 2 Kosakonia sp. Analysis and application of GDP-L-fucose biosynthesis flux in engineered recombinant strains: The specific experimental methods are as follows: 1) Flux Balance Analysis (FBA) The previously successfully constructed Kosakonia sp. The metabolic model fuc101 of the engineered recombinant strain (the code of which is stored on: https: / / github.com / y1zh2 / the-GEM-of-Kosakonia) was imported into the CAVE online platform (https: / / cave.biodesign.ac.cn / ), using glucose as the carbon source and GDP-L-fucose as the final product. gltA The on / off state of gene-guided citric acid synthesis was used as a metabolic node for FBA metabolic analysis, and the results are as follows: Picture 2 As shown, gltA The gene knockout shut down the TCA cycle, significantly reducing glucose consumption and increasing the proportion of its metabolic flux to GDP-L-fucose from 0.14% to 0.67%.

[0032] 2) Wet test verification Using the crispr-cas9 toolbox Kosakonia sp. Recombinant strains of engineered bacteria gltA Gene knockout yielded the knockout strain K2. Using the shake-flask fermentation method described in Example 1, the knockout strain and strain K1 were co-transferred into TB medium with glucose as the carbon source for shake-flask fermentation to verify the results. Calcium carbonate was added to maintain the pH of the fermentation broth. The fermentation results are shown in the attached figure. Picture 3As shown, compared to K1, K2 had almost the same 2′-fucosylated lactose production, but glucose consumption decreased by 32% and yield per cell increased by 46%.

[0033] Example 3 gltA Construction and application of adaptive dynamic metabolic regulation strains with genes as key nodes The specific experimental method is as follows: 1) Construction of strains with dynamic metabolic regulation gltA Gene-guided citric acid synthesis is the initial reaction in the TCA cycle. While static knockout can reduce carbon source loss, it also has a significant negative impact on strain growth and weakens carbon source uptake. Dynamic metabolic regulation strategies, such as... Picture 4 As shown, the crispr-cas9 toolbox is used to... Kosakonia sp. Among engineered recombinant strains gltA The initial promoter of the gene is replaced with σ² 8 Dependency promoter P fliC (nucleotide sequence: GACTAACAAAAAATGGCTGTTTTTGAAAAAAATTCTAAAGGTTGTTTTACGACAGACGATAACAGGGTTGACGGCGATTGAGCCGACGGGTGGAAACCCAATACGTAATCAACGACTTGCAATATAGGATAACGAATC), resulting in the dynamic regulation of strain K3, which adapts the TCA cycle to the strain's growth (σ²). 8 Dependency promoter P fliC The target gene is expressed efficiently from the growth phase to the stationary phase and stops being expressed after the end of the stationary phase, thus achieving dynamic regulation of growth in the early stage of fermentation and production in the later stage.

[0034] 2) Shake-flask fermentation The dynamically regulated strain K3 and the knockout strain K2 were subjected to shake-flask fermentation using the method described in Example 1 to verify the results, as shown below. Picture 5 As shown, compared with the knockout strain K2, the dynamic regulation strain K3 showed a 44% increase in 2′-fucosylated lactose production, reaching 17.2 g / L, indicating improved strain growth and complete glucose uptake.

[0035] Example 4: 5L fermentation process of strain K3 under dynamic regulation 1) Seed culture of microorganisms: The dynamically regulated strain K3 was inoculated into seed culture medium, and 100 mL of liquid was added to a 500 mL shake flask. The culture temperature was 30℃, the shaker speed was 220 rpm, and the culture time was 24 hours.

[0036] 2) Upper-tank fermentation process: Inoculate the inoculum culture from step 1) at 10% into a 5L fermentation tank. Aeration is set to 5, pH is controlled to 6.9 with ammonia, rotation speed is 500 rpm, fermentation temperature is 30℃, and fermentation time is 96 h. A 600 g / L glucose solution is prepared in the feeding tank. When the glucose concentration in the feeding tank reaches 10 g / L, feeding begins at a uniform rate, maintaining this concentration until fermentation is complete. The fermentation results are as follows: Picture 6 As shown, the final yield of 2′-fucosylated lactose reached 119.21 g / L, the glucose conversion rate reached 85%, and the production rate was 1.24 g / L / h.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A dynamically regulated bacterial strain for the efficient production of 2′-fucosylated lactose from glucose, characterized in that, The dynamically regulated strain controls the host strain gltA The initial promoter of the gene is replaced with σ² 8 Dependency promoter P fliC The result.

2. The dynamically regulated strain according to claim 1, characterized in that, The host strain includes Kosakonia sp. Strains, Escherichia coli, or yeast.

3. The dynamically regulated strain according to claim 1, characterized in that, The σ² 8 Dependency promoter P fliC The nucleotide sequence is shown in SEQ ID NO.

1.

4. The use of the dynamically regulated strain according to any one of claims 1-3 in the preparation of 2′-fucosylated lactose or products containing 2′-fucosylated lactose.

5. A method for efficiently producing 2′-fucosylated lactose, characterized in that, The method uses glucose as a substrate and the dynamically regulated strain described in any one of claims 1-3 as the fermentation strain to produce 2′-fucosylated lactose.

6. The method according to claim 5, characterized in that, The method involves inoculating the dynamically regulated strain according to any one of claims 1-3 into a fermentation system and fermenting it for 96 h at 30-32℃, pH 6.9-7.2, rotation speed 400-600 rpm, and aeration value 5-7.

7. The method according to claim 6, characterized in that, The fermentation system contains 40-60 g / L glucose, 3.6-4.2 g / L tryptone, 7.2-8.4 g / L yeast extract, 0.7-1.0 g / L KH2PO4, 0.4-0.6 g / L (NH4)2HPO4, 1-1.4 g / L MgSO4·7H2O, 0.5-0.8 g / L NaCl, 0.8-1.0 g / L citric acid monohydrate, 3-4 mL Tween 80, 1-2 mL defoamer, and 30-40 g / L lactose.

8. The method according to claim 5, characterized in that, During fermentation, when glucose is consumed to 10 g / L, glucose is added to maintain a glucose concentration of not less than 10 g / L.

9. A method for improving the production of 2′-fucosylated lactose by host bacteria, characterized in that, By using the host strain gltA The initial promoter of the gene is replaced with σ² 8 Dependency promoter P fliC .

10. The method according to claim 9, characterized in that, The host strain includes Kosakonia sp. Strains, Escherichia coli, or yeast.

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