Recombinant escherichia coli for producing 2'-fucosyllactose and construction method and application thereof

Abstract

The present application relates to a kind of 2'-fucosyllactose producing recombinant escherichia coli and its construction method and application, belong to the field of bioengineering technology.The present application expresses 2'-fucosyllactose remediation synthesis pathway related gene in recombinant escherichia coli, including the gene fkp of coding GDP-fucose pyrophosphorylase FKP and the gene futC of coding fucosyltransferase FutC, respectively in the upstream of fkp gene and futC gene expression macromolecular coagulation material protein FUSLCD after the modification of coagulation tag GCN4, and replaces replicon, constructs a strain of high-yield 2'-fucosyllactose recombinant escherichia coli, yield and production intensity reach 38.68g / L and 0.35g / L / h respectively.It has good application prospect, has the potential of industrial application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, in particular to a recombinant Escherichia coli for producing 2'-fucosyllactose and a construction method and application thereof. BACKGROUND

[0002] 2'-fucosyllactose (2'-FL) is an important component of human milk oligosaccharides, which accounts for 20% of human milk carbohydrates, and has good effects on regulating the intestinal flora of infants, stimulating the maturation of the immune system, and resisting the invasion of intestinal pathogenic microorganisms. Microbial fermentation has the advantages of environmental friendliness and mild production conditions, and is widely used in the pharmaceutical, cosmetic, and food additive industries.

[0003] At present, an important direction of green biological manufacturing is to efficiently synthesize 2'-fucosyllactose through synthetic biology technology and metabolic engineering strategy. Therefore, microbial manufacturing based on metabolic engineering and synthetic biology methods has become an important means for constructing 2'-fucosyllactose cell factories, which modifies and transforms the cell metabolic pathway, changes the cell characteristics, and combines with cell gene regulation, metabolic regulation, and biochemical engineering, significantly improving the synthesis efficiency of target products. Traditional metabolic engineering strategies mainly focus on the expression of metabolic pathways, the knockout of byproduct pathways, and the dynamic regulation method of gene circuits. Although these methods can significantly improve the yield of products, they are easy to cause imbalance of metabolic pathways and metabolic networks, and ultimately cause the synthesis efficiency of products to decrease. At present, the research on microbial synthesis of 2'-fucosyllactose lacks the related research on co-localization catalysis between enzymes and substrates. SUMMARY

[0004] To solve the above technical problems, the present application provides a recombinant Escherichia coli for producing 2'-fucosyllactose and a construction method and application thereof. The present application expresses 2'-fucosyllactose salvage synthesis pathway related genes in the recombinant Escherichia coli, including the gene fkp encoding GDP-fucose pyrophosphorylase FKP and the gene futC encoding fucosyltransferase FutC, and expresses the macromolecular coagulation protein FUSLCD modified by the coagulation tag GCN4 upstream of the fkp gene and the futC gene, and replaces the replicon, thereby realizing the strengthening of the 2'-fucosyllactose salvage synthesis pathway.

[0005] The first object of the present application is to provide a recombinant E. coli for producing 2'-fucosyllactose, wherein a macromolecular coagulation protein FUSLCD modified by a coagulation tag GCN4 is connected to a gene fkp encoding GDP-fucose pyrophosphatase FKP and a gene futC encoding fucosyltransferase FutC respectively to obtain a recombinant gene, and the recombinant gene is connected to a free expression vector to obtain a recombinant genetically engineered bacterium.

[0006] Further, the free expression vector contains a replicon as shown in any one of SEQ ID NO. 1-3.

[0007] Further, the nucleotide sequence encoding the gene fkp is as shown in SEQ ID NO. 4, the nucleotide sequence encoding the gene futC is as shown in SEQ ID NO. 5, the nucleotide sequence encoding the macromolecular coagulation protein FUSLCD is as shown in SEQ ID NO. 6, and the nucleotide sequence encoding the coagulation tag GCN4 is as shown in SEQ ID NO. 7.

[0008] Further, the starting strain is knocked out of lacZ and wcaJ genes.

[0009] The second object of the present application is to provide a microbial preparation containing the above-mentioned recombinant genetically engineered bacterium.

[0010] The third object of the present application is to provide a construction method of the above-mentioned recombinant E. coli, comprising the following steps:

[0011] (1) a macromolecular coagulation protein FUSLCD modified by a coagulation tag GCN4 is connected to a gene fkp encoding GDP-fucose pyrophosphatase FKP and a gene futC encoding fucosyltransferase FutC respectively to obtain a recombinant gene;

[0012] (2) the recombinant gene is connected to a free expression vector containing a replicon as shown in any one of SEQ ID NO. 1-3 to obtain a recombinant free expression vector;

[0013] (3) the lacZ and wcaJ genes of the starting strain are knocked out, and the recombinant free expression vector is transformed into the starting strain to obtain the recombinant E. coli.

[0014] The fourth object of the present application is to provide a method for producing 2'-fucosyllactose, wherein the above-mentioned recombinant genetically engineered bacterium or the above-mentioned microbial preparation is added to a fermentation system.

[0015] Further, the initial OD of the strain fermentation is 0.04-0.3. 600

[0016] ​Further, the fermentation medium is DM medium.

[0017] Further, the fermentation time is 70-110 hours.

[0018] The above technical solutions of the present application have the following advantages compared with the prior art:

[0019] The present application uses a simple design method to strengthen the catalytic efficiency of Li Jinmei by expressing the macromolecular condensate protein FUSLCD, assembles the related genes of the 2'-fucosyllactose salvage synthesis pathway, and constructs a recombinant E. coli strain with high yield of 2'-fucosyllactose. The present application enriches and develops the theory and practice of regulatory element design and construction, and provides a new idea for dynamic balance of complex microbial metabolic networks. The recombinant strain provided by the present application is applied to fermentation production, and the yield and production intensity of 2'-fucosyllactose reach 38.68 g / L and 0.35 g / L / h, respectively. It has good application prospect and potential for industrial application. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0021] Figure 1 is the construction of the macromolecular condensate system in Example 1 of the present application;

[0022] Figure 2 is the plasmid map of pET28a-GCN4-FUSLCD-GFP in Example 1 of the present application;

[0023] Figure 3 is the plasmid map of pET28a-GFP in Example 1 of the present application;

[0024] Figure 4 is the salvage synthesis path of 2'-fucosyllactose in Example 2 of the present application;

[0025] Figure 5 is the comparison of the 2'-fucosyllactose peak time in the standard sample and the sample in Example 2 of the present application;

[0026] Figure 6 is the comparison of the 2'-fucosyllactose yield of different recombinant strains in Example 2 of the present application;

[0027] Figure 7 is the change of 2'-fucosyllactose content in the fermentation tank of FL-G2 strain in Example 6 of the present application. DETAILED DESCRIPTION

[0028] The present application will be further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand and implement the present application, but the embodiments are not intended to limit the present application.

[0029] (I) Seed medium

[0030] Seed medium: LB medium, ingredients include 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride.

[0031] Fermentation medium: ingredients include standard fermentation medium (DM medium, 1 L): 13.5 g / L of potassium dihydrogen phosphate, 4.0 g / L of ammonium phosphate, 1.7 g / L of citric acid, 1.4 g / L of magnesium sulfate heptahydrate, 10 mL / L of trace metal solution (10 g / L of ferric chloride, 2.25 g / L of zinc sulfate heptahydrate, 1.0 g / L of copper sulfate pentahydrate, 0.35 g / L of manganese sulfate monohydrate, 0.23 g / L of sodium borate decahydrate, 0.11 g / L of ammonium molybdate, 2.0 g / L of calcium chloride dihydrate, pH 6.8); add glucose of corresponding concentration (40 g / L of glucose for shake flask fermentation, 40 g / L of glycerol for fermentation tank fermentation). Autoclave glucose and glycerol, adjust pH to 7 by KOH before adding to the fermentation medium, and sterilize by 0.22 μm membrane. Add defoamer (Sigma 204) as needed.

[0032] (II) Fermentation production of 2'-fucosyllactose

[0033] (1) Shake flask fermentation

[0034] Inoculate the recombinant strain into 10 mL of seed medium, cultivate at 37°C, 200-220 rpm for 12-18 h to obtain seed liquid, inoculate the seed liquid into 250 mL shake flask with 50 mL of DM medium at an inoculation amount of 2% (v / v), pH 6.8, constant temperature 30°C, 200-220 rpm, and fermentation period 110 h.

[0035] (2) 5L fermentation tank fermentation

[0036] Inoculate the recombinant strain into 10 mL of seed medium, cultivate at 37°C, 200 rpm for 12-18 h to obtain seed liquid, inoculate the seed liquid into a fermentation tank with 3.5 L of DM medium at an inoculation amount of 5% (v / v), pH 7.1, pressure 1 mpa, constant temperature 30°C, 450-650 rpm, aeration amount 1 vvm, and fermentation period 110 h.

[0037] (III) Determination of 2'-fucosyllactose content

[0038] Fermentation sample preparation: Take the fermentation broth sample, centrifuge at 12000 rpm for 5 min, dilute the supernatant, filter through a 0.22 μm water system membrane, and the filtrate is used for liquid chromatography analysis.

[0039] 2'-Fucosyllactose content determination: Shimadzu high performance liquid chromatography (with differential detector), using Phenomenex Carbohydrate Analysis (Rezex ROA-organic acid H+(8%)) chromatographic column to analyze the sample, the mobile phase is 0.005M H2SO4, the mobile phase is filtered through a 0.22 μm filter membrane, ultrasonic degassing, the flow rate is 0.6 mL / min, the column temperature is 60℃; injection volume: 10 μL.

[0040] Example 1: Construction and verification of macromolecular condensate system

[0041] For specific methods, refer to Gao, C., Hou, J., Xu, P. et al. Programmable biomolecular switches for rewiring flux in Escherichia coli. Nature Communications: 10, 3751 (2019).

[0042] Using pET28a engineering plasmid as a template, linearized vector was recovered by double digestion with EcoR I and Hind III, and GCN4-FUSLCD-GFP fragment was amplified from FUSLCD protein containing GCN4 tag codon-optimized by Anhui General Biotech Co., Ltd. and fused with GFP protein. GCN4-FUSLCD-GFP fragment was inserted into pET28a engineering plasmid by Nuaidian homologous recombination to obtain pET28a-GCN4-FUSLCD-GFP plasmid (A), wherein the nucleotide sequence encoding FUSLCD protein is shown in SEQ ID NO. 6, and the nucleotide sequence encoding GCN4 tag is shown in SEQ ID NO. 7. Figure 1

[0043] Using pET28a engineering plasmid as a template, linearized vector was recovered by double digestion with EcoR I and Hind III, and GFP protein was amplified and homologously recombined with linearized vector, and GFP fragment was inserted into pET28a engineering plasmid to obtain pET28a-GFP plasmid (B). Figure 1

[0044] ​​The obtained recombinant plasmids pET28a-GCN4-FUSLCD-GFP and pET28a-GFP were introduced into competent cells E. coli BL21 to obtain strains E. coli BL21 / pET28a-GCN4-FUSLCD-GFP and BL21 / pET28a-GFP containing pET28a-GCN4-FUSLCD-GFP and pET28a-GFP plasmids, respectively. Figure 2 and Figure 3 ).

[0045] The strains E. coli BL21 / pET28a-GCN4-FUSLCD-GFP and E. coli BL21 / pET28a-GFP were inoculated in LB medium and cultured at 30°C and 200 rpm for 20 h, and the expression of GCN4-FUSLCD-GFP protein and GFP protein was detected by protein gel. The results are shown in Figure 1 B, which shows two additional bands compared with the control BL21, wherein the protein sizes of GCN4-FUSLCD-GFP and GFP are about 73 and 32 kDa, respectively.

[0046] As shown in C and D in Figure 1 , E. coli BL21 / pET28a-GCN4-FUSLCD-GFP forms a multi-enzyme complex with GFP green fluorescence in the cell, i.e., there is a macromolecular condensate at both ends and no macromolecular condensate in the middle. The control group BL21 / pET28a-GFP cannot form a macromolecular condensate in the cell, and GFP is uniformly distributed in the cell.

[0047] Example 2: Detection of 2'-fucosyllactose content in a shake flask

[0048] As shown in Figure 4The shown 2'-fucosyllactose synthesis pathway, using Bacteroides fragilis genome as template, amplifying GDP-fucose pyrophosphorylase FKP gene fragment (as shown in SEQ ID NO. 4), using Helicobacter pylori genome as template to amplify fucosyltransferase FUTC gene fragment (as shown in SEQ ID NO. 5), and using T7 promoter expression, two fragments are fused to obtain FKP-T7-FUTC fragment, inserted into Xho I single enzyme cut pJ01 vector, obtain pJ01-FKP-T7-FUTC plasmid, reference from the literature Gao, C., Hou, J., Xu, P. et al. Programmable biomolecular switches for rewiring flux in Escherichia coli. Nature Communications: 10, 3751 (2019).

[0049] Further, on the basis of pET28a-GCN4-FUSLCD-GFP plasmid, remove GFP protein by whole plasmid PCR method, obtain pET28a-GCN4-FUSLCD plasmid. Then, linearize pET28a-GCN4-FUSLCD plasmid by the same whole plasmid PCR method, amplify the fragment containing FKP, and obtain pET28a-GCN4-FUSLCD-FKP through homologous recombination. Next, linearize pET28a-GCN4-FUSLCD plasmid by the same whole plasmid PCR method, amplify the fragment containing FUTC, and obtain pET28a-GCN4-FUSLCD-FUTC through homologous recombination. Then, using pET28a-GCN4-FUSLCD-FUTC plasmid as template, amplifying GCN4-FUSLCD-FUTC fragment containing T7 promoter, and obtaining pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUTC plasmid through homologous recombination.

[0050] The replicon was obtained by amplifying the plasmids pRSF, pACYC and pCDF from the commercialized plasmid pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC, respectively, and named G1, G2 and G3. The G1, G2 and G3 replicons were replaced in pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC by homologous recombination to obtain the plasmids pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC-G1, pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC-G2 and pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC-G3. The intensities of G1, G2 and G3 replicons were G1 > G2 > G3. The nucleotide sequence of G1 is shown in SEQ ID NO. 1, the nucleotide sequence of G2 is shown in SEQ ID NO. 2, and the nucleotide sequence of G3 is shown in SEQ ID NO. 3.

[0051] The plasmid pJ01-FKP-T7-FUTC was transformed into E. coli BL (21) with lacZ and wcaJ genes knocked out to obtain a 2'-fucosyllactose production chassis strain FL. Then the above constructed plasmids were introduced into the FL competent cells to obtain 2'-fucosyllactose production strains FL-G1, FL-G2 and FL-G3 with the plasmids pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC-G1, pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC-G2 and pET28a-GCN4-FUSLCD-FKP-T7-GCN4-FUSLCD-FUC-G3, i.e. macromolecular agglomerate systems with different intensities, to verify the influence on 2'-fucosyllactose production. As shown in FIG. 2, the peak time of the sample was the same as that of the standard 2'-fucosyllactose in HPLC, indicating that the fermentation broth contained 2'-fucosyllactose. Figure 5

[0052] The above 2'-fucosyllactose production strains were placed in DM medium for shake flask culture, and the content of 2'-fucosyllactose in the fermentation broth was identified. The results are shown in FIG. 3. Figure 6 ​As shown, with the extension of cultivation time, the 2'-fucosylated lactose yield of the control group FL was 1.51 g / L, with a production intensity of 0.01 g / L / h, while the 2'-fucosylated lactose yields of the experimental groups FL-G1, FL-G2, and FL-G3 reached 1.65 g / L, 3.35 g / L, and 2.31 g / L, respectively, with production intensities of 0.02 g / L / h, 0.03 g / L / h, and 0.02 g / L / h. The yields were 9.27%, 121.85%, and 52.98% higher than those of the control group, respectively.

[0053] Example 3: Detection of 2'-fucosylated lactose content in fermenters

[0054] The fermentation performance of strain FL-G2 was tested in a 5L fermenter.

[0055] The FL-G2 strain constructed in Example 2 was inoculated into 10 mL of seed culture medium and cultured at 37°C and 200 rpm for 12-18 h to obtain seed liquid. The seed liquid was then inoculated into a fermenter containing 3.5 L of DM culture medium at an inoculation rate of 5% (v / v). The pH value was 7.1, the pressure was 1 MPa, the temperature was constant at 30°C, the rpm was 550 rpm, the aeration rate was 1 vvm, and the fermentation cycle was 110 h.

[0056] like Figure 7 As shown, at the end of fermentation, the yield and production intensity of 2'-fucosylated lactose reached 38.68 g / L and 0.35 g / L / h, respectively.

[0057] 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 Escherichia coli producing 2'-fucosyllactose, characterized in that, The recombinant genetically engineered bacteria is a recombinant gene GCN4-FUSLCD-FKP obtained by connecting a macromolecular coagulation protein FUSLCD modified by a coagulation tag GCN4 with a gene encoding GDP-fucose pyrophosphorylase FKP fkp The recombinant gene GCN4-FUSLCD-FKP is connected with a gene encoding fucosyltransferase FutC to obtain a recombinant gene GCN4-FUSLCD-FUTC futC The recombinant gene GCN4-FUSLCD-FUTC is connected to a free expression vector, and the recombinant gene is transformed into a starting strain to obtain the recombinant Escherichia coli. The episomal expression vector contains a replicon as shown in any one of SEQ ID NO. 1-3; Encoding fkp The nucleotide sequence of the gene is shown in SEQ ID NO. 4, encoding futC The nucleotide sequence of the gene is shown in SEQ ID NO. 5, encoding the nucleotide sequence of the macromolecular agglomerate protein FUSLCD is shown in SEQ ID NO. 6, encoding the nucleotide sequence of the coagulation promoting tag GCN4 is shown in SEQ ID NO.

7.

2. The recombinant E. coli of claim 1, wherein: The starting strain is knocked out lacZ and wcaJ genes.

3. A microbial preparation comprising the recombinant E. coli of claim 1 or 2.

4. A method for constructing the recombinant E. coli of claim 1 or 2, characterized by, comprising the following steps: (1) The recombinant gene is obtained by connecting the coagulation tag GCN4 modified macromolecular coagulation protein FUSLCD with the gene encoding GDP-fucose pyrophosphorylase FKP fkp and the gene encoding fucosyltransferase FutC futC respectively. (2) linking the recombinant gene to an episomal expression vector containing a replicon as shown in any one of SEQ ID NO. 1-3, to obtain a recombinant episomal expression vector; (3) knocking out the lacZ and wcaJ gene of the starting strain, and transforming a recombinant episomal expression vector into the starting strain to obtain the recombinant Escherichia coli.

5. A method for producing 2'-fucosyllactose, characterized by, adding the recombinant E. coli of claim 1 or 2 or the microbial preparation of claim 3 to a fermentation system.

6. The method of claim 5, wherein: Strain fermentation initial OD 600 was 0.04-0.

3.

7. The method of claim 5, wherein: The fermentation medium is DM medium.

8. The method of claim 5, wherein: The fermentation time is 70-110 hours.

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