A method for enhancing the production performance of 2'-fucosyllactose by a protein self-assembly system

Abstract

The present invention discloses a method for enhancing the production performance of 2'-fucosyllactose by a protein self-assembly system, and belongs to the field of bioengineering technology. The present invention successfully constructs a high-yield 2'-fucosyllactose production engineered strain by expressing an expression cassette for the protein self-assembly protein Mi3 and combining the expression of genes related to the 2'-fucosyllactose synthesis pathway. In addition, the method is simple in design and can enhance the catalytic efficiency of the enzymes in the synthesis pathway. The present invention constructs a 2'-fucosyllactose production engineered strain and a protein self-assembly regulation method, and after fermentation for 105 hours, the 2'-fucosyllactose yield and production intensity reach 44.66g / L and 0.41g / L / h, respectively. It has good application prospects and the potential for industrial application.

Description

Technical Field

[0001] The invention relates to a method for strengthening the production performance of 2'-fucosyllactose by a protein self-assembly system, and belongs to the technical field of bioengineering. Background Art

[0002] 2'-Fucosyllactose (2'-FL), a key component of human milk oligosaccharides, has been shown to modulate immunity, aid brain development, and regulate intestinal flora. It is widely used as a food additive in foods such as infant formula. Compared to traditional chemical synthesis methods, which require multiple protective reactions, have low product yields, and pose significant pollution risks, biosynthesis of 2'-fucosyllactose holds strategic importance for environmental protection and optimizing the structure of the food ingredient industry.

[0003] Therefore, metabolic engineering strategies such as pathway design, byproduct blocking, and cofactor regeneration have been developed for the production of 2'-fucosyllactose. Current strategies primarily focus on pathway construction or enhancement, but lack research on the carbon flux competition between 2'-fucosyllactose synthesis and cell growth. Fructose-6-phosphate, the key precursor of 2'-fucosyllactose, enters glycolysis and the tricarboxylic acid cycle under the catalysis of phosphofructokinase (PfkA) for cell growth. Traditional metabolic engineering strategies primarily include modular optimization of pathway enzymes, truncation of byproduct pathways, regulation of cofactor metabolic balance, and global transcription factor regulation. These approaches primarily focus on conventional expression control of local pathways. While these approaches can significantly improve product yield and production intensity, they also suffer from limited catalytic properties of pathway enzymes, ultimately limiting product synthesis efficiency. Therefore, to address the issue of pathway enzyme catalytic efficiency in complex microbial metabolic networks, microbial scaffold proteins offer a way to improve the catalytic distance between pathway enzymes and substrates, further enhancing chemical production efficiency. Summary of the Invention

[0004] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for enhancing the production performance of 2-fucosyllactose by a protein self-assembly system. The method aims to utilize the protein self-assembly protein Mi3 in combination with the RIAD / RIDD polypeptide tag; realize multi-enzyme assembly through the protein self-assembly system and 2'-fucosyllactose, thereby enhancing the 2'-fucosyllactose synthesis pathway. This will enrich and develop the theory and practice of metabolic regulatory element design and construction, and provide new ideas for enhancing the catalytic efficiency of pathway enzymes in complex microbial metabolic networks.

[0005] The first technical solution provided by the present invention is a genetically engineered bacterium, which expresses a gene encoding a self-assembly protein Mi3 and a gene encoding an enzyme related to the de novo synthesis pathway of 2'-fucosyllactose; the gene encoding the self-assembly protein Mi3 is connected to a gene encoding an RD (RIDD) tag, and the gene related to the de novo synthesis pathway of 2'-fucosyllactose is connected to a gene encoding a RA (RIAD) tag; the enzyme related to the 2'-fucosyllactose synthesis pathway is assembled with the self-assembly protein Mi3 through the RIAD tag and the RIDD tag; the genes encoding the enzymes related to the 2'-fucosyllactose synthesis pathway include the gene manB encoding phosphomannose mutase ManB, the gene manC encoding mannose-1-phosphate guanylyltransferase ManC, the gene gmd encoding GDP mannose 4,6-dehydratase Gmd, the gene wcaG encoding GDP-L-fucose synthase WcaG, and the gene futC encoding fucosyltransferase FutC.

[0006] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the self-assembly protein Mi3 is shown as SEQ ID NO.1.

[0007] In one embodiment of the present invention, the self-assembling protein Mi3 is connected to the RD tag via a connecting peptide (GGGGS) 3, and the enzymes related to the 2'-fucosyllactose synthesis pathway are connected to the RA tag via a connecting peptide (GGGGS) 3.

[0008] In one embodiment of the present invention, the nucleotide sequence of the gene manB encoding phosphomannose mutase is shown in SEQ ID NO.2.

[0009] In one embodiment of the present invention, the nucleotide sequence of the gene manC encoding mannose-1-phosphate guanylyltransferase is shown in SEQ ID NO.3.

[0010] In one embodiment of the present invention, the nucleotide sequence of the gene gmd encoding GDP mannose 4,6-dehydratase is shown in SEQ ID NO.4.

[0011] In one embodiment of the present invention, the nucleotide sequence of the gene wcaG encoding GDP-L-fucose synthase is shown in SEQ ID NO.5.

[0012] In one embodiment of the present invention, the nucleotide sequence of the gene futC encoding fucosyltransferase is shown in SEQ ID NO.6.

[0013] In one embodiment of the present invention, the nucleotide sequence of the tag RA is shown as SEQ ID NO.7.

[0014] In one embodiment of the present invention, the nucleotide sequence of the tag RD is shown as SEQ ID NO.8.

[0015] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli as a host.

[0016] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli with knocked-out lacZ gene and wcaJ gene as a host.

[0017] In one embodiment of the present invention, the genetically engineered bacteria co-expresses ManB, ManC, Gmd, WcaG and FutC using the pJ01 vector.

[0018] Furthermore, the genetically engineered bacteria co-express BC-RA fusion protein, DG-RA fusion protein and F-RA fusion protein using the pJ01 vector, wherein the BC-RA fusion protein comprises the RA tag protein, the ManB and the ManC connected in sequence by a connecting peptide (GGGGS) 3, the DG-RA fusion protein comprises the RA tag protein and the Gmd and the WcaG connected in sequence by a connecting peptide (GGGGS) 3, and the F-RA fusion protein comprises the RA tag protein and the FutC connected by a connecting peptide (GGGGS) 3.

[0019] In one embodiment of the present invention, the pJ01 vector is constructed by inserting a T7Te terminator sequence after the rrnB T1 terminator using the pTargetF plasmid as a base, and removing the sgRNA expression cassette to obtain plasmid pJ01 containing only the Pj23119 constitutive promoter and terminator. The pJ01 vector is disclosed in Chinese patent application publication number CN 113929787 A.

[0020] In one embodiment of the present invention, the genetically engineered bacteria uses the pET22b plasmid as a vector to express the self-assembly protein Mi3 connected to a RIDD tag.

[0021] Furthermore, the original RBS sequence of the pET22b plasmid is replaced by the M1, M2 or M3 gene fragment.

[0022] Furthermore, the nucleotide sequence of the M1 gene fragment is shown in SEQ ID NO.10.

[0023] Furthermore, the nucleotide sequence of the M2 gene fragment is shown in SEQ ID NO.11.

[0024] Furthermore, the nucleotide sequence of the M3 gene fragment is shown in SEQ ID NO.12.

[0025] The second technical solution provided by the present invention is a method for constructing a genetically engineered bacterium that produces 2'-fucosyllactose. The method uses Escherichia coli as a chassis strain, and expresses a gene encoding a self-assembly protein Mi3 and genes encoding enzymes related to the de novo synthesis pathway of 2'-fucosyllactose on the chassis strain; the gene encoding the self-assembly protein Mi3 is connected to a gene encoding an RD tag, and the gene encoding the enzymes related to the de novo synthesis pathway of 2'-fucosyllactose is connected to a gene encoding an RA tag; the genes encoding the enzymes related to the synthesis pathway of 2'-fucosyllactose include the gene manB encoding phosphomannose mutase ManB, the gene manC encoding mannose-1-phosphate guanylyltransferase ManC, the gene gmd encoding GDP mannose 4,6-dehydratase Gmd, the gene wcaG encoding GDP-L-fucose synthase WcaG, and the gene futC encoding fucosyltransferase FutC.

[0026] In one embodiment of the present invention, the genetically engineered bacteria co-expresses ManB, ManC, Gmd, WcaG and FutC using the pJ01 vector.

[0027] Furthermore, the genetically engineered bacteria co-express BC-RA fusion protein, DG-RA fusion protein and F-RA fusion protein using the pJ01 vector, wherein the BC-RA fusion protein comprises the RA tag protein, the ManB and the ManC connected in sequence by a connecting peptide (GGGGS) 3, the DG-RA fusion protein comprises the RA tag protein and the Gmd and the WcaG connected in sequence by a connecting peptide (GGGGS) 3, and the F-RA fusion protein comprises the RA tag protein and the FutC connected in sequence by a connecting peptide (GGGGS) 3.

[0028] In one embodiment of the present invention, the pJ01 vector is constructed by inserting a T7Te terminator sequence after the rrnB T1 terminator using the pTargetF plasmid as a base, and removing the sgRNA expression cassette to obtain plasmid pJ01 containing only the Pj23119 constitutive promoter and terminator. The pJ01 vector is disclosed in Chinese patent application publication number CN 113929787 A.

[0029] In one embodiment of the present invention, the genetically engineered bacteria uses the pET22b plasmid as a vector to express the self-assembly protein Mi3 connected to a RIDD tag.

[0030] The third technical solution provided by the present invention is a method for producing 2'-fucosyllactose, which utilizes the genetically engineered bacteria described in the first technical solution to carry out fermentation production of 2'-fucosyllactose.

[0031] In one embodiment of the present invention, the fermentation medium includes DM medium.

[0032] In one embodiment of the present invention, the DM medium includes a carbon source, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L ammonium phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, 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, and 2.0 g / L calcium chloride dihydrate.

[0033] In one embodiment of the present invention, the carbon source comprises 40 g / L glucose.

[0034] In one embodiment of the present invention, the fermentation conditions are 30-38°C, 200-220rpm, and the initial OD of the strain is 600 0.04-0.1, fermentation 70-110h;

[0035] Or the fermentation conditions are 30-38°C, 480-530rpm, inoculation volume of 5-10%, liquid volume of 30-50%, pH of 6.0-7.0, and initial OD of the strain fermentation. 600 The humidity is 0.04-0.3, the ventilation volume is 1-2vvm, and the fermentation time is 70-120h.

[0036] The fourth technical solution provided by the present invention is the use of the genetically engineered bacteria described in the first technical solution, or the method described in the second technical solution, or the method described in the third technical solution in the preparation of 2'-fucosyllactose or a product containing 2'-fucosyllactose.

[0037] The fifth technical solution provided by the present invention is the application of the genetically engineered bacteria described in the first technical solution, or the method described in the second technical solution, or the method described in the third technical solution in the fields of biology, pharmaceuticals, food or chemical industry.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention uses a genetically engineered bacterium to express a gene encoding the self-assembly protein Mi3 and genes for enzymes involved in the de novo 2'-fucosyllactose synthesis pathway. The self-assembly protein Mi3 is linked to an N-terminal fusion RD tag, and the enzymes involved in the 2'-fucosyllactose synthesis pathway are linked to a C-terminal fusion RA tag. The self-assembly protein Mi3, through the RA and RD tags, recruits and encapsulates the enzymes involved in the de novo 2'-fucosyllactose synthesis pathway to form a multi-enzyme complex, which helps concentrate the enzyme concentration and enhances the catalytic efficiency between the substrate and the enzymes. This successfully constructs a high-yield 2'-fucosyllactose production strain. Furthermore, the method is simple in design and enhances the catalytic efficiency of the enzymes involved in the synthesis pathway. By constructing a 2'-fucosyllactose production strain and introducing a protein self-assembly control method, the present invention achieved a 2'-fucosyllactose yield and production intensity of 44.66 g / L and 0.41 g / L / h, respectively, after 105 hours of fermentation. This invention has promising application prospects and potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 For the construction of protein self-assembly system and protein expression.

[0041] Figure 2 This is the plasmid map of the protein self-assembly system.

[0042] Figure 3 Construction of a de novo synthesis pathway for 2'-fucosyllactose.

[0043] Figure 4 is the peak time of 2'-fucosyllactose in the standard sample and fermentation broth.

[0044] Figure 5 represents the change of 2'-fucosyllactose content in the shake flask.

[0045] Figure 6 The changes in 2'-fucosyllactose content in a 5-L fermentor. DETAILED DESCRIPTION

[0046] The plasmid construction involved in the present invention is carried out using classical molecular biological methods.

[0047] (1) Culture medium

[0048] Seed culture medium: LB medium, containing 10 g / L peptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0049] Fermentation medium: The composition includes standard fermentation medium (DM medium, 1 L): 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L ammonium phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, 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); and the corresponding concentration of glucose (40 g / L glucose for shake flask fermentation and 40 g / L glucose for fermentation in fermenter). Glucose was sterilized by autoclaving, adjusted to pH 7 with KOH, and sterilized by passing through a 0.22 μm membrane before adding to the fermentation medium. Defoamer (Sigma 204) was added as needed.

[0050] (2) Fermentation production of 2'-fucosyllactose

[0051] (1) Shake flask fermentation

[0052] The recombinant strain was inoculated into 10 mL of seed culture medium and cultured at 37°C, 200-220 rpm for 12-18 h to obtain seed liquid. The seed liquid was inoculated into a 250 mL shake flask containing 50 mL of DM culture medium at an inoculum size of 4% (v / v), pH 6.8, constant pressure temperature at 30°C, 200-220 rpm, and the fermentation cycle was 110 h.

[0053] (2) Fermentation in a 5-L fermenter

[0054] The recombinant strain 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 inoculated into a fermenter containing 3.5 L of DM culture medium at a 5% (v / v) inoculation rate, pH 7.1, pressure 1 MPa, constant temperature 30°C, 450-650 rpm, ventilation volume 1 vvm, and a fermentation cycle of 110 h.

[0055] (III) Determination of 2'-fucosyllactose content

[0056] Fermentation sample preparation: Take a fermentation broth sample, centrifuge it at 12000 rpm for 5 min, take the supernatant and dilute it, filter it through a 0.22 μm water system membrane, and use the filtrate for liquid chromatography analysis.

[0057] Determination of 2'-fucosyllactose content: Shimadzu high performance liquid chromatography (with differential detection) using Finomei Carbohydrate Analysis (Rezex ROA-organic acid H +The sample was analyzed by a chromatographic column (8%), with a mobile phase of 0.005 M H2SO4 filtered through a 0.22 μm filter membrane and ultrasonically degassed at a flow rate of 0.6 mL / min and a column temperature of 60°C; injection volume: 10 μL.

[0058] Example 1 Construction and verification of protein self-assembly system

[0059] Using the pET22b plasmid as a template, the self-assembly protein Mi3 encoding gene (as shown in SEQ ID NO.1) that has been codon-optimized from Anhui General Biotechnology Co., Ltd. was amplified by double enzyme digestion with Nde1 and Bamh1, and the Mi3 protein was inserted into the pET22b plasmid by Novogene recombination to obtain the pET22b-Mi3 plasmid. Using the pET22b-Mi3 plasmid as a template, a RD tag sequence was connected to the N-terminus of the Mi3 protein through a linker peptide (GGGGS) 3 by whole-plasmid PCR to obtain the pET22b-RD-Mi3 plasmid ( Figure 1 B).

[0060] Furthermore, using the pET28a plasmid as a template, the green fluorescent protein GFP gene fragment (as shown in SEQ ID NO.9) was amplified by double enzyme digestion with Bamh1 and Xho1, and the linearized vector was recovered by gel recovery. The GFP protein gene was inserted into the pET28a plasmid by the Norwegian homologous recombination method to obtain the pET28a-GFP plasmid. Using the pET28a-GFP plasmid as a template, a RA tag sequence was fused to the C-terminus of the GFP protein through a connecting peptide (GGGGS) 3 by whole plasmid PCR to obtain the pET28a-GFP-RA plasmid ( Figure 1 A).

[0061] Finally, the pET22b-RD-Mi3 engineering plasmid was used as a template, and the linearized vector pET22b-RD-Mi3 was recovered by double enzyme digestion with Sal1 and Hind111. The GFP-RA fragment containing the T7 promoter was amplified using the pET28a-GFP-RA plasmid as a template. By single fragment homologous recombination, the GFP-RA fragment containing the T7 promoter was connected to the linearized vector pET22b-RD-Mi3 to obtain the pET22b-RD-Mi3-T7-GFP-RA plasmid ( Figure 2 ).

[0062] The obtained recombinant plasmid pET22b-RD-Mi3-T7-GFP-RA was introduced into competent cells E. coli BL21 to obtain the strain E. coli BL21 / pET22b-RD-Mi3-T7-GFP-RA containing the pET22b-RD-Mi3-T7-GFP-RA plasmid.

[0063] The strain E. coli BL21 / pET22b-RD-Mi3-T7-GFP-RA was inoculated into LB medium and cultured at 37°C and 200 rpm for 20 h. The expression of RD-Mi3 protein and GFP-RA protein was detected by protein gel. Figure 1 As shown in C.

[0064] Depend on Figure 1 AB shows that Mi3 protein combines with RIAD / RIDD tags and undergoes protein self-assembly with GFP protein to form Mi3 protein-enzyme complex in the cell, which is beneficial to concentrate the concentration of enzyme and enhance the catalytic efficiency between substrate and enzyme. Figure 1 C shows that the expression of RD-Mi3 protein and GFP-RA protein in pET22b-RD-Mi3-T7-GFP-RA plasmid can be detected by protein gel, which obviously has two more bands than the control Escherichia coli BL (21). The protein sizes of RD-Mi3 and GFP-RA are about 30 and 28 kDa respectively.

[0065] Example 2 Detection of 2'-fucosyllactose content in shake flasks

[0066] like Figure 3 The de novo synthesis pathway of 2'-fucosyllactose shown in the figure was used. The Escherichia coli K12 genome was used as a template to amplify the gene encoding the BC-RA fusion protein (the BC-RA fusion protein comprises 3RA tag protein, ManB and ManC, sequentially connected by a connecting peptide (GGGGS)). The gene encoding the BC-RA fusion protein was inserted into the pJ01 vector (the pJ01 vector is disclosed in the Chinese patent application publication No. CN113929787A, specifically based on the commercial plasmid pTargetF (Addgene Plasmid #62226), using whole-plasmid PCR to insert a T7Te terminator sequence after the rrnB T1 terminator, and remove the sgRNA expression cassette to obtain a plasmid PJ01 containing only the Pj23119 constitutive promoter and terminator) after single enzyme digestion with Xho1, thereby obtaining the pJ01-BC-RA plasmid.

[0067] Using the same method, the gene encoding the DG-RA fusion protein (the DG-RA fusion protein contains the RA tag protein, Gmd, and WcaG, linked sequentially by the linker peptide (GGGGS)) was amplified and inserted into the pJ01 vector after single-enzyme digestion with Spe1, generating the pJ01-DG-RA plasmid. Using the pJ01-DG plasmid as a template, the pJ01-DG-RA fragment carrying the promoter was amplified and inserted into the pJ01-BC-RA vector after single-enzyme digestion with Sal1 via single-fragment homologous recombination, generating the pJ01-BC-RA-DG-RA plasmid.

[0068] Using the same method, the gene encoding the F-RA fusion protein (RA tag protein and FutC connected by a connecting peptide (GGGGS) 3) was amplified and inserted into the pJ01-BC-RA-DG-RA vector to obtain the pJ01-BC-RA-DG-RA-F-RA plasmid.

[0069] The construction of three plasmids, pET22b-M1-RD-Mi3, pET22b-M2-RD-Mi3 and pET22b-M3-RD-Mi3, all used pET22b-RD-Mi3 as template.

[0070] By using whole plasmid PCR, the original RBS sequence was replaced with three gene fragments of different strengths: M1, M2, and M3.

[0071] The plasmid pJ01-BC-RA-DG-RA was transformed into Escherichia coli BL(21)ΔlacZΔcaJ (derived from Pathway Optimization of 2′-Fucosyllactose Production in Engineered Escherichia coli, J Agric Food Chem, 2021, DOI: 10.1021 / acs.jafc.0c07224), and obtained the 2'-fucosyllactose production chassis strain FL-0, and then the plasmid pET22b-RD-Mi3 constructed in Example 1 was introduced into the FL competent cells to obtain 2'-fucosyllactose production strains FL-M1, FL-M2 and FL-M3 with pJ01-BC-RA-DG-RA and pET22b-RD-Mi3 (pET22b-M1-RD-Mi3, pET22b-M2-RD-Mi3, pET22b-M3-RD-Mi3), wherein M1, M2 and M3 are RBSs of different strengths, i.e., protein self-assembly systems of different strengths are obtained to verify the effect on 2'-fucosyllactose production. Figure 4As shown, the peak time of the standard 2'-fucosyllactose in HPLC is the same as that of the 2'-fucosyllactose in the sample, indicating that the fermentation broth contains 2'-fucosyllactose.

[0072] The above-mentioned 2'-fucosyllactose producing strain was placed in DM medium for shake flask culture, and the 2'-fucosyllactose content in the fermentation broth was identified. Figure 5 As shown, with the extension of culture time, the 2'-fucosyllactose production of the control group FL-0 strain was 1.7 g / L, and the production intensity reached 0.02 g / L / h, while the 2'-fucosyllactose production of the experimental groups FL-M1, FL-M2 and FL-M3 could reach 2.58 g / L, 2.49 g / L and 1.99 g / L, respectively, and the production intensity reached 0.025 g / L / h, 0.024 g / L / h, and 0.019 g / L / h, respectively, and the production was increased by 51.76%, 46.47% and 17.06% compared with the control group FL-0 strain.

[0073] Example 3 Optimization of the start codon of the nucleotide sequence of Mi3

[0074] The start codon TTG of the nucleotide sequence of the self-assembling protein Mi3 in Example 1 was replaced with ATG and GTG by whole plasmid PCR based on the strain M1 with the highest yield, to obtain strains FL-M1-1 and FL-M1-2. Other conditions or parameters were the same as in Example 1. The results are shown in Figure 2. Figure 6 As shown, the 2'-fucosyllactose production of the experimental group strains FL-M1-1 and FL-M1-2 reached 2.88 g / L and 2.79 g / L, respectively, which were 11.63% and 8.13% higher than the production of the control group FL-M1. Therefore, the strain FL-M1-1 was selected as the strain for the next fermentation tank production.

[0075] Example 4 Detection of 2'-fucosyllactose content in a fermenter

[0076] The fermentation performance of the FL-M1-1 strain was tested in a 5 L fermenter.

[0077] The FL-M1-1 strain constructed in Example 2 was inoculated into 10 mL of seed culture medium and cultured at 30°C and 200 rpm for 12-18 h to obtain seed liquid. The seed liquid was inoculated into a fermenter containing 3.5 L of DM culture medium at an inoculum size of 5% (v / v), with a pH of 7.1, a pressure of 1 MPa, a constant temperature of 30°C, 500 rpm, a ventilation volume of 1 vvm, and a fermentation cycle of 105 h.

[0078] like Figure 7As shown, at the end of fermentation, the yield and production intensity of 2'-fucosyllactose reached 44.6 g / L and 0.41 g / L / h, respectively.

[0079] Comparative Example

[0080] Referring to the method in Example 2, the C-termini of the five genes on the pJ01-BCDGF plasmid were connected to RA through a connecting peptide (GGGGS) 3 by whole-plasmid PCR to construct the pJ01-RA-B-RA-C-RA-D-RA-G-RA-F plasmid. pJ01-RA-B-RA-C-RA-D-RA-G-RA-F and pET22b-RD-Mi3 (pET22b-M1-RD-Mi3, pET22b-M2-RD-Mi3, pET22b-M3-RD-Mi3) were co-transformed to form strains FL-M1', FL-M2', and FL-M3'. In addition, a control group without RIAD / RIDD was constructed, that is, the MI3 protein was fused with the five proteins in pJ01-BCDGF respectively to form the pJ01-MI3-BCDGF plasmid, and the strain FL' was obtained. The above strains FL-M1', FL-M2', FL-M3' and FL' were fermented in DM medium.

[0081] result Figure 8 The control strain FL' produced only 1.2 g / L of 2'-fucosyllactose, a 29.41% decrease compared to the experimental strain FL-0. This suggests that direct fusion of the Mi3 protein with the pathway protein impairs the activity of the pathway enzyme, mitigating the decrease in 2'-fucosyllactose production. Furthermore, the RIAD / RIDD peptide pair can prevent direct fusion with the pathway enzyme, significantly increasing production. Furthermore, the 2'-fucosyllactose production of the control strains FL-M1', FL-M2', and FL-M3' was 2.18 g / L, 2.09 g / L, and 1.87 g / L, respectively. These yields were 15.5%, 16.06%, and 6.03% lower than those of the experimental strains FL-M1, FL-M2, and FL-M3, respectively. This suggests that adding three RA peptides to the C-termini of the ManB-ManC fusion protein, GmD-WcaG fusion protein, and FutC protein in the experimental group was more effective than adding the RA peptide pair to all five genes.

[0082] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria uses BL21 (DE3) ΔlacZ ΔcaJ as a starting strain, and expresses a gene encoding a self-assembly protein Mi3 and a gene encoding an enzyme related to the 2'-fucosyllactose de novo synthesis pathway; the gene encoding the self-assembly protein Mi3 is connected to a gene encoding a RIDD tag, and the gene encoding the enzyme related to the 2'-fucosyllactose de novo synthesis pathway is connected to a gene encoding a RIAD tag; the enzyme related to the 2'-fucosyllactose de novo synthesis pathway is assembled with the self-assembly protein Mi3 through the RIAD tag and the RIDD tag; the gene encoding the enzyme related to the 2'-fucosyllactose synthesis pathway includes a gene encoding a phosphomannose mutase ManB manB , gene encoding mannose-1-phosphate guanylyltransferase ManC manC , Gene encoding GDP-mannose 4,6-dehydratase Gmd gmd , the gene encoding GDP-L-fucose synthase WcaG wcaG and the gene encoding the fucosyltransferase FutC futC ; The genetically engineered bacteria co-express BC-RA fusion protein, DG-RA fusion protein and F-RA fusion protein using a pJ01 vector, wherein the BC-RA fusion protein comprises the RIAD tag protein, the ManB and the ManC sequentially connected via a connecting peptide (GGGGS) 3, the DG-RA fusion protein comprises the RIAD tag protein, the Gmd and the WcaG sequentially connected via a connecting peptide (GGGGS) 3, the F-RA fusion protein comprises the RIAD tag protein and the FutC connected via a connecting peptide (GGGGS) 3, and the nucleotide sequence of the self-assembly protein Mi3 is shown in SEQ ID NO.1; futC The nucleotide sequence is shown in SEQ ID NO.

6.

2. The genetically engineered bacterium according to claim 1, characterized in that The self-assembling protein Mi3 is connected to the RIDD tag through a linker peptide (GGGGS)3.

3. The genetically engineered bacterium according to claim 1, characterized in that The genetically engineered bacteria uses the pET22b plasmid as a vector to express the self-assembly protein Mi3 connected with a RIDD tag.

4. The genetically engineered bacterium according to claim 3, characterized in that The original RBS sequence of the pET22b plasmid is replaced with an M1, M2 or M3 gene fragment, and the nucleotide sequences of the M1, M2 and M3 gene fragments are shown in SEQ ID NOs. 10 to 12.

5. A method for constructing a genetically engineered bacterium producing 2'-fucosyllactose, characterized in that: The method comprises the following steps: using Escherichia coli BL21 (DE3) ΔlacZΔcaJ as a chassis strain, expressing a gene encoding a self-assembly protein Mi3 and a gene encoding an enzyme related to the de novo synthesis pathway of 2'-fucosyllactose on the chassis strain; the gene encoding the self-assembly protein Mi3 is connected to a gene encoding a RIDD tag, and the gene encoding the enzyme related to the de novo synthesis pathway of 2'-fucosyllactose is connected to a gene encoding a RIAD tag; the gene encoding the enzyme related to the de novo synthesis pathway of 2'-fucosyllactose includes a gene encoding a phosphomannose mutase ManB. manB , gene encoding mannose-1-phosphate guanylyltransferase ManC manC , Gene encoding GDP-mannose 4,6-dehydratase Gmd gmd , the gene encoding GDP-L-fucose synthase WcaG wcaG and the gene encoding the fucosyltransferase FutC futC ; The genetically engineered bacteria co-express BC-RA fusion protein, DG-RA fusion protein and F-RA fusion protein using the pJ01 vector, wherein the BC-RA fusion protein comprises the RIAD tag protein, the ManB and the ManC connected in sequence by a connecting peptide (GGGGS) 3, the DG-RA fusion protein comprises the RIAD tag protein, the Gmd and the WcaG connected in sequence by a connecting peptide (GGGGS) 3, and the F-RA fusion protein comprises the RIAD tag protein and the FutC connected by a connecting peptide (GGGGS) 3.

6. A method for producing 2'-fucosyllactose, characterized in that: The genetically engineered bacteria according to any one of claims 1 to 4 are used to ferment and produce 2'-fucosyllactose.

7. The method according to claim 6, characterized in that The fermentation conditions are 30-38°C, 200-220 rpm, and the initial OD of the strain is 600 0.04-0.1, fermentation 70-110 h; Or the fermentation conditions are 30-38°C, 480-530 rpm, inoculation volume of 5-10%, liquid volume of 30-50%, pH of 6.0-7.0, and initial OD of the strain fermentation. 600 The pH value is 0.04-0.3, the aeration rate is 1-2 vvm, and the fermentation time is 70-120 h.

8. Use of the genetically engineered bacterium according to any one of claims 1 to 4 in the preparation of 2'-fucosyllactose or a product containing 2'-fucosyllactose.

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