An engineered bacterium with high fucose production, and its construction method and application

Abstract

The present application relates to the field of microbial genetic engineering technology, and in particular to an engineered bacterium with high fucose production, and its construction method and application. The present application provides an engineered bacterium with high fucose production, wherein the engineered bacterium overexpresses wbgL, afcA, BCGW gene clusters, self-assembling short peptides RIAD-RIDD and RGG domains. The present application introduces a protein (RGG) sequence formed by membraneless organelles and a self-assembling short peptide (RIAD-RIDD) to mediate the assembly and combination of key enzymes WbgL and AcfA in the pathway into an enzyme complex, thereby reducing the loss of metabolic intermediates, improving product production efficiency, and increasing the yield and yield of product fucose.

Description

Technical Field

[0001] The present application relates to the technical field of microbial genetic engineering, and in particular to an engineered bacterium with high fucose production, a construction method thereof, and an application thereof. Background Art

[0002] Fucose (FUC), also known as 6-deoxygalactose, is a ubiquitous natural sugar. L-fucose is one of the five monosaccharides that make up human milk oligosaccharides and one of the eight essential sugars for the human body. It is widely found in animals, plants, and algae, playing important physiological roles. L-fucose can regulate intestinal flora and maintain intestinal health; it can bind to viruses, bacteria, and toxins, preventing them from infecting cells and thus enhancing the body's immune system. L-fucose has skin moisturizing properties, delaying skin aging, stimulating fibroblast proliferation, and protecting fibroblasts from damage caused by radiation and ultraviolet rays. Currently, commercial production of FUC primarily involves extraction and purification of fucoidan using alginate hydrolysis, chemical conversion using hexose as a raw material, and enzymatic hydrolysis of the extracellular polysaccharides of fucose-producing bacteria. These L-fucose production methods suffer from low economic efficiency, environmental impact, and limited application scenarios. Therefore, the development of new L-fucose production methods has potential application value. The biosynthesis method only requires cheap carbon sources and renewable donors within cells as raw materials, and can achieve high economic output at a low environmental cost, so it has broader application prospects.

[0003] Currently, the yield of Fucoxanthin (FUC) synthesized by microorganisms remains low, unable to meet the needs of large-scale industrial production. Previous studies have shown that plasmid-based strains synthesizing FUC often experience plasmid loss, preventing stable and efficient FUC synthesis, and the rate-limiting factor for FUC synthesis remains unknown. Furthermore, excessive 2'-FL residues can impair FUC product purification, necessitating the development of engineered bacteria that can reduce 2'-FL residues to increase FUC levels. Summary of the Invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide an engineered bacterium that utilizes membraneless organelles to increase FUC production while reducing 2'-FL, as well as a construction method and application thereof.

[0005] To achieve the above objectives, the technical solutions adopted in this application are:

[0006] In a first aspect, the present application provides an engineered bacterium with high fucose production, which overexpresses α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA, BCGW gene cluster, self-assembling short peptide RIAD-RIDD and RGG domain.

[0007] The engineered bacteria of the present application overexpress α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA, and BCGW gene clusters to be able to biosynthesize fucose in vivo. The biosynthesis process of fucose is as follows: fructose-6-phosphate is synthesized into GDP-L-fucose under the action of genes manA, manB, manC, gmd, and wcaG, extracellular lactose is transported into the cell by β-galactoside permease LacY, and then reacts with GDP-L-fucose to synthesize 2'-FL, which is decomposed into lactose and fucose under the action of α-L-fucosidase. The present application introduces a protein (RGG) sequence formed by membraneless organelles and a self-assembling short peptide (RIAD-RIDD) to mediate the assembly and combination of the key enzymes WbgL and AcfA in the pathway into an enzyme complex, thereby reducing the loss of metabolic intermediates, improving product production efficiency, and increasing the yield and yield of the product fucose.

[0008] As a preferred embodiment of the engineered bacteria described in this application, the amino acid sequence of the α-1,2-fucosyltransferase wbgL is shown in SEQ ID NO.4;

[0009] The amino acid sequence of the α-L-fucosidase afcA is shown in SEQ ID NO.5;

[0010] The BCGW gene cluster is a gene cluster that integrates phosphomannose mutant enzyme manB, mannose 1-1 phosphate guanosine transferase manC, GDP-D-mannose-4,6-dehydratase gmd and GDP-L-fucose synthase wcaG. The amino acid sequence of the BCGW gene cluster is shown in SEQ ID NO.6.

[0011] As a preferred embodiment of the engineered bacteria described in the present application, the nucleotide sequence of RIAD in the self-assembling short peptide RIAD-RIDD is shown as SEQ ID NO.7, and the nucleotide sequence of RIDD in the self-assembling short peptide RIAD-RIDD is shown as SEQ ID NO.8;

[0012] The amino acid sequence of the RGG domain is shown in SEQ ID NO. 9. The RGG domain of the present application is derived from the LAF-1 protein of Caenorhabditis elegans and can form protein aggregates in Escherichia coli. The RIDD connected to the RGG domain is integrated into the E. coli genome, reducing the risk of decreased fucose production due to plasmid loss during fucose synthesis.

[0013] As a preferred embodiment of the engineered bacteria described in this application, the RIAD in the self-assembling short peptide is located at the N-terminus and / or C-terminus of the α-1,2-fucosyltransferase wbgL, and the RIAD in the self-assembling short peptide is located at the C-terminus of the α-L-fucosidase afcA. During the experiment, the inventors found that connecting the RIAD to the N-terminus or C-terminus of wbgL and to the C-terminus of afcA can effectively increase the production of fucose. However, when the RIAD was connected to the N-terminus of afcA, the production of fucose decreased significantly.

[0014] As a preferred embodiment of the engineered bacteria described in the present application, the copy number ratio of α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA and BCGW gene cluster in the engineered bacteria is α-1,2-fucosyltransferase wbgL: α-L-fucosidase afcA: BCGW gene cluster = (1-3): (1-3): (1-5).

[0015] As a preferred embodiment of the engineered bacteria described in the present application, the copy number ratio of the α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA and BCGW gene cluster in the engineered bacteria is α-1,2-fucosyltransferase wbgL: α-L-fucosidase afcA: BCGW gene cluster = 3: (2-3): (4-5).

[0016] This application optimizes the copy number of the key protein α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA and BCGW gene cluster for fucose synthesis. The results show that when the copy number ratio of wbgL, afcA and BCGW is wbgL:afcA:BCGW=3:(2-3):(4-5), the fucose production of the prepared engineered bacteria increases with the increase of the copy number of wbgL, afcA and BCGW, and the maximum fucose production can reach 95.88g / L.

[0017] As a preferred implementation of the engineered bacteria described in the present application, the integration sites of the α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA, BCGW gene cluster, self-assembling short peptide RIAD-RIDD and RGG domain include at least one of caiB, lacA, hlyE, wzxC, intQ, ykgH, araA, ydeU, yjiV, yjipQ, ybeQ and rhaA.

[0018] As a preferred embodiment of the engineered bacteria described in the present application, the promoter used by the gene integrated into the engineered bacteria is a strong promoter, and the strong promoter is at least one of a T7 promoter, a CaMV promoter, a SV40 promoter, and a SFFV promoter.

[0019] As a preferred embodiment of the engineered bacteria described in the present application, the engineered bacteria is prepared by integrating α-1,2-fucosyltransferase wbgL with a strong promoter, α-L-fucosidase afcA, BCGW gene cluster, self-assembling short peptide RIAD-RIDD and RGG domain with engineered bacteria A as the chassis strain.

[0020] As a preferred embodiment of the engineered bacteria described in the present application, the engineered bacteria A has knocked out the genes of β-galactosidase lacZ, fucose isomerase / fucokinase gene cluster fucIK and undecyl phosphate glucose phosphotransferase wcaj.

[0021] In the present application, the engineered bacteria A is obtained by modifying the chassis strain Escherichia coli BL21 (DE3) and knocking out the genes of β-galactosidase lacZ, fucose isomerase / fucokinase gene cluster fucIK and undecyl phosphate glucose phosphotransferase wcaj.

[0022] In a second aspect, the present application provides a method for constructing the above-mentioned engineered bacteria, comprising the following steps:

[0023] S1. Using gene editing technology, the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK, and the undecyl-phosphate glucose phosphotransferase wcaj genes in Escherichia coli BL21 (DE3) were knocked out to obtain engineered strain A.

[0024] S2, integrating the α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA, and BCGW gene clusters into the engineered bacteria A obtained in step S1 to obtain engineered bacteria B;

[0025] S3. Integrate the fragment of the RIAD in the self-assembling short peptide connected to the N-terminus and / or C-terminus of α-1,2-fucosyltransferase wbgL, the fragment of the RIAD in the self-assembling short peptide connected to the C-terminus of α-L-fucosidase afcA, and the fragment connected to the RGG domain of the self-assembling short peptide into the engineered bacteria B obtained in step S2 to obtain an engineered bacteria with high fucose production.

[0026] As a preferred embodiment of the construction method described in the present application, in step S1, the amino acid sequence of the β-galactosidase lacZ is as shown in SEQ ID NO.1;

[0027] The amino acid sequence of the fucose isomerase / fucokinase gene cluster fucIK is shown in SEQ ID NO.2;

[0028] The amino acid sequence of the undecyl-glucose phosphate phosphotransferase wcaj is shown in SEQ ID NO.3.

[0029] In a third aspect, the present application provides the use of the above-mentioned engineered bacteria in the production of fucose.

[0030] In a fourth aspect, the present application provides a method for producing fucose, which is mainly obtained by fed-batch fermentation of the above-mentioned engineered bacteria.

[0031] Compared with the prior art, this application has the following beneficial effects:

[0032] 1. This application transforms the chassis strain engineered bacteria A to overexpress the α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA, and BCGW gene cluster, so that Escherichia coli can biosynthesize fucose in vivo.

[0033] 2. The present application connects a self-assembling short peptide RIAD to the N-terminus or C-terminus of wbgL and to the C-terminus of afcA, and connects the RGG domain on the LAF-1 protein from Caenorhabditis elegans to the self-assembling short peptide RIDD, thereby enabling WbgL and AfcA to assemble into membraneless organelles, thereby reducing the loss of intermediate products (2'-FL, etc.) to increase the fucose production and yield, and achieving the purpose of high fucose production.

[0034] 3. This application optimizes the copy number of the key proteins for fucose synthesis, α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA and BCGW gene cluster. The results show that when the copy number ratio of wbgL, afcA and BCGW is wbgL:afcA:BCGW = (1-3): (1-3): (1-5), the fucose production of the prepared engineered bacteria increases with the increase of the copy number of wbgL, afcA and BCGW, and the maximum fucose production can reach 95.88 g / L. DETAILED DESCRIPTION

[0035] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below in conjunction with specific embodiments.

[0036] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0037] In the following examples, the gene information used is shown in Table 1.

[0038] Table 1 Genes and their information used in the following examples

[0039]

[0040] In the following examples and comparative examples, unless otherwise specified, the methods used for transferring plasmids into competent cells or for transformation were all chemical transformations; the resistance plates used were kanamycin resistance plates; and the positive transformants screened were identified by PCR detection and cloned fragment sequencing.

[0041] In the following examples and comparative examples, unless otherwise specified, the cloning described herein refers to cloning using a ligase-independent single-fragment one-step cloning kit, and the operation was performed according to the kit instructions. The ligase-independent single-fragment one-step cloning kit was provided by Nanjing Novozymes Biotech Co., Ltd., with the product number C112-02.

[0042] The trace element solution mainly consists of 13.74 g / L nitrilotriacetic acid sodium salt, 5.6 g / L ammonium ferric citrate, 0.9 g / L ZnSO2·7H2O, 0.2 g / L CoCl2·6H2O, 1.0 g / L MnCl2·4H2O, 0.10 g / L CuCl2·2H2O, 0.2 g / L H3BO3, and 0.2 g / L Na2MoO4·2H2O, and is sterilized by filtration using a 0.22 μm pore size filter membrane.

[0043] The fermentation medium used in the fed-batch fermentation experiment contained 10 g / L initial glycerol, 4.0 g / L (NH4)2SO4, 9.2 g / L K2HPO4, 8.2 g / L KH2PO4, 0.3 g / L citric acid, 6.0 g / L tryptone, 2.0 g / L yeast extract, 10 mg / L thiamine, 2.0 g / L MgSO4·7H2O, 0.02 g / L CaCl2 and 10 mL / L trace element solution.

[0044] The feed solution used in the fed-batch fermentation experiment included 800 g / L carbon source and 5 g / LMgSO4·7H2O. The carbon source was glucose and glycerol, and the mass ratio of glucose to glycerol was glucose:glycerol=4:6.

[0045] The pEcCas vector, pSPIN plasmid, pCDFDuet plasmid, pRSF plasmid, pETDuet plasmid, and pEcgRNA plasmid were all provided by Addgene.

[0046] The method for preparing the expression cassette fragments of T7-BCGW, T7-wbgL and T7-afcA is as follows: phosphomannose mutase manB (GeneID: 946574), α-D-mannose 1-phosphate guanylyltransferase manC (GeneID: 946580), GDP-mannose 4,6-dehydrogenase gmd (GeneID: 946562) and GDP-L-fucose synthase wcaG (Gene ID: 946563) from Escherichia coli str. K-12 substr. MG1655 (GenBank: NC_000913.3) were PCR amplified respectively, and inserted into the vector (pRSFDuet-1) through the corresponding restriction sites to construct the recombinant plasmid pRSFDuet-manC-manB-gmd-wcaG;

[0047] Based on the sequence of the α-1,2-fucosyltransferase gene wbgL in Escherichia coli O126, the sequence of the α-1,2-fucosyltransferase gene wbGL was obtained through codon optimization and chemical synthesis. The wbgL gene fragment was amplified by PCR using the wbgL-F / R primer pair (see Table 1) and inserted into the vector pETDuet-1 through the corresponding restriction sites to obtain the recombinant vector pETDuet-wbgL.

[0048] The afcA-specific sequence was obtained through codon optimization and chemical synthesis, and the expression cassette fragment containing afcA was cloned into the pETDuet-1 plasmid to obtain the pETD-afcA plasmid;

[0049] The expression cassette fragments of T7-BCGW, T7-wbgL and T7-afcA were obtained in the above-mentioned plasmids (pRSFDuet-manC-manB-gmd-wcaG, pETDuet-wbgL and pETD-afcA) by PCR technology, and the PCR primers used were BCGW-transF / BCGW-transR, WbgL-transF / WbgL-tra nsR and AfcA-transF / AfcA-transR, respectively.

[0050] The techniques not described in detail in the following embodiments and effect examples are all commonly used techniques in the art. Please refer to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiment" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0051] Example 1

[0052] This example provides an engineered bacterium that produces high fucose and a method for constructing the same. The method comprises the following steps:

[0053] 1.1 The lacZ, fucIK, and wcaj genes in Escherichia coli star BL21(DE3) were knocked out using a modified CRISPR-Cas9 technique to generate strain A. The knockout vector used was the pEcCas vector containing Cas9 and λ-Red recombinase, and targeted gene editing was performed using a pEcgRNA containing an sgRNA sequence and an N20-specific sequence.

[0054] 1.2 The caiB-N32-specific sequence was cloned into the pSL1765 plasmid to obtain the pSPIN-caiB(N32) plasmid. The T7-manB-manC-T7-Gmd-wcaG expression cassette was obtained by PCR using the pRSFDuet-T7-manB-manC-Gmd-wcaG plasmid as a template. The T7-manB-manC-T7-Gmd-wcaG expression cassette fragment was cloned into the pSPIN-caiB(N32) plasmid to obtain the pSPIN-caiB(N32)-BCGW plasmid, which was transformed into the strain A obtained in step 1.1. Positive transformants were identified by caiB-DF / caiB-DR primers. After removing the plasmid from the positive transformants, strain B was obtained.

[0055] 1.3 Follow the procedure in step 1.2 to integrate the T7-wbgL expression cassette into the lacA site of strain B obtained in step 1.2 to obtain strain C;

[0056] 1.4 Follow the procedure in step 1.2 to integrate the T7-afcA expression cassette into the hlyE site of strain C obtained in step 1.3 to obtain strain D;

[0057] 1.5 Entrust a biotechnology company to construct the pCDFDuet plasmid containing the T7 promoter, the RGG domain, and the self-assembling short peptide RIDD, and connect the self-assembling short peptide RIDD and the RGG domain using a connecting peptide (GGGGS)3 to obtain the pCDFDuet-RGG-RIDD plasmid;

[0058] 1.6 A biotechnology company was commissioned to construct the self-assembling short peptide RIAD sequence at the N-terminus of wbgL and the C-terminus of afcA and cloned into the pETDuet plasmid to obtain the pETDuet-RIAD-wbgL and pETDuet-RIAD-afcA plasmids, respectively;

[0059] 1.7 Use PCR to obtain the T7-RGG-RIDD expression cassette fragment from the pCDFDuet-RGG-RIDD plasmid obtained in step 1.5. Also, obtain the T7-RIAD-wbgL and T7-afcA-RIAD expression cassette fragments from the pETDuet-RIAD-wbgL and pETDuet-RIAD-afcA plasmids obtained in step 1.6, respectively.

[0060] 1.8 Integrate the T7-RGG-RIDD, T7-RIAD-wbgL, and T7-afcA-RIAD expression cassette fragments obtained in step 1.7 into strain D obtained in step 1.7, wherein T7-RGG-RIDD is integrated into the nagB site, T7-RIAD-wbgL is integrated into the lacA site, and T7-afcA-RIAD is integrated into the hlyE site. Positive transformants are screened by resistance plate screening. After removing the plasmid from the positive transformants, the engineered strain FUC-R-02 is obtained. At this time, the copy number ratio of BCGW, wbgL, and afcA in FUC-R-02 is BCGW:wbgL:afcA = 2:1:1;

[0061] The primer sequences used in the above steps are shown in Table 2.

[0062] Table 2 Primers used to construct E. coli FUC04

[0063]

[0064]

[0065] Example 2

[0066] This example provides an engineered bacterium that produces high fucose and a method for constructing the same. The construction method is similar to that of Example 8, except that in step 1.6, the self-assembling short peptide RIAD sequence is constructed at the C-terminus of wbgL to obtain the pETDuet-wbgL-RIAD plasmid. The remaining steps and parameter conditions remain unchanged, and the engineered bacterium obtained in step 8.4 is named FUC-R-04.

[0067] Example 3

[0068] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0069] 3.1 According to the operation in step 1.2 of Example 1, the expression cassette fragment of T7-BCGW was integrated into the wzxC site of the engineered bacteria FUC-R-04 obtained in Example 2 to obtain the engineered bacteria FUC2C-1. At this time, the copy number ratio of BCGW, wbgL and afcA in FUC2C-1 was BCGW:wbgL:afcA=2:1:1;

[0070] The primers used in the above steps are shown in Table 3.

[0071] Table 3 Primers used to construct engineered bacteria FUC2C-1

[0072]

[0073] Example 4

[0074] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0075] 4.1 According to the procedure of step 1.2 of Example 1, the expression cassette fragment of T7-wbgL was integrated into the ydeU site of the engineered strain FUC2C-1 obtained in Example 3 to obtain the engineered strain FUC2C-2. At this time, the copy number ratio of BCGW, wbgL and afcA in FUC2C-2 was BCGW:wbgL:afcA=2:2:1;

[0076] The primers used in the above steps are shown in Table 4.

[0077] Table 4 Primers used to construct engineered bacteria FUC2C-2

[0078]

[0079] Example 5

[0080] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0081] 5.1 According to the procedure of step 1.2 of Example 1, the expression cassette fragment of T7-BCGW was integrated into the intQ site of the engineered strain FUC2C-1 obtained in Example 3 to obtain the engineered strain FUC2C-3. At this time, the copy number ratio of BCGW, wbgL and afcA in FUC2C-3 was BCGW:wbgL:afcA=3:1:1;

[0082] The primers used in the above steps are shown in Table 5.

[0083] Table 5 Primers used to construct engineered bacteria FUC2C-3

[0084]

[0085] Example 6

[0086] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0087] 6.1 Following the procedure in step 1.2 of Example 1, the T7-wbgL expression cassette fragment was integrated into the yjipQ site of the engineered strain FUC2C-3 obtained in Example 5 to obtain engineered strain FUC2C-4. The copy number ratio of BCGW, wbgL, and afcA in FUC2C-4 was BCGW:wbgL:afcA = 3:2:1.

[0088] The primers used in the above steps are shown in Table 6.

[0089] Table 6 Primers used to construct engineered bacteria FUC2C-4

[0090]

[0091] Example 7

[0092] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0093] 7.1 Following the procedure in step 1.2 of Example 1, the T7-afcA expression cassette fragment was integrated into the ybeQ site of the engineered strain FUC2C-4 obtained in Example 6 to obtain engineered strain FUC2C-5. The copy number ratio of BCGW, wbgL, and afcA in FUC2C-5 was BCGW:wbgL:afcA = 3:2:2.

[0094] The primers used in the above steps are shown in Table 7.

[0095] Table 7 Primers used to construct engineered bacteria FUC2C-5

[0096]

[0097] Example 8

[0098] This embodiment provides a high-fucose-producing Escherichia coli and a construction method thereof, wherein the construction method comprises the following steps:

[0099] 8.1 Following the procedure in step 1.2 of Example 1, the T7-BCGW expression cassette fragment was integrated into the araA site of the engineered strain FUC2C-5 obtained in Example 7 to obtain engineered strain FUC2C-6. The copy number ratio of BCGW, wbgL, and afcA in FUC2C-6 was BCGW:wbgL:afcA = 4:2:2.

[0100] The primers used in the above steps are shown in Table 8.

[0101] Table 8 Primers used to construct engineered bacteria FUC2C-6

[0102]

[0103] Example 9

[0104] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0105] 9.1 Following the procedure in step 1.2 of Example 1, the T7-wbgL expression cassette fragment was integrated into the yjiV site of the engineered strain FUC2C-6 obtained in Example 8 to obtain engineered strain FUC2C-7. The copy number ratio of BCGW, wbgL, and afcA in FUC2C-7 was BCGW:wbgL:afcA = 4:3:2.

[0106] The primers used above are shown in Table 9.

[0107] Table 9 Primers used to construct engineered bacteria FUC2C-7

[0108]

[0109] Example 10

[0110] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0111] 10.1 Following the procedure in step 1.2 of Example 1, the T7-afcA expression cassette fragment was integrated into the rhaA site of the engineered strain FUC2C-7 obtained in Example 9 to obtain engineered strain FUC2C-8. The copy number ratio of BCGW, wbgL, and afcA in FUC2C-8 was BCGW:wbgL:afcA = 4:3:3.

[0112] The primers used above are shown in Table 10.

[0113] Table 10 Primers used to construct engineered bacteria FUC2C-8

[0114]

[0115]

[0116] Example 11

[0117] This embodiment provides an engineered bacterium with high fucose production and a method for constructing the same. The method comprises the following steps:

[0118] 11.1 Following the procedure in step 1.2 of Example 1, the T7-BCGW expression cassette fragment was integrated into the ykgH site of the engineered strain FUC2C-7 obtained in Example 9 to obtain engineered strain FUC2C-9. The copy number ratio of BCGW, wbgL, and afcA in FUC2C-9 was BCGW:wbgL:afcA = 5:3:2.

[0119] The primers used above are shown in Table 11.

[0120] Table 11 Primers used to construct engineered bacteria FUC2C-9

[0121]

[0122] Comparative Example 1

[0123] This comparative example provides an engineered bacterium with high fucose production and a method for constructing the same. The construction method is similar to that of Example 1, except that in step 1.6, the self-assembling short peptide RIAD sequence is constructed at the N-terminus of afcA to obtain the pETDuet-RIAD-afcA plasmid. The remaining steps and parameter conditions remain unchanged, and the engineered bacterium obtained in step 8.4 is named FUC-R-01.

[0124] Comparative Example 2

[0125] This comparative example provides an engineered bacterium with high fucose production and a method for constructing the same. The construction method is similar to that of Example 1, except that in step 1.6, the self-assembling short peptide RIAD sequence is constructed at the N-terminus of afcA to obtain the pETDuet-RIAD-afcA plasmid. The remaining steps and parameter conditions remain unchanged, and the engineered bacterium obtained in step 8.4 is named FUC-R-03.

[0126] Effect Examples

[0127] 1. The engineered bacteria of Examples 1-11 and Comparative Examples 1-2 were subjected to a fed-batch fermentation experiment, and the fucose production in the fermentation broth was detected by high performance liquid chromatography.

[0128] 1. Fed-batch fermentation experiment

[0129] (1) Escherichia coli was cultured in a 1 L shake flask containing 150 mL of LB medium for 6 h to obtain a secondary seed solution;

[0130] (2) 150 mL of the seed solution obtained in step (1) was transferred to a 5 L fermentor containing 2.5 L of fermentation medium, and 28% (v / v) NH4OH was automatically added to adjust the pH to 6.8. The dissolved oxygen was maintained at 30-50% by automatically controlling the stirring speed. The culture temperature was 37°C, the ventilation rate was 2 VVM, and the rotation speed was 900 rpm;

[0131] (3) When the initial glycerol in the fermentation medium was completely consumed, the feed solution was added to the fermenter at a constant rate of 4 g / L / h, the fermentation temperature was adjusted to 29.5°C, and the fermentation was continued for 4 h;

[0132] (4) adding isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.2 mM and 40 g of lactose, supplemented with 17.78 g / L / h of glycerol solution, and culturing for 90 h before fermentation to obtain a fermentation broth.

[0133] 2. Post-treatment of fermentation broth and HPLC detection

[0134] 1 mL of fermentation broth was placed in a boiling water bath for 10 min to kill the strain, and then centrifuged at 12000 g for 10 min. The supernatant was filtered using a 0.22 μm filter, and the filtrate was used to detect FUC and 2'-FL.

[0135] FUC and 2'-FL in the samples were determined using an HPX.87H column (Bio-Rad) and a high-performance liquid chromatograph (LC-16, Shimadzu, Japan). The FUC and 2'-FL contents in the samples were calculated using the external standard method. The chromatographic conditions were a mobile phase of 5 mM aqueous sulfuric acid, a flow rate of 0.5 mL / min, a column temperature of 60°C, and an injection volume of 10 μL.

[0136] A FUC standard with a gradient concentration of 0-2.0 g / L was taken, and a standard curve was drawn with FUC concentration as the horizontal axis and liquid chromatography peak area as the vertical axis; a 2'-FL standard curve was drawn according to the FUC method.

[0137] The calculated FUC and 2'-FL concentrations in different fermentation broths were converted to the yields obtained by fermentation in a 5 L fermenter. The results are shown in Table 12.

[0138] Table 12 Calculation results of FUC and 2'-FL production of different engineered bacteria

[0139]

[0140]

[0141] The present application transforms the chassis strain Escherichia coli BL21 (DE3), knocks out the genes of β-galactosidase lacZ, fucose isomerase / fucokinase gene cluster fucIK and undecyl phosphate glucose phosphotransferase wcaj, and integrates phosphomannose mutant enzyme manB, mannose 1-1 phosphate guanyl transferase manC, GDP-D-mannose-4,6-dehydratase gmd and GDP-L-fucose synthase wcaG gene cluster BCGW, α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA, BCGW gene cluster, so that Escherichia coli can biosynthesize fucose in vivo. As shown in Table 1, the present application connects a self-assembling short peptide RIAD to the N-terminus or C-terminus of wbgL and to the C-terminus of afcA, and connects the RGG domain on the LAF-1 protein from Caenorhabditis elegans to the self-assembling short peptide RIDD, thereby enabling WbgL and AfcA to be assembled into membraneless organelles, thereby reducing the loss of intermediate products (2'-FL, etc.) to increase the fucose production and yield, and achieving the purpose of high fucose production. On this basis, the present application optimized the copy numbers of the key proteins for fucose synthesis, α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA and BCGW gene cluster. The results showed that when the copy number ratio of wbgL, afcA and BCGW was wbgL:afcA:BCGW = (1-3): (1-3): (1-5), the fucose production of the prepared engineered bacteria increased with the increase of the copy number of wbgL, afcA and BCGW, and the maximum fucose production could reach 95.88 g / L.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. An engineered bacterium with high fucose production, characterized in that: The engineered bacteria is prepared by using engineered bacteria A as a base strain and integrating α-1,2-fucosyltransferase wbgL with a strong promoter, α-L-fucosidase afcA, BCGW gene cluster, self-assembling short peptide RIAD-RIDD and RGG domain; The engineered bacteria A is obtained by knocking out the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK, and the undecyl-phosphate glucose phosphotransferase wcaj genes in Escherichia coli BL21 (DE3) using gene editing technology; The amino acid sequence of the α-1,2-fucosyltransferase wbgL is shown in SEQ ID NO.4; The amino acid sequence of the α-L-fucosidase afcA is shown in SEQ ID NO.5; The BCGW gene cluster is a gene cluster that integrates phosphomannose mutant enzyme manB, mannose 1-1 phosphate guanosyltransferase manC, GDP-D-mannose-4,6-dehydratase gmd and GDP-L-fucose synthase wcaG. The amino acid sequence of the BCGW gene cluster is shown in SEQ ID NO.6; The RIAD nucleotide sequence in the self-assembling short peptide RIAD-RIDD is shown in SEQ ID NO.7, and the RIDD nucleotide sequence in the self-assembling short peptide RIAD-RIDD is shown in SEQ ID NO.8; The amino acid sequence of the RGG domain is shown in SEQ ID NO.9; The RIAD in the self-assembling short peptide is located at the N-terminus and / or C-terminus of α-1,2-fucosyltransferase wbgL, or the RIAD in the self-assembling short peptide is located at the C-terminus of α-L-fucosidase afcA; The copy number ratio of the α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA and BCGW gene cluster in the engineered bacteria is α-1,2-fucosyltransferase wbgL:α-L-fucosidase afcA:BCGW gene cluster=3:(2-3):(4-5); The amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO.1; The amino acid sequence of the fucose isomerase / fucokinase gene cluster fucIK is shown in SEQ ID NO.2; The amino acid sequence of the undecyl-glucose phosphate phosphotransferase wcaj is shown in SEQ ID NO.

3.

2. The method for constructing an engineered bacterium according to claim 1, wherein: The following steps are involved: S1. Using gene editing technology, the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK, and the undecyl-phosphate glucose phosphotransferase wcaj genes in Escherichia coli BL21 (DE3) were knocked out to obtain engineered strain A. S2, integrating the α-1,2-fucosyltransferase wbgL, α-L-fucosidase afcA, and BCGW gene clusters into the engineered bacteria A obtained in step S1 to obtain engineered bacteria B; S3. Integrate the fragment of the RIAD in the self-assembling short peptide connected to the N-terminus and / or C-terminus of α-1,2-fucosyltransferase wbgL, the fragment of the RIAD in the self-assembling short peptide connected to the C-terminus of α-L-fucosidase afcA, and the fragment connected to the RGG domain of the self-assembling short peptide into the engineered bacteria B obtained in step S2 to obtain an engineered bacteria with high fucose production.

3. The construction method according to claim 2, wherein: In step S1, the amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO.1; The amino acid sequence of the fucose isomerase / fucokinase gene cluster fucIK is shown in SEQ ID NO.2; The amino acid sequence of the undecyl-glucose phosphate phosphotransferase wcaj is shown in SEQ ID NO.

3.

4. Use of the engineered bacteria as claimed in claim 1 in producing fucose.

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