Engineering bacterium for synthesizing fucose by taking 3-fucosyllactose as precursor as well as construction method and application of engineering bacterium

Abstract

The invention relates to the technical field of microbial genetic engineering, in particular to an engineering bacterium for synthesizing fucose by taking 3-fucosyllactose as a precursor as well as a construction method and application of the engineering bacterium. The invention provides an engineering bacterium capable of realizing high yield of fucose, and the engineering bacterium is used for expressing alpha-1, 3-fucosyltransferase and alpha-L-fucosidase. According to the application, firstly, alpha-L-fucosidase of different sources is obtained through heterologous expression, the alpha-L-fucosidase with the activity of catalyzing conversion of 3-FL into FUC is obtained through screening, on the basis, in order to further increase the yield of FUC, endogenous sugar transporter is knocked out, so that 3-FL is left in cells as much as possible to react with the alpha-L-fucosidase, and the yield of FUC is increased. Therefore, the yield of the FUC is improved.

Description

Technical Field

[0001] The present application relates to the technical field of microbial genetic engineering, and in particular to an engineering bacterium for synthesizing fucose using 3-fucosyllactose as a precursor, and a construction method and application thereof. Background Art

[0002] Fucose (Fucose, FUC) is a widely existing natural sugar, also known as 6-deoxy-galactose. L-fucose is one of the five monosaccharides that constitute human milk oligosaccharides. It is widely present in animals, plants, and algae, and plays an important physiological function. L-fucose has the effects of regulating intestinal health, enhancing body resistance, and delaying skin aging. The commercial production of FUC mainly includes methods such as extracting fucoidan by hydrolysis of alginate and then purifying it, chemical methods using hexose as raw material conversion, and enzymatic hydrolysis of extracellular polysaccharides of fucose-producing bacteria. These L-fucose production methods have low economic benefits, are unfriendly to the environment, and have limited application scenarios. Therefore, the invention of a new L-fucose production method has potential application value. The biosynthesis method only requires cheap carbon sources and intracellular renewable donors as raw materials, and obtains high economic output at a lower environmental cost, so it has a broader application prospect. However, the microbial synthesis yield of FUC is still low, and the synthesis path is single, which cannot break through the yield bottleneck. Therefore, it is urgent to develop a new synthesis path to increase the yield of FUC. Summary of the invention

[0003] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide an engineering bacterium capable of synthesizing fucose using 3-fucosyllactose, and a construction method and application thereof.

[0004] To achieve the above purpose, the technical solution adopted by this application is:

[0005] In a first aspect, the present application provides an engineered bacterium that synthesizes fucose using 3-fucosyllactose as a precursor, wherein the engineered bacterium expresses α-1,3-fucosyltransferase and α-L-fucosidase.

[0006] The present application first obtains α-L-fucosidase from different sources through heterologous expression to screen for α-L-fucosidase with the activity of catalyzing the conversion of 3-FL to FUC. On this basis, in order to further increase the yield of FUC, the endogenous sugar transporter is knocked out so that 3-FL remains in the cell as much as possible to react with α-L-fucosidase, thereby increasing the yield of FUC.

[0007] As a preferred embodiment of the engineering bacteria described in the present application, the α-L-fucosidase is α-L-fucosidase AfcA or α-L-fucosidase AfcB. In order to confirm the 3-FL catalytic activity of α-L-fucosidase, the inventors prepared α-L-fucosidases from different sources to conduct 3-FL catalytic experiments, and screened out α-L-fucosidase AfcA (derived from Bifidobacterium) and AfcB (derived from Bifidobacterium bifidum JCM 1254) that catalyze 3-FL to FUC, so that the prepared engineering bacteria have the activity of converting 3-FL into FUC, and realize the total biosynthesis of FUC in the engineering bacteria.

[0008] As a preferred embodiment of the engineered bacteria described in the present application, the amino acid sequence of the α-1,3-fucosyltransferase is shown in SEQ ID NO.1;

[0009] The amino acid sequence of the α-L-fucosidase AfcA is shown in SEQ ID NO.3;

[0010] The amino acid sequence of the α-L-fucosidase AfcB is shown in SEQ ID NO.4.

[0011] As a preferred embodiment of the engineered bacteria described in the present application, the engineered bacteria also express the BCGW gene cluster and knock out the sugar transporter.

[0012] As a preferred embodiment of the engineered bacteria described in the present application, the BCGW gene cluster is a gene cluster integrating phosphomannose mutant enzyme manB, mannose-1-phosphate guanosine transferase manC, GDP-D-mannose-4,6-dehydratase gmd and GDP-L-fucose synthase wcaG, and the amino acid sequence of the BCGW gene cluster is shown in SEQ ID NO.2.

[0013] In the present application, fructose-6-phosphate is used as a substrate to synthesize GDP-L-fucose under the action of genes manA, manB, manC, gmd and wcaG, and extracellular lactose is transported into the cell by β-galactoside permease LacY, and then 3-FL is synthesized with GDP-L-fucose under the action of α-1,3-fucosyltransferase, and 3-FL is decomposed into fucose under the action of α-L-fucosidase, thereby realizing the total synthesis of fucose.

[0014] As a preferred embodiment of the engineered bacteria described in the present application, the sugar transporter includes at least one of a multidrug efflux system protein MdfA, a MFS transporter protein MdtD and an outer membrane porin C OmpC.

[0015] During the experiment, the present application found that the catalytic efficiency of α-L-fucosidase with catalytic 3-FL activity was not ideal. In order to improve the catalytic efficiency, starting from increasing the substrate concentration, the endogenous sugar transporters of Escherichia coli BL21 were searched and knocked out one by one to screen for sugar transporters related to 3-FL transport. The results showed that MdfA, MdtD and OmpC in the sugar transporters were closely related to the efficiency of 3-FL transport to the extracellular space, and when MdfA, MdtD and / or OmpC were knocked out, the extracellular concentration of 3-FL decreased significantly (from 7.6 g / L to 0.3 g / L). Therefore, knocking out MdfA, MdtD and / or OmpC increased the 3-FL concentration in the engineered bacteria, thereby achieving an increase in FUC production.

[0016] As a preferred implementation of the engineered bacteria described in the present application, the amino acid sequence of the multidrug efflux system protein MdfA is shown in SEQ ID NO.5;

[0017] The amino acid sequence of the MFS transporter MdtD is shown in SEQ ID NO.6;

[0018] The amino acid sequence of the outer membrane porin C OmpC is shown in SEQ ID NO.7.

[0019] As a preferred embodiment of the engineered bacteria described in the present application, the engineered bacteria also knock out the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the phosphate undecyl glucose phosphotransferase wcaj gene;

[0020] The amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO.8;

[0021] The amino acid sequence of the fucose isomerase / fucokinase gene cluster fucIK is shown in SEQ ID NO.9;

[0022] The amino acid sequence of the undecapryl glucose phosphotransferase wcaj is shown in SEQ ID NO.10.

[0023] The present application integrates the BCGW gene cluster and α-1,3-fucosyltransferase into the engineered bacteria, and simultaneously knocks out the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the 11-carbonyl glucose phosphotransferase wcaj gene, thereby constructing a 3-FL biosynthetic pathway to provide precursors for the synthesis of FUC.

[0024] In a second aspect, the present application provides a method for constructing the above-mentioned engineered bacteria, comprising the following steps:

[0025] S1. The β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the 11-carbonyl glucose phosphotransferase wcaj gene in Escherichia coli BL21 (DE3) were knocked out using Cripsr-Cas9 technology to obtain Escherichia coli I.

[0026] S2, using enzyme ligation method to integrate the vector carrying α-1,3-fucosyltransferase, α-L-fucosidase, and BCGW gene cluster into the Escherichia coli I obtained in step S1 to obtain Escherichia coli II;

[0027] S3. Use Cripsr-Cas9 technology to knock out the sugar transporter in Escherichia coli II obtained in step S2, and obtain an engineered bacterium that synthesizes fucose using 3-fucosyllactose as a precursor.

[0028] In a third aspect, the present application provides the use of the above-mentioned engineered bacteria in the production of fucose.

[0029] 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.

[0030] Compared with the prior art, this application has the following beneficial effects:

[0031] 1. The present application constructs an intracellular biosynthetic pathway of FUC by integrating the BCGW gene cluster, α-1,3-fucosyltransferase and α-L-fucosidase with catalytic 3-FL activity into Escherichia coli, and knocking out the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the 11-carbonyl glucose phosphotransferase wcaj gene, thus opening up a new biosynthetic pathway for FUC biosynthesis.

[0032] 2. The present application prepared α-L-fucosidases from different sources to conduct 3-FL catalysis experiments, and screened out α-L-fucosidases AfcA and AfcB that catalyze 3-FL to FUC, so that the prepared engineered bacteria have the activity of converting 3-FL to FUC.

[0033] 3. During the experiment, the present application found that the catalytic efficiency of α-L-fucosidase with catalytic 3-FL activity was low. In order to improve the catalytic efficiency, the substrate concentration was increased. The endogenous sugar transporters of Escherichia coli BL21 were searched and knocked out one by one to screen for sugar transporters related to 3-FL transport. The results showed that MdfA, MdtD and OmpC in the sugar transporters were closely related to the efficiency of 3-FL transport to the extracellular space, and when MdfA, MdtD and / or OmpC were knocked out, the extracellular concentration of 3-FL decreased significantly (from 7.6 g / L to 0.3 g / L). Therefore, MdfA, MdtD and / or OmpC were knocked out, so that the 3-FL concentration in the engineered bacteria was increased, thereby achieving an increase in FUC production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a liquid chromatogram of 3-fucosyllactose, lactose and fucose in the fermentation broth of FUC01A and FUC02B in the effect example of the present application, in which 3FA-3 refers to FUC01A, and 3FB-3 refers to FUC01B;

[0035] Figure 2 This is the fucose fermentation curve of FUC01A and FUC02B in the effect example of this application. DETAILED DESCRIPTION

[0036] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0037] 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.

[0038] In the following examples, the gene information used is shown in Table 1.

[0039] Table 1 Genes and their information used in the following examples

[0040]

[0041]

[0042] In the following examples and comparative examples, unless otherwise specified, the methods used to transfer plasmids into competent cells or to transform them 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.

[0043] In the following examples and comparative examples, unless otherwise specified, the cloning refers to cloning using a non-ligase-dependent single-fragment one-step cloning kit, and the operation is performed according to the kit instructions. The non-ligase-dependent single-fragment one-step cloning kit is provided by Nanjing Novezan Biotech Co., Ltd., and the product number is C112-02.

[0044] The trace element solution is mainly composed of 13.74g / L nitrilotriacetic acid sodium salt, 5.6g / L ammonium ferric citrate, 0.9g / LZnSO2·7H2O, 0.2g / L CoCl2·6H2O, 1.0g / L MnCl2·4H2O, 0.10g / L CuCl2·2H2O, 0.2g / LH3BO3, and 0.2g / L Na2MoO4·2H2O, and is filtered and sterilized with a filter membrane with a pore size of 0.22μm.

[0045] 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.

[0046] The feed solution used in the fed-batch fermentation experiment included 800 g / L glycerol and 5 g / LMgSO4·7H2O.

[0047] pEcCas vector, pSPIN plasmid, pCDFDuet plasmid, pRSF plasmid, pETDuet plasmid and pEcgRNA plasmid were all provided by Addgene.

[0048] The techniques not described in detail in the following embodiments and effect examples are all commonly used techniques in the art, and reference may be made to "Handbook of Molecular Biology Experiments" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiments (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiments" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0049] Example 1

[0050] In order to verify whether α-L-fucosidases from different sources have the effect of catalyzing 1,3-glycosidic bonds on 3-fucosyllactose (3-FL), in this example, α-L-fucosidases A1, A2, B1, B2, and B3 were respectively transferred into Escherichia coli BL21 for heterologous expression to obtain α-L-fucosidases, and then the enzyme catalytic reaction was verified. The specific scheme is as follows:

[0051] 1.1 α-L-fucosidase A1, A2, B1, B2, B3 were amplified by PCR to obtain afcA1, afcA2, afcB1, afcB2, afcB3 fragments, which were then linked to the pETD vector by restriction enzymes to obtain pETD-afcA, pETD-afcA2, pETD-afcB, pETD-afcB2 and pETD-afcB3 respectively;

[0052] 1.2 pETD-afcA, pETD-afcA2, pETD-afcB, pETD-afcB2 and pETD-afcB3 were respectively transformed into Escherichia coli BL21 to obtain strains AfcA1, AfcA2, AfcB1, AfcB2 and AfcB3;

[0053] 1.3 The strains AfcA1, AfcA2, AfcB1, AfcB2, and AfcB3 obtained in step 1.2 were cultured in LB liquid medium to OD 600 =0.6, IPTG was added for induction, and the cells were collected by centrifugation after induction at 28°C for 16 h;

[0054] 1.4 The bacteria obtained in step 1.3 were subjected to ultrasonic disruption. 3-FL solution was added at a ratio of 1L of 50g / L 3-FL solution per 100g of wet weight of bacteria. The total system was 50mL. The system was placed at 37°C for catalysis for 4h. Samples were taken at 0.1, 0.5, 1, 2 and 4h of catalysis to determine the contents of FUC and 3-FL in the system. The results are shown in Table 2.

[0055] Table 2 Determination results of 3-FL catalytic activity of different α-L-fucosidases

[0056]

[0057]

[0058] As shown in Table 2, after 4 h of catalysis, AfcA1 and AfcB1 catalyzed almost all of the 3-FL, producing 16.2 g / L and 15.0 g / L of FUC, respectively. This indicates that α-L-fucosidase AfcA1 and AfcB1 have strong activities in catalyzing the conversion of 3-FL to FUC, and the catalytic activity of AfcA1 is superior to that of AfcB1. Therefore, AfcA1 and AfcB1 are used as one of the key enzymes for the conversion of 3-FL to FUC. Hereinafter, AfcA1 is named AfcA and AfcB1 is named AfcB.

[0059] Example 2

[0060] This embodiment provides an engineering bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof, wherein the construction method comprises the following steps:

[0061] 2.1 The lacZ, fucIK and wcaj genes in Escherichia coli star BL21 (DE3) were knocked out using the improved CRISPR-Cas9 technology to obtain Escherichia coli I; the knockout vector used was the pEcCas vector containing Cas9 and λ-Red recombinase, and the pEcgRNA containing the sgRNA sequence and the N20 specific sequence was used for targeted gene editing;

[0062] 2.2 Phosphomannose mutase manB (Gene ID: 946574), α-D-mannose 1-phosphoguanyl transferase manC (Gene ID: 946580), GDP-mannose 4,6-dehydrogenase gmd (Gene ID: 946562) and GDP-L-fucose synthase wcaG (Gene ID: 946563) from Escherichia coli str. K-12 substr. MG1655 (GenBank: NC_000913.3) were amplified by PCR respectively, and inserted into the vector pRSFDuet-1 in sequence through the corresponding restriction sites, and the T7 promoter was inserted before the start site of manB to obtain the recombinant plasmid pRSFDuet-manB-manC-gmd-wcaG (i.e., BCGW gene cluster) with T7 promoter, and the T7-BCGW expression cassette was obtained by PCR;

[0063] 2.3 The fragments of α-1,3-fucosyltransferase bacfut1 and α-L-fucosidase afcA were obtained by PCR amplification, and inserted into the vector pETD through the restriction sites in sequence, and the T7 promoter was inserted before the start site of bacfut1 to obtain the recombinant plasmid pETD-bacfut-afcA with T7 promoter, and the T7-bacfut-afcA expression cassette was obtained by PCR;

[0064] 2.4 The caiB-N32 specific sequence was cloned into the pSL1765 plasmid to obtain the pSPIN-caiB(N32) plasmid, and the T7-BCGW expression cassette fragment was cloned into the pSPIN-caiB(N32) plasmid to obtain the pSPIN-caiB(N32)-BCGW plasmid, which was transformed into the E. coli I obtained in step 2.1 to obtain E. coli a;

[0065] 2.5 According to the method of step 2.4, the T7-bacfut-afcA expression cassette obtained in step 2.2 was integrated into the E. coli a obtained in step 2.4, wherein bacfut1 was integrated and expressed at the lacA site, and afcA was integrated and expressed at the hlyE site, to obtain E. coli II, named strain FUC01A;

[0066] 2.6 Knock out the multidrug efflux system mdfA in the Escherichia coli II obtained in step 2.5 according to the method of step 2.1 to obtain strain FUCA03;

[0067] The primer sequences used in the above steps are shown in Table 3.

[0068] Table 3 Primers used to construct strain FUCA03

[0069]

[0070]

[0071] Example 3

[0072] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA with the MFS transporter mdtD, and the resulting strain is named FUCA04, and the remaining steps and parameter conditions remain unchanged.

[0073] The primers used in the above steps are shown in Table 4.

[0074] Table 4 Primers used to construct engineered bacteria FUCA04

[0075]

[0076] Example 4

[0077] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA with the outer membrane porin C ompC, and the resulting strain is named FUCA05, and the remaining steps and parameter conditions remain unchanged.

[0078] The primers used in the above steps are shown in Table 5.

[0079] Table 5 Primers used to construct engineered bacteria FUCA05

[0080]

[0081] Example 5

[0082] This embodiment provides an engineering bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof, wherein the construction method comprises the following steps:

[0083] 5.1 According to the method of step 2.1 of example 2, the MFS transport protein mdtD and the outer membrane porin CompC in the engineered bacteria FUCA03 obtained in step 2.6 of example 2 were knocked out to obtain the engineered bacteria FUCA08. The primers used are shown in Table 4 of example 3 and Table 5 of example 4.

[0084] Example 6

[0085] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that: the α-L-fucosidase afcA in step 2.3 is replaced by α-L-fucosidase afcB; afcA in step 2.5 is replaced by afcB, the obtained Escherichia coli II is named strain FUC01B, the strain obtained in step 2.6 is named FUCB03, and the remaining steps and parameter conditions remain unchanged.

[0086] The primers used in the above steps are shown in Table 6.

[0087] Table 6 Primers used to construct engineered bacteria FUC01B

[0088]

[0089] Example 7

[0090] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that: the α-L-fucosidase afcA in step 2.3 is replaced by α-L-fucosidase afcB, and in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA by the MFS transporter mdtD. The obtained strain is named FUCB04, and the remaining steps and parameter conditions remain unchanged.

[0091] The primers used in the above steps are shown in Table 4 and Table 6.

[0092] Example 8

[0093] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that: the α-L-fucosidase afcA in step 2.3 is replaced by α-L-fucosidase afcB, and in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA by the outer membrane porin C ompC. The obtained strain is named FUCB05, and the remaining steps and parameter conditions remain unchanged.

[0094] The primers used in the above steps are shown in Tables 5 and 6.

[0095] Example 9

[0096] This embodiment provides an engineering bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof, wherein the construction method comprises the following steps:

[0097] 9.1 According to the method of step 2.1 of example 2, the MFS transporter mdtD and the outer membrane porin CompC in the engineered bacteria FUCB03 obtained in step 6 were knocked out to obtain the engineered bacteria FUCB08. The primers used are shown in Table 4 of example 3 and Table 5 of example 4.

[0098] Comparative Example 1

[0099] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA with the sugar efflux transporter setA, and the resulting strain is named FUCA02, and the remaining steps and parameter conditions remain unchanged.

[0100] The primers used in the above steps are shown in Table 7.

[0101] Table 7 Primers used to construct engineered bacteria FUCA02

[0102]

[0103] Comparative Example 2

[0104] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA with the outer membrane porin F ompF, and the resulting strain is named FUCA06, and the remaining steps and parameter conditions remain unchanged.

[0105] The primers used in the above steps are shown in Table 8.

[0106] Table 8 Primers used to construct engineered bacteria FUCA06

[0107]

[0108] Comparative Example 3

[0109] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA with the glycine betaine / L-proline transporter proP, and the resulting strain is named FUCA07, and the remaining steps and parameter conditions remain unchanged.

[0110] The primers used in the above steps are shown in Table 9.

[0111] Table 9 Primers used to construct engineered bacteria FUCA07

[0112]

[0113] Comparative Example 4

[0114] This embodiment provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that: the α-L-fucosidase afcA in step 2.3 is replaced by α-L-fucosidase afcB; in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA by the sugar efflux transporter setA, and the obtained strain is named FUCB02, and the remaining steps and parameter conditions remain unchanged.

[0115] The primers used in the above steps are shown in Tables 6 and 7.

[0116] Comparative Example 5

[0117] This example provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that: the α-L-fucosidase afcA in step 2.3 is replaced by α-L-fucosidase afcB; in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA by the outer membrane porin F ompF, and the obtained strain is named FUCB06, and the remaining steps and parameter conditions remain unchanged.

[0118] The primers used in the above steps are shown in Table 6 and Table 8.

[0119] Comparative Example 6

[0120] This example provides an engineered bacterium for synthesizing FUC using 3-FL as a precursor and a construction method thereof. The construction method is similar to that of Example 2, except that: the α-L-fucosidase afcA in step 2.3 is replaced by α-L-fucosidase afcB; in step 2.6, the knocked-out gene is replaced by the multidrug system efflux mdfA by the glycine betaine / L-proline transporter proP, and the obtained strain is named FUCA07, and the remaining steps and parameter conditions remain unchanged.

[0121] The primers used in the above steps are shown in Table 6 and Table 9.

[0122] Effect example

[0123] 1. The strains FUC01A, FUC01B, FUCA02-FUCA08 and FUCB02-FUCB08 of Examples 1-8 and Comparative Examples 1-6 were subjected to fed-batch fermentation experiments, and the yield of fucose in the fermentation broth was detected by high performance liquid chromatography.

[0124] 1. Fed-batch fermentation experiment

[0125] (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;

[0126] (2) 150 mL of the seed solution obtained in step (1) was transferred into a 5 L fermentation tank containing 2.5 L of fermentation medium, 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;

[0127] (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;

[0128] (4) adding isopropyl β-d-1-thiogalactopyranoside to a final concentration of 0.2 mM and 40 g of lactose, supplementing with 17.78 g / L / h of glycerol solution, culturing for 90 h before fermentation is terminated to obtain a fermentation broth.

[0129] 2. Post-treatment of fermentation broth and HPLC detection

[0130] 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 3-FL.

[0131] HPX.87H chromatographic column (Bio-Rad) and high performance liquid chromatograph (LC-16, Shimadzu, Japan) were used to detect FUC and 3-FL in the test samples, and the FUC and 3-FL contents in the test samples were calculated according to the external standard method. The chromatographic conditions were 5 mM sulfuric acid aqueous solution as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 60 °C, and an injection volume of 10 μL.

[0132] Take 0-2.0g / L gradient concentration of FUC standard, and draw a standard curve with FUC concentration as the horizontal axis and liquid chromatography peak area as the vertical axis; 3-FL standard curve is drawn according to the FUC method.

[0133] The calculated FUC and 3-FL concentrations in different fermentation broths were converted into the yields obtained by fermentation in a 5L fermenter. The yield calculation results are shown in Table 10, and the liquid chromatograms of 3-FL and FUC are shown in Figure 1 The fermentation curves of FUC01A and FUC01B are shown in Figure 2 .

[0134] Table 10 Calculation results of FUC and 3-FL production of different engineered bacteria

[0135] source Strain name Total FUC concentration (g / L) Total 3-FL concentration (g / L) Extracellular 3-FL concentration (g / L) Example 1 FUC01A 22.76 9.9 7.6 Comparative Example 1 FUCA02 23.51 14.88 12.35 Example 2 FUCA03 29.10 1.6 1.6 Example 3 FUCA04 26.61 3.1 2.5 Example 4 FUCA05 27.96 2.1 2.0 Comparative Example 2 FUCA06 23.82 9.0 7.3 Comparative Example 3 FUCA07 23.40 9.2 8.7 Example 4 FUCA08 31.40 0.4 0.3 Example 6 FUC01B 13.5 35.2 32.1 Comparative Example 4 FUCB02 14.9 30.8 28.8 Example 6 FUCB03 18.2 19.7 17.2 Example 7 FUCB04 15.8 27.3 24.6 Example 8 FUCB05 16.6 24.0 21.8 Comparative Example 5 FUCB06 14.0 33.1 31.6 Comparative Example 6 FUCB07 14.7 31.5 29.2 Example 9 FUCB08 20.8 14.2 12.5

[0136] like Figure 1 and Figure 2 As shown, 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 phosphate undecyl glucose phosphotransferase wcaj, integrates phosphomannose mutant enzyme manB, mannose-1-phosphate guanosyltransferase manC, GDP-D-mannose-4,6-dehydratase gmd and GDP-L-fucose synthase wcaG, α-1,3-fucosyltransferase bacfut1, α-L-fucosidase afcA and BCGW gene cluster, so that Escherichia coli can biosynthesize 3-fucosyllactose in vivo, and converts 3-fucosyllactose as a precursor to obtain fucose under the catalysis of α-L-fucosidase.

[0137] The present application first obtains α-L-fucosidases from different sources by heterologous expression to screen and obtain α-L-fucosidases AfcA and AfcB having the activity of catalyzing the conversion of 3-FL to FUC. On this basis, in order to further increase the FUC yield, the endogenous sugar transporter is knocked out so that 3-FL remains in the cell as much as possible to react with AfcA or AfcB. As shown in Table 10, after MdfA, MdtD and / or OmpC are knocked out, the extracellular 3-FL concentration of the strain is greatly reduced, indicating that after the sugar transporter MdfA, MdtD and / or OmpC are knocked out, 3-FL can remain in the cell to react with AfcA or AfcB, so that the utilization rate of intracellular 3-FL after the fermentation is improved, thereby achieving an increase in FUC yield.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than 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, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. An engineered bacterium that synthesizes fucose using 3-fucosyllactose as a precursor, characterized in that: The engineered bacteria express α-1,3-fucosyltransferase and α-L-fucosidase.

2. The engineered bacteria according to claim 1, characterized in that The α-L-fucosidase is α-L-fucosidase AfcA or α-L-fucosidase AfcB.

3. The engineered bacteria according to claim 1 or 2, characterized in that The amino acid sequence of the α-1,3-fucosyltransferase is shown in SEQ ID NO.1; The amino acid sequence of the α-L-fucosidase AfcA is shown in SEQ ID NO.3; The amino acid sequence of the α-L-fucosidase AfcB is shown in SEQ ID NO.

4.

4. The engineered bacteria according to any one of claims 1 to 3, characterized in that The engineered bacteria also express the BCGW gene cluster and knock out the sugar transporter.

5. The engineered bacteria according to claim 4, characterized in that The BCGW gene cluster is a gene cluster integrating phosphomannose mutant enzyme manB, mannose 1-1 phosphate guanosine transferase manC, GDP-D-mannose-4,6-dehydratase gmd and GDP-L-fucose synthase wcaG, and the amino acid sequence of the BCGW gene cluster is shown in SEQ ID NO.

2.

6. The engineered bacteria according to claim 4, characterized in that The sugar transporter comprises at least one of a multidrug efflux system protein MdfA, a MFS transporter protein MdtD and an outer membrane porin C OmpC.

7. The engineered bacteria according to claim 6, characterized in that The amino acid sequence of the multidrug efflux system protein MdfA is shown in SEQ ID NO.5; The amino acid sequence of the MFS transporter MdtD is shown in SEQ ID NO.6; The amino acid sequence of the outer membrane porin C OmpC is shown in SEQ ID NO.

7.

8. The engineered bacteria according to any one of claims 1 to 7, characterized in that The engineered bacteria also knock out the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the phosphate undecyl glucose phosphotransferase wcaj gene; The amino acid sequence of the β-galactosidase lacZ is shown in SEQ ID NO.8; The amino acid sequence of the fucose isomerase / fucokinase gene cluster fucIK is shown in SEQ ID NO.9; The amino acid sequence of the undecapryl glucose phosphotransferase wcaj is shown in SEQ ID NO.

10.

9. The method for constructing an engineered bacterium according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. The β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the 11-carbonyl glucose phosphotransferase wcaj gene in Escherichia coli BL21 (DE3) were knocked out using Cripsr-Cas9 technology to obtain Escherichia coli I. S2, using enzyme ligation method to integrate the vector carrying α-1,3-fucosyltransferase, α-L-fucosidase, and BCGW gene cluster into the Escherichia coli I obtained in step S1 to obtain Escherichia coli II; S3. Use Cripsr-Cas9 technology to knock out the sugar transporter in Escherichia coli II obtained in step S2, and obtain an engineered bacterium that synthesizes fucose using 3-fucosyllactose as a precursor.

10. Use of the engineered bacteria according to any one of claims 1 to 8 in producing fucose.

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