An engineered bacterium for reducing lactose residue during fucose synthesis, 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 that reduces lactose residues during fucose synthesis, and its construction method and application. The present application provides an engineered bacterium that reduces lactose residues during fucose synthesis, wherein the engineered bacterium expresses enzymes for synthesizing fucose and enzymes for metabolizing lactose. The present application integrates GalM, LacZ, GalE, GalT, GalK, and BLUSP into the engineered bacterium FUCΔZ that is capable of fully synthesizing fucose, constructs a lactose metabolic pathway, and obtains enzymes capable of metabolizing lactose by regulating temperature through transcription and translation, thereby metabolizing lactose into glucose-1-phosphate, so as to achieve the purpose of low or no lactose residue in the fermentation broth.

Description

Technical Field

[0001] The present application relates to the field of microbial genetic engineering technology, and in particular to an engineering bacterium for reducing lactose residue during fucose synthesis, and a construction method and application thereof. Background Art

[0002] Fucose (Fucose, FUC), also known as 6-deoxy-galactose, is a widely occurring natural sugar. L-fucose is one of the five monosaccharides that make up human milk oligosaccharides. It is widely found in animals, plants, and algae, and plays an important physiological role. L-fucose has been shown to regulate intestinal health, enhance immunity, and slow skin aging.

[0003] At present, the applicant's preliminary research has successfully biosynthesized FUC in Escherichia coli. The pathway is: 2'-fucosyllactose is synthesized through a series of enzyme reactions and under the mediation of lactose using fructose-6-phosphate as a substrate. 2'-fucosyllactose is decomposed into fucose and lactose under the action of α-L-fucosidase. According to preliminary studies, the addition of lactose as a precursor is necessary in the fucose pathway, but lactose will not be consumed, and lactose will inevitably remain in the fermented liquid. In the presence of lactose, due to the close molecular weight of lactose and fucose, fucose cannot be separated using ultrafiltration membranes or nanofiltration membranes. The inability to separate lactose reduces the purity of the prepared fucose. Therefore, there is an urgent need to construct an engineered bacterium that can maintain fucose production while reducing lactose residues to solve the problem of being unable to separate and purify fucose. 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 for reducing lactose residue during fucose synthesis, 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 the first aspect, the present application provides an engineered bacterium for reducing lactose residue during fucose synthesis.

[0007] The engineered bacteria express an enzyme for synthesizing fucose and an enzyme for metabolizing lactose;

[0008] The enzyme that synthesizes fucose includes at least one of phosphomannose mutant enzyme ManB, mannose-1-phosphate guanyl transferase ManC, GDP-D-mannose-4,6-dehydratase Gmd, GDP-L-fucose synthase WcaG, α-1,2-fucosyltransferase WbgL and α-L-fucosidase;

[0009] The enzyme that metabolizes lactose includes at least one of β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronyltransferase GalT, galactokinase GalK, galactose-1-epimerase GalM and UTP-glucose-1-phosphate uridyltransferase.

[0010] As a preferred embodiment of the engineered bacteria described in the present application, the engineered bacteria integrates the coding genes of β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK, galactose-1-epimerase GalM and UTP-glucose-1-phosphate uridyltransferase BLUSP into the engineered bacteria FUCΔZ that synthesizes fucose.

[0011] The present application integrates galactose-1-epimerase GalM, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronyltransferase GalT, galactokinase GalK and UTP-glucose-1-phosphate uridyltransferase BLUSP into an engineered bacterium capable of fully synthesizing fucose, constructs a lactose metabolic pathway (lactose → galactose → α-galactose → α-galactose-1-phosphate → UDP-galactose → UDP-glucose → glucose-1-phosphate), obtains enzymes capable of metabolizing lactose through transcription and translation, and ultimately metabolizes lactose into glucose-1-phosphate, thereby achieving the purpose of low or no residual lactose in the fermentation broth.

[0012] Glucose-1-phosphate, the final product of the lactose metabolic pathway, can be converted to glucose-6-phosphate by phosphoglucomutase in E. coli and metabolized in the glycolysis pathway. In the lactose metabolic pathway, the BLUSP-mediated conversion of UDP-glucose to glucose-1-phosphate is a reversible reaction. Based on the chemical equilibrium principle of reversible reactions, as glucose-1-phosphate is metabolized, its concentration decreases. Therefore, to achieve equilibrium, BLUSP mediates the conversion of UDP-glucose to glucose-1-phosphate.

[0013] As a preferred embodiment of the engineered bacteria described in the present application, the engineered bacteria FUCΔZ is based on Escherichia coli BL21 (DE3), and the β-galactosidase lacZ, fucose isomerase / fucokinase gene cluster fucIK and 11-carbonyl glucose phosphotransferase wcaj genes are knocked out, and the phosphomannose mutant enzyme manB, mannose-1-phosphate guanosyltransferase manC, GDP-D-mannose-4,6-dehydratase Gmd, GDP-L-fucose synthase wcaG, α-1,2-fucosyltransferase wbgL and α-L-fucosidase afcA genes are integrated.

[0014] As a preferred embodiment of the engineered bacteria described in the present application, the β-galactosidase LacZ is derived from the γ-Proteobacteria, the UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK and galactose-1-epimerase GalM are derived from Escherichia coli K-12 substr.MG1655, and the UTP-glucose-1-phosphate uridyltransferase BLUSP is derived from Bifidobacterium longum subsp.

[0015] As a preferred embodiment of the engineered bacteria described in the present application, the Bifidobacterium longum subspecies is Bifidobacterium longum subsp. longum ATCC 55813.

[0016] As a preferred embodiment of the engineered bacteria described in the present application, the amino acid sequence of the β-galactosidase LacZ is shown in SEQ ID NO.1;

[0017] The amino acid sequence of the UDP-galactose-4-epimerase GalE is shown in SEQ ID NO.2;

[0018] The amino acid sequence of the galactose-1-phosphate uronyltransferase GalT is shown in SEQ ID NO.3;

[0019] The amino acid sequence of the galactokinase GalK is shown in SEQ ID NO.4;

[0020] The amino acid sequence of the galactose-1-epimerase GalM is shown in SEQ ID NO.5;

[0021] The amino acid sequence of the UTP-glucose-1-phosphate uridyltransferase is shown in SEQ ID NO.6.

[0022] The present application constructs a lactose metabolic pathway by integrating galactose-1-epimerase GalM, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK and UTP-glucose-1-phosphate uridyltransferase BLUSP into an engineered bacterium, which can be expressed at a suitable temperature to metabolize lactose.

[0023] As a preferred embodiment of the engineered bacteria described in the present application, the expression of β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK, galactose-1-epimerase GalM and UTP-glucose-1-phosphate uridyltransferase in the engineered bacteria is controlled by a promoter with a temperature-sensitive element.

[0024] As a preferred embodiment of the engineered bacteria described in the present application, the temperature-sensitive element is the temperature-sensitive mutant clts857 of the cl protein, and the nucleotide sequence of the temperature-sensitive element is shown in SEQ ID NO.7.

[0025] The temperature-sensitive element used in this application is cl protein, which can inhibit the promoters PR and PL at 30°C and release the inhibition of the promoters at 42°C. It can control the expression of galactose-1-epimerase GalM, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK and UTP-glucose-1-phosphate uridyl transferase BLUSP by regulating temperature. The optimal fermentation temperature for the engineering bacteria of this application to produce FUC is 30°C. At this time, The engineered bacteria do not metabolize lactose, and the synthesis pathway of fucose is not affected. After the fermentation is completed, the fermentation broth is heated twice to activate the promoters of galactose-1-epimerase GalM, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK and UTP-glucose-1-phosphate uridyl transferase BLUSP, thereby constructing a lactose metabolic pathway. This enables the strain to metabolize lactose by itself, saving the step of separating lactose and FUC.

[0026] As a preferred embodiment of the engineered bacteria described in the present application, the engineered bacteria further comprises overexpressing β-galactosidase permease LacY.

[0027] As a preferred embodiment of the engineered bacteria described herein, the overexpression of the β-galactosidase LacY is achieved by replacing the promoter of the β-galactosidase LacY with a strong promoter; the strong promoter includes at least one of a T7 promoter, a Tac promoter, a Lac promoter, and a Trp promoter. To improve lactose transport capacity, the present invention replaces the promoter of the β-galactosidase LacY with a strong promoter, thereby increasing the intracellular concentration of lactose, thereby facilitating fucose synthesis and enabling complete lactose metabolism during the two heat treatments.

[0028] As a preferred embodiment of the engineered bacteria described in the present application, the strong promoter is the Tac promoter, and the nucleotide sequence of the Tac promoter is shown in SEQ ID NO.8.

[0029] In a second aspect, the present application provides a method for constructing the above-mentioned engineered bacteria, comprising the following steps:

[0030] S1. Using Cripsr-Cas9 and enzyme ligation technology, the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK, and the undecyl-glucose phosphotransferase wcaj genes in Escherichia coli BL21(DE3) were knocked out, and the phosphomannose mutant manB, mannose-1-phosphate guanyltransferase manC, GDP-D-mannose-4,6-dehydratase Gmd, GDP-L-fucose synthase wcaG, α-1,2-fucosyltransferase wbgL, and α-L-fucosidase afcA genes were integrated to obtain the engineered strain FUCΔZ.

[0031] S2, synthesize a β-galactosidase LacZ expression cassette containing a thermosensitive element in the promoter, and transfer it into the engineered bacteria FUCΔZ obtained in step S1 to obtain engineered bacteria A;

[0032] S3, replacing the promoter of β-galactosidase LacY in the engineered bacteria A obtained in step S2 with a strong promoter to obtain engineered bacteria B;

[0033] S4, synthesize the expression cassettes of UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronyltransferase GalT, and galactokinase GalK, whose promoters contain temperature-sensitive elements, and transfer them into the engineered bacteria B obtained in step S3 to obtain engineered bacteria C;

[0034] S5. Using Cripsr-Cas9 technology to knock out the galactose-1-epimerase GalM in the engineered bacteria C obtained in step S4, and integrating the galactose-1-epimerase GalM whose promoter contains a temperature-sensitive element;

[0035] S6. A UTP-glucose-1-phosphate uridyltransferase BLUSP expression cassette containing a thermosensitive element in the promoter was synthesized to obtain an engineered bacterium that reduces lactose residue during fucose synthesis.

[0036] In a third aspect, the present application provides the use of the above-mentioned engineered bacteria in the production of fucose.

[0037] In a fourth aspect, the present application provides a method for producing fucose with reduced lactose residue, comprising the following steps:

[0038] (1) subjecting the engineered bacteria capable of reducing lactose residue during fucose synthesis to fed-batch fermentation for 78-90 hours to obtain a fermentation broth;

[0039] (2) The fermentation liquid obtained in step (1) is treated at 40-42° C. for 2-4 hours, and then the temperature is adjusted to 37-42° C. for fermentation for 4-10 hours to obtain a fermentation stock liquid.

[0040] In the present application, the fermentation broth is first subjected to a first heating treatment at 40-42°C for 2-4 hours to release the inhibition of the promoter by the temperature-sensitive element, thereby allowing the enzyme in the lactose metabolic pathway to be expressed. The temperature is then adjusted to 37-40°C for a second heating treatment for 4-10 hours to allow the lactose to fully react with the enzyme and complete the metabolism of the lactose.

[0041] As a preferred embodiment of the production method described in the present application, in step (1), the fed-batch fermentation is to inoculate the seed liquid of the engineered bacteria into a fermentation tank containing a fermentation medium, adjust the pH to 6.8-7.2, maintain the dissolved oxygen at 30-50%, the culture temperature to 35-37°C, the aeration rate to 2-3 VVM, and the rotation speed to 800-900 rpm until the glycerol in the fermentation medium is exhausted, add the feed solution to the fermentation tank at a constant rate of 4-5 g / L / h, adjust the culture temperature to 29-30°C, ferment for 4-5 h, add IPTG with a final concentration of 0.2-0.3 mM and 35-40 g lactose, supplement with 17-18 g / L / h of glycerol solution, and culture for 78-90 h;

[0042] The fermentation medium comprises 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 / LMgSO4·7H2O, 0.02 g / L CaCl2 and 10 mL / L trace element solution;

[0043] The feed solution includes 750-800 g / L of a carbon source and 4-5 g / L of MgSO4·7H2O, wherein the carbon source is glycerol.

[0044] As a preferred embodiment of the production method described in the present application, in step (1), the fed-batch fermentation is to inoculate the seed liquid of the engineered bacteria into a fermentation tank containing a fermentation medium, adjust the pH to 6.8, maintain the dissolved oxygen at 30-50%, the culture temperature to 37°C, the aeration rate to 2VVM, and the rotation speed to 900 rpm until the glycerol in the fermentation medium is exhausted, add the feed solution to the fermentation tank at a constant rate of 4 g / L / h, adjust the culture temperature to 29.5°C, ferment for 4 h, add IPTG with a final concentration of 0.2 mM and 40 g lactose, supplement with 17.78 g / L / h of glycerol solution, and culture for 78-90 h;

[0045] The fermentation medium comprises 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 / LMgSO4·7H2O, 0.02 g / L CaCl2 and 10 mL / L trace element solution;

[0046] The feed solution includes 800 g / L of a carbon source and 5 g / L of MgSO 4 ·7H 2 O, wherein the carbon source is glycerol.

[0047] As a preferred embodiment of the production method described in the present application, in step (2), the obtained fermentation broth is treated at 42°C for 2 hours and then maintained at 40°C for 4-10 hours.

[0048] Compared with the prior art, this application has the following beneficial effects:

[0049] 1. The present application integrates galactose-1-epimerase GalM, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronyltransferase GalT, galactokinase GalK and UTP-glucose-1-phosphate uridyltransferase BLUSP with temperature-sensitive elements into the engineered bacterium FUCΔZ capable of fully synthesizing fucose, constructing a lactose metabolic pathway (lactose → galactose → α-galactose → α-galactose-1-phosphate → UDP-galactose → UDP-glucose → glucose-1-phosphate). By regulating the temperature to obtain enzymes capable of metabolizing lactose through transcription and translation, lactose is ultimately metabolized into glucose-1-phosphate, thereby achieving the purpose of low or no residual lactose in the fermentation broth.

[0050] 2. The temperature-sensitive element used in this application is cl protein, which can inhibit the promoters PR and PL at 30°C and release the inhibition of the promoters at 42°C. It can control the expression of galactose-1-epimerase GalM, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK and UTP-glucose-1-phosphate uridyl transferase BLUSP by regulating temperature. The optimal fermentation temperature for the engineering bacteria of this application to produce FUC is 30°C, at which time the lactose metabolism of the engineering bacteria is The metabolic pathway is closed, and the synthesis pathway of fucose is not affected. After the fermentation is completed, the fermentation broth is heated twice to activate the promoters of galactose-1-epimerase GalM, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK and UTP-glucose-1-phosphate uridyl transferase BLUSP. The lactose metabolic pathway is activated, allowing the strain to metabolize lactose by itself, saving the step of separating lactose and FUC, and further improving the purity of FUC. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This application discloses a process for degradation of lactose by the engineered bacteria FUCB to reduce lactose residue during fucose synthesis. DETAILED DESCRIPTION

[0052] 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.

[0053] 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.

[0054] In the following examples, the gene information used is shown in Table 1.

[0055] Table 1 Genes and their information used in the following examples

[0056] Chinese name Coding genes source β-galactosidase lacZ SEQ ID NO.1 of this application UDP-galactose-4-epimerase galE SEQ ID NO.2 of this application Galactose-1-phosphate uronyltransferase galT SEQ ID NO.3 of this application Galactokinase galK SEQ ID NO.4 of this application Galactose-1-epimerase galM SEQ ID NO.5 of this application UTP-glucose-1-phosphate uridyltransferase BLUSP SEQ ID NO.6 of this application Temperature sensitive element clts857 <![CDATA[cl ts -p R -p L ]]> SEQ ID NO.7 of this application Tac promoter tacP SEQ ID NO.8 of this application

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The feed solution used in the fed-batch fermentation experiment included 800 g / L carbon source and 5 g / L MgSO 4 ·7H 2 O, wherein the carbon source was glycerol.

[0062] The pEcCas vector, pSPIN plasmid, pCDFDuet plasmid, pRSF plasmid, pETDuet plasmid, and pEcgRNA plasmid were all provided by Addgene.

[0063] 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).

[0064] HPLC detection steps for lactose, galactose and FUC:

[0065] (1) 1 mL of fermentation broth was placed in a boiling water bath for 10 min to kill the strain, and then centrifuged at 12,000 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.

[0066] (2) FUC and 3-FL in the test samples were detected using an HPX.87H column (Bio-Ra) and a high-performance liquid chromatograph (LC-16, Shimadzu, Japan). The lactose, galactose, and FUC contents in the test 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.

[0067] 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; lactose and galactose standard curves are drawn according to the FUC method.

[0068] Example 1

[0069] This embodiment provides an engineered bacterium for reducing lactose residue during fucose synthesis and a method for constructing the same. The method comprises the following steps:

[0070] 1.1 Strain D was constructed according to steps 1.1 to 1.4 of Example 1 in the specification of patent number 2024110034987 and named FUCΔZ;

[0071] 1.2 According to the enzyme cutting site, cl ts -p R -p L The promoter and lacZ were connected to the pETDuet-1 vector to form pETDuet-cl ts -p R -p L -lacZ vector was used as template to amplify the cl ts -p R -p L -lacZ expression cassette, cl ts -p R -p L The -lacZ expression cassette fragment was integrated into the strain FUCΔZ obtained in step 1.1. Positive transformants were screened on resistance plates and the plasmid was removed from the positive transformants to obtain strain I.

[0072] 1.3 Construct a Tac-lacY expression cassette with a Tac promoter according to the method in step 1.2, integrate it into strain I obtained in step 1.2, screen positive transformants and remove the plasmid to obtain strain II;

[0073] 1.4 Name the galK-galT-galE gene cluster galKTE and construct the cl ts -p R -p L promoter cl ts -pR -p L The galKTE expression cassette was integrated into strain II obtained in step 1.3. After screening positive transformants, the plasmid was removed to obtain strain III.

[0074] 1.5 Follow the steps in step 1.2 to build a ts -p R -p L promoter cl ts -p R -p L -galM was integrated into the strain obtained by knocking out the galM gene in strain III obtained in step 1.4 using CRISPR-Cas9 technology. After screening positive transformants, the plasmid was removed to obtain strain IV. The knockout vector used was the pEcCas vector containing Cas9 and λ-Red recombinase, and targeted gene editing was performed using pEcgRNA containing the sgRNA sequence and the N20-specific sequence;

[0075] 1.6 Follow the steps in step 1.2 to build a ts -p R -p L promoter cl ts -p R -p L -BLUSP was integrated into strain IV obtained in step 1.5. After screening positive transformants, the plasmid was removed to obtain strain V, which was named FUCB.

[0076] The primers used in the above steps are shown in Table 2

[0077] Table 2 Primers and their sequences used in the construction of engineered bacteria FUCB

[0078]

[0079] Example 2

[0080] Since a temperature-sensitive element was inserted into the engineered bacteria FUCB, the cl protein can inhibit the promoters PR and PL and is also sensitive to temperature (the cl protein inhibits the promoter at 30°C and releases the inhibition of the promoter at 42°C). Since the optimal activity of most enzymes in E. coli is at 37°C, it is necessary to explore the effect of increasing temperature on enzyme activity. The specific plan is as follows:

[0081] 1. Fermentation broth prepared by batch fermentation

[0082] (1) The engineered bacteria obtained in Example 1 were cultured in a 1 L shake flask containing 150 mL of LB medium for 6 h to obtain a secondary seed solution;

[0083] (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;

[0084] (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 30°C, and the fermentation was continued for 4 h;

[0085] (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.

[0086] 2. Exploring post-treatment conditions for fermentation broth

[0087] The fermentation broth was heated twice according to the conditions in Table 3. The lactose content in the fermentation broth was measured at 0, 1, 2, 4, and 8 h after the second heating treatment. The results are shown in Table 3. The first heating was performed to release the inhibition of the promoter and enable the expression of enzymes involved in the lactose metabolic pathway. The second heating was performed to allow the lactose-metabolizing enzymes to react with lactose, allowing it to be fully metabolized into glucose-1-phosphate.

[0088] Table 3 Effects of different treatments on lactose residue in fermentation broth

[0089]

[0090]

[0091] As shown in Table 3, the residual lactose content of the fermentation broth of groups A, B, C, and D after the second heating treatment for 8 h was significantly reduced compared with the residual lactose content before the second heating treatment, indicating that the second heating treatment of the fermentation broth in the present application can regulate the temperature-sensitive element to release the inhibition of the promoter, so that lactose is metabolized into glucose-1-phosphate in the body through the processing of LacZ, GalM, GalK, GalT, GalE, and BLUSP (metabolic process see Figure 1 ), reducing residual lactose, significantly improving the purity of FUC, and saving the FUC separation and purification step. Furthermore, since heating causes some liquid to evaporate, slight changes in the FUC and lactose contents may occur. The data in Table 3 show that the two heating steps do not significantly affect the fucose yield. This demonstrates that the fucose production method of the present application can reduce residual lactose without affecting FUC yield, thus offering high economic benefits.

[0092] Example 3

[0093] This embodiment provides a method for producing fucose with reduced lactose residue, the production method comprising the following steps:

[0094] 3.1 The engineered bacteria FUCB obtained in Example 1 was used to prepare a fermentation broth according to the fed-batch fermentation experiment in Example 2;

[0095] 3.2 The fermentation liquid obtained in step 3.1 was treated at 42°C for 2 hours, and the temperature was adjusted to 40°C for fermentation and culture for 8 hours to obtain the fermentation stock liquid.

[0096] The HPLC method was used to measure the lactose content, 0 galactose content, and 41.42 g / L of the fermentation liquid. The lactose content, 0 galactose content, and 41.42 g / L of FUCΔZ were respectively obtained. The results indicate that the modification of FUCΔZ in the present application does not affect the yield of FUC, and that a FUC fermentation liquid free of lactose residue can be obtained by two heating treatments.

[0097] 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. A method for producing fucose with reduced lactose residue, characterized in that: The following steps are involved: (1) The engineered bacteria that reduces lactose residue during fucose synthesis are subjected to fed-batch fermentation for 78-90 hours to obtain a fermentation broth; (2) The fermentation liquid obtained in step (1) was treated at 42°C for 2 hours, and then the temperature was adjusted to 40°C for fermentation for 8 hours, and the fermentation was terminated; In step (1), the engineered bacteria for reducing lactose residue during fucose synthesis express phosphomannose mutant enzyme ManB, mannose-1-phosphate guanylyltransferase ManC, GDP-D-mannose-4,6-dehydratase Gmd, GDP-L-fucose synthase WcaG, α-1,2-fucosyltransferase WbgL, α-L-fucosidase, β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK, galactose-1-epimerase GalM and UTP-glucose-1-phosphate uridyltransferase; The expression of LacZ, GalE, GalT, GalK, GalM and UTP-glucose-1-phosphate uridyltransferase in the engineered bacteria for reducing lactose residue during fucose synthesis is controlled by a promoter with a temperature-sensitive element; In step (1), the engineered bacteria for reducing lactose residues during fucose synthesis integrates the coding genes of β-galactosidase LacZ, UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronic acid transferase GalT, galactokinase GalK, galactose-1-epimerase GalM and UTP-glucose-1-phosphate uridyltransferase BLUSP into the engineered bacteria FUCΔZ for synthesizing fucose; The engineered bacterium FUCΔZ is based on Escherichia coli BL21 (DE3) and is characterized by knocking out the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK, and the 11-carbonyl glucose phosphotransferase wcaj genes, and integrating the phosphomannose mutant enzyme manB, the mannose-1-phosphate guanyltransferase manC, the GDP-D-mannose-4,6-dehydratase Gmd, the GDP-L-fucose synthase wcaG, the α-1,2-fucosyltransferase wbgL, and the α-L-fucosidase afcA genes; In step (1), the temperature-sensitive element is the temperature-sensitive mutant clts857 of the cl protein, and the nucleotide sequence of the temperature-sensitive element is shown in SEQ ID NO.

7.

2. The production method according to claim 1, wherein The amino acid sequence of the knockout and integrated β-galactosidase LacZ is shown in SEQ ID NO.1; The amino acid sequence of the UDP-galactose-4-epimerase GalE is shown in SEQ ID NO.2; The amino acid sequence of the galactose-1-phosphate uronyltransferase GalT is shown in SEQ ID NO.3; The amino acid sequence of the galactokinase GalK is shown in SEQ ID NO.4; The amino acid sequence of the galactose-1-epimerase GalM is shown in SEQ ID NO.5; The amino acid sequence of the UTP-glucose-1-phosphate uridyltransferase is shown in SEQ ID NO.

6.

3. The production method according to any one of claims 1 to 2, characterized in that In step (1), the engineered bacteria further comprises overexpressing β-galactosidase permease LacY.

4. The production method according to claim 3, wherein In step (1), the method for constructing the engineered bacteria includes the following steps: S1. Using Cripsr-Cas9 and enzyme ligation 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, and the phosphomannose mutant manB, mannose-1-phosphate guanyltransferase manC, GDP-D-mannose-4,6-dehydratase Gmd, GDP-L-fucose synthase wcaG, α-1,2-fucosyltransferase wbgL, and α-L-fucosidase afcA genes were integrated to obtain the engineered strain FUCΔZ. S2, synthesize a β-galactosidase LacZ expression cassette containing a thermosensitive element in the promoter, and transfer it into the engineered bacteria FUCΔZ obtained in step S1 to obtain engineered bacteria A; S3, replacing the promoter of β-galactosidase LacY in the engineered bacteria A obtained in step S2 with a strong promoter to obtain engineered bacteria B; S4, synthesize the expression cassettes of UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronyltransferase GalT, and galactokinase GalK, whose promoters contain temperature-sensitive elements, and transfer them into the engineered bacteria B obtained in step S3 to obtain engineered bacteria C; S5. Using Cripsr-Cas9 technology to knock out the galactose-1-epimerase GalM in the engineered bacteria C obtained in step S4, and integrating the galactose-1-epimerase GalM whose promoter contains a temperature-sensitive element; S6. A UTP-glucose-1-phosphate uridyltransferase BLUSP expression cassette containing a thermosensitive element in the promoter was synthesized to obtain an engineered bacterium that reduces lactose residue during fucose synthesis.

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