Engineering bacterium for reducing lactose residue in fucose synthesis process 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 reducing lactose residues in the fucose synthesis process and a construction method and application of the engineering bacterium. The invention provides an engineering bacterium for reducing lactose residues in a fucose synthesis process. The engineering bacterium expresses an enzyme for synthesizing fucose and an enzyme for metabolizing lactose. GalM, LacZ, GalE, GalT, GalK and BLUSP are integrated in the engineering bacterium FUCdeltaZ capable of realizing total synthesis of fucose, a lactose metabolism pathway is constructed, an enzyme capable of metabolizing lactose is obtained through transcription translation by regulating and controlling the temperature, and lactose is metabolized into glucose-1-phosphoric acid, so that the purpose of no lactose and low residue or no residue in fermentation liquor is achieved.

Description

Technical Field

[0001] The present application relates to the technical field of microbial genetic engineering, and in particular to an engineering bacterium for reducing lactose residues during fucose synthesis, 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 make up human milk oligosaccharides. It is widely found 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.

[0003] At present, the applicant's preliminary research has successfully synthesized FUC in Escherichia coli, and its pathway is: fructose-6-phosphate is used as a substrate to synthesize 2'-fucosyllactose through a series of enzyme reactions and under the mediation of lactose, and 2'-fucosyllactose is decomposed into fucose and lactose under the action of α-L-fucosidase. According to previous studies, adding 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, it is urgent 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 engineering bacterium for reducing lactose residues during fucose synthesis, and a construction method and application thereof.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] In a first aspect, the present application provides an engineering 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 synthesizing fucose includes at least one of 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;

[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-isomerase 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 metabolic pathway of lactose (lactose→galactose→α-galactose→α-galactose-1-phosphate→UDP-galactose→UDP-glucose→glucose-1-phosphate), obtains an enzyme capable of metabolizing lactose through transcription and translation, and finally metabolizes lactose into glucose-1-phosphate, so as to achieve the purpose of low or no residual lactose in the fermentation broth.

[0012] The final product of the lactose metabolic pathway, glucose-1-phosphate, can be converted into glucose-6-phosphate by phosphoglucomutase in Escherichia coli and enter the glycolysis pathway for metabolism. In the lactose metabolic pathway, the conversion of UDP-glucose into glucose-1-phosphate mediated by BLUSP is a reversible reaction. Based on the chemical reaction equilibrium principle of reversible reactions, when glucose-1-phosphate is metabolized, its concentration will also decrease. Therefore, in order to achieve reaction equilibrium, BLUSP will mediate more UDP-glucose into 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), in which the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the phosphate undecyl glucose phosphotransferase wcaj gene are knocked out, and the phosphomannose mutant enzyme manB, the mannose-1-phosphate guanosyltransferase 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 are integrated.

[0014] As a preferred embodiment of the engineered bacteria described in the present application, the β-galactosidase LacZ is derived from 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 subsp. is Bifidobacterium longum subsp. longum ATCC 55813.

[0016] As a preferred embodiment of the engineering 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 uronyl transferase 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 a 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 the present application is cl protein, which can inhibit the promoters PR and PL at 30°C, release the inhibition of the promoter at 42°C, and 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 the present 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, and construct a lactose metabolic pathway, so that the strain itself can metabolize lactose, 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-galactoside permease LacY.

[0027] As a preferred embodiment of the engineering bacteria described in the present application, the overexpression of β-galactosidase LacY is to replace the promoter of β-galactosidase LacY with a strong promoter; the strong promoter includes at least one of T7 promoter, Tac promoter, Lac promoter and Trp promoter. In order to improve the transport capacity of lactose, the present application increases the intracellular concentration of lactose by replacing the promoter of β-galactosidase LacY with a strong promoter, so that the synthesis of fucose and the lactose can be completely metabolized during the two heating treatments.

[0028] As a preferred embodiment of the engineered bacteria described in the present application, the strong promoter is 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. The β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the phosphate undecyl glucose phosphotransferase wcaj gene in Escherichia coli BL21 (DE3) were knocked out by using Cripsr-Cas9 and restriction ligation technology, and the phosphomannose mutant 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 were integrated to obtain the engineered bacteria FUCΔZ.

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

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

[0033] S4, synthesizing the expression cassettes of UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronyl transferase GalT and galactokinase GalK containing the thermosensitive element in the promoter, and transferring them into the engineered bacteria B obtained in step S3 to obtain the 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 temperature-sensitive element in the synthetic promoter was obtained to obtain an engineered bacterium that reduces lactose residues 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 for 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 solution.

[0040] The present application performs a first heating treatment on the fermentation broth at 40-42°C for 2-4 hours to allow the temperature-sensitive element to release the inhibition of the promoter and express the enzyme in the lactose metabolic pathway, and then adjusts the temperature to 37-40°C for a second heating treatment for 4-10 hours to allow lactose to fully react with the enzyme and complete the metabolism of lactose.

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

[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, and the carbon source is glycerol.

[0044] As a preferred embodiment of the production method described in the present application, in step (1), the batch fed 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 ventilation rate to 2VVM, the rotation speed to 900rpm, and culture until the glycerol in the fermentation medium is exhausted, add the feed solution to the fermentation tank at a constant rate of 4g / L / h, adjust the culture temperature to 29.5°C, ferment for 4h, add IPTG with a final concentration of 0.2mM and 40g lactose, supplement with 17.78g / L / h of glycerol solution, and culture for 78-90h;

[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 MgSO4·7H2O, and 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 bacteria FUCΔZ 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 by transcription and translation through temperature regulation, and finally metabolizes lactose into glucose-1-phosphate, so as to achieve the purpose of low or no residual lactose in the fermentation broth.

[0050] 2. The temperature-sensitive element used in the present application is cl protein, which can inhibit the promoters PR and PL at 30°C, release the inhibition of the promoter at 42°C, and 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 the present 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, so that the strain itself can metabolize lactose, 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 of degrading lactose by using the engineered bacteria FUCB to reduce lactose residues during fucose synthesis. DETAILED DESCRIPTION

[0052] 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.

[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 uronosyltransferase 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 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.

[0058] 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.

[0059] 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.

[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 of carbon source and 5 g / L of MgSO4·7H2O, wherein the carbon source was glycerol.

[0062] 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, 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).

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

[0065] (1) 1 mL of fermentation broth was boiled in water 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.

[0066] (2) 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 sample to be tested, and the contents of lactose, galactose and FUC in the sample to be tested were calculated according to the external standard method. The chromatographic conditions were as follows: mobile phase: 5 mM aqueous sulfuric acid solution, flow rate: 0.5 mL / min, column temperature: 60 °C, injection volume: 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 engineering bacterium for reducing lactose residue during fucose synthesis and a construction method thereof, wherein the construction 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 the strain D was named FUCΔZ;

[0071] 1.2 According to the restriction 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 a template to obtain 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, and positive transformants were obtained by resistance plate screening. After the plasmid was removed from the positive transformants, strain I was obtained;

[0072] 1.3 According to the method of step 1.2, a Tac-lacY expression cassette with a Tac promoter was constructed and integrated into the strain Ⅰ obtained in step 1.2. After screening positive transformants, the plasmid was removed to obtain strain Ⅱ;

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

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

[0075] 1.6 Follow the method in step 1.2 to build a ts -p R -p L The promoter cl ts -p R -p L -BLUSP was integrated into strain IV obtained in step 1.5, and the plasmid was removed after screening positive transformants 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 sensitive to temperature (the cl protein inhibits the promoter at 30°C and releases the inhibition of the promoter at 42°C). The optimal activity of most enzymes in E. coli is at 37°C, so it is necessary to explore the effect of increased temperature on enzyme activity. The specific plan is as follows:

[0081] 1. Fermentation broth obtained by batch feeding 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 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;

[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, supplementing with 17.78 g / L / h of glycerol solution, culturing for 90 h before fermentation is terminated to obtain a fermentation broth.

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

[0087] The fermentation broth was heated twice according to the conditions in Table 3, and the lactose content in the fermentation broth was detected at 0, 1, 2, 4 and 8 h of the second heating treatment, respectively. The results are shown in Table 3. The first heating was to release the inhibition of the promoter and express the enzyme involved in the lactose metabolic pathway, and the second heating was to react the enzyme that metabolizes lactose with lactose, so that lactose is 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 in the fermentation broth of groups A, B, C and D after the second heating treatment for 8 hours was significantly reduced compared with the residual lactose before the second heating treatment, indicating that the present application can regulate the temperature-sensitive element to release the inhibition of the promoter by performing the second heating treatment on the fermentation broth, so that lactose is metabolized into glucose-1-phosphate in vivo through the treatment of LacZ, GalM, GalK, GalT, GalE and BLUSP (the metabolic process is shown in Figure 1 ), reducing the residual lactose, greatly improving the purity of FUC, and saving the steps of FUC separation and purification. At the same time, since the heating treatment will cause a part of the liquid to evaporate, there will be slight changes in the content of FUC and lactose. According to the data in Table 3, the two heatings will not cause much difference in the yield of fucose, which shows that the fucose production method of the present application can reduce the residual lactose without affecting the yield of FUC, and has 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 culture for 8 hours to obtain the fermentation stock liquid.

[0096] The HPLC method was used to measure the lactose content in the fermentation stock solution to be 0, the galactose content to be 0, and the FUC content to be 41.42 g / L, while the lactose content of FUCΔZ was 15.05 g / L, the galactose content to be 0, and the FUC content to be 41.42 g / L, indicating that the modification of FUCΔZ in the present application does not affect the yield of FUC, and a FUC fermentation solution without 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 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 for reducing lactose residue during fucose synthesis, characterized in that: The engineered bacteria express an enzyme for synthesizing fucose and an enzyme for metabolizing lactose; The enzyme synthesizing fucose includes at least one of 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; 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.

2. The engineered bacteria according to claim 1, characterized in that The engineering 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 uridyl transferase BLUSP into the engineering bacteria FUCΔZ for synthesizing fucose.

3. The engineered bacteria according to claim 2, characterized in that The engineered bacteria FUCΔZ is based on Escherichia coli BL21 (DE3), in which the β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the phosphate undecyl glucose phosphotransferase wcaj gene are knocked out, and the phosphomannose mutant enzyme manB, the mannose-1-phosphate guanosyltransferase 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 gene are integrated.

4. The engineered bacteria according to claim 3, characterized in that The amino acid sequence of the 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 uronyl transferase 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.

5. The engineered bacteria according to any one of claims 1 to 4, characterized in that The expression of beta-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 uridyl transferase in the engineering bacteria is controlled by a promoter with a temperature-sensitive element.

6. The engineered bacteria according to claim 5, characterized in that The temperature-sensitive element is a temperature-sensitive mutant of the cl protein, clts857, and the nucleotide sequence of the temperature-sensitive element is shown in SEQ ID NO.

7.

7. The engineered bacteria according to any one of claims 1 to 6, characterized in that The engineered bacteria also include overexpression of β-galactosidase permease LacY.

8. The method for constructing an engineered bacterium according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. The β-galactosidase lacZ, the fucose isomerase / fucokinase gene cluster fucIK and the phosphate undecyl glucose phosphotransferase wcaj gene in Escherichia coli BL21 (DE3) were knocked out by using Cripsr-Cas9 and restriction ligation technology, and the phosphomannose mutant 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 were integrated to obtain the engineered bacteria FUCΔZ. S2, synthesizing a β-galactosidase LacZ expression cassette containing a thermosensitive element in the promoter, and transferring it into the engineered bacteria FUCΔZ obtained in step S1 to obtain engineered bacteria A; S3, replacing the promoter of β-galactoside permease LacY in the engineered bacteria A obtained in step S2 with a strong promoter to obtain engineered bacteria B; S4, synthesizing the expression cassettes of UDP-galactose-4-epimerase GalE, galactose-1-phosphate uronyl transferase GalT and galactokinase GalK containing the thermosensitive element in the promoter, and transferring them into the engineered bacteria B obtained in step S3 to obtain the 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 temperature-sensitive element in the synthetic promoter was obtained to obtain an engineered bacterium that reduces lactose residues during fucose synthesis.

9. Use of the engineered bacteria according to any one of claims 1 to 7 in producing fucose.

10. A method for producing fucose with reduced lactose residue, characterized in that: The following steps are involved: (1) subjecting the engineered bacteria for reducing lactose residue in the fucose synthesis process according to any one of claims 1 to 7 to fed-batch fermentation for 78 to 90 hours to obtain a fermentation broth; (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, and the fermentation is terminated.

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