WO2022148008A1 - 产塔格糖的枯草芽孢杆菌基因工程菌及制备塔格糖的方法 - Google Patents

产塔格糖的枯草芽孢杆菌基因工程菌及制备塔格糖的方法 Download PDF

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WO2022148008A1
WO2022148008A1 PCT/CN2021/108983 CN2021108983W WO2022148008A1 WO 2022148008 A1 WO2022148008 A1 WO 2022148008A1 CN 2021108983 W CN2021108983 W CN 2021108983W WO 2022148008 A1 WO2022148008 A1 WO 2022148008A1
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tagatose
phosphate
gene
thermostable
glucose
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French (fr)
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马延和
石婷
李运杰
韩平平
李元
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中国科学院天津工业生物技术研究所
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Priority to JP2023540968A priority patent/JP2024503821A/ja
Priority to EP21917051.1A priority patent/EP4276171A1/en
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Definitions

  • the invention relates to the technical field of bioengineering, in particular to a tagatose-producing genetically engineered bacteria and a method for preparing tagatose.
  • Tagatose is a rare monosaccharide occurring in nature, the ketose form of galactose and the epimer of fructose.
  • the sweetness properties of tagatose are similar to that of sucrose, with only one-third the calories of sucrose, and are known as low-calorie sweeteners.
  • Natural tagatose mainly exists in dairy products such as yogurt and milk powder. Tagatose offers a very fresh and pure sweetness with a taste profile similar to fructose.
  • tagatose has important physiological functions such as low calorie, low glycemic index, anti-caries, anti-oxidation, prebiotics, improving intestinal function, immune regulation, drug precursors, etc., and can be widely used in food, beverage, medicine, Health care and other fields, with huge economic value (Oh D-K: Tagatose: properties, applications, and biotechnological processes. App. Microbiol. Biotechnol. 2007, 76: 1-8).
  • the U.S. Food and Drug Administration confirmed the safety of tagatose and approved it as a GRAS (Generally Regarded As Safe) product.
  • the Food and Agriculture Organization of the United Nations and the World Health Organization Joint Committee on Food Additives (JECFA) recommended tagatose as a new low-calorie sweetener that can be used as a food additive.
  • the production methods of tagatose mainly include chemical synthesis and biotransformation.
  • galactose is used as raw material, and it is formed by chemical method or biological transformation through isomerization reaction.
  • the chemical synthesis method uses soluble alkali metal salts or alkaline earth metal salts as catalysts to promote galactose to generate tagatose under alkaline conditions, and form a metal hydroxide-tagatose complex, which is then neutralized with acid to obtain tagatose. sugar.
  • Chemical methods have high energy consumption, complex products, difficult purification, many side reactions, and chemical pollution.
  • the biological method uses galactitol or galactose to convert the corresponding substrate into tagatose under the catalysis of enzymes or microorganisms.
  • Galactitol is expensive and limited in sources, making it unsuitable for use as a raw material for industrial production.
  • the mainstream production method of tagatose is to make pure tagatose through the steps of galactose isomerization, desalination, decolorization, separation, concentration and crystallization.
  • South Korea's CJ company excavated and transformed hexuronate 4-epimerase to convert fructose into tagatose (CN105431541B, CN109415715A), and the modified hexuronate 4-epimerase enzyme activity was extremely low , does not have industrial application value.
  • South Korea's CJ Company also excavated a new source of 6-phosphate tagatose phosphatase and applied it to the conversion of substrates such as starch, maltodextrin and sucrose to produce tagatose (WO2018004310A1, CN 109790524A), but this 6-phosphate tagatose The enzyme activity of sugar phosphatase is very low, and it does not have industrial application value at this stage.
  • the fermentation production host of the key enzymes in this method is Escherichia coli BL21 (DE3).
  • Escherichia coli is not suitable for industrial production as a production strain for food preparation production-related enzymes.
  • the in vitro enzyme separation and purification steps are cumbersome, and the enzyme recovery and utilization rate Low cost, difficult to recycle, unable to further reduce production costs, and unable to achieve mass production and application of industrialized high-concentration products.
  • the main purpose of the present invention is to provide a method for preparing and producing high-concentration tagatose by utilizing Bacillus subtilis whole cells to catalyze high-concentration starch to prepare and produce high-concentration tagatose.
  • the present invention first provides a tagatose-producing Bacillus subtilis genetically engineered bacteria, which co-express ⁇ -glucan phosphorylase gene, glucose phosphate mutase gene, glucose phosphate isomerase gene, 6-phosphate tower Bacillus subtilis genetically engineered bacteria expressing glycoepimerase gene and 6-phosphate tagatose phosphatase gene, or expressing ⁇ -glucan phosphorylase gene, glucose phosphate mutase gene, glucose phosphate isomerase gene respectively
  • the principle of the above technical solution is to make full use of the relevant catalytic pathways to express at the level of living cells and realize an effective catalytic reaction, which includes converting the substrate starch into the intermediate glucose-1-glucose by ⁇ -glucan phosphorylase in the presence of inorganic phosphorus.
  • G1P intermediate glucose-1-phosphate
  • G1P is mutated to another intermediate glucose-6-phosphate (G6P) by glucose phosphate mutase
  • intermediate glucose-6 is converted by glucose phosphate isomerase - Phosphate (G6P) is mutated to another intermediate fructose-6-phosphate (F6P)
  • intermediate fructose-6-phosphate (F6P) is isomerized to another by 6-phosphate tagatose epimerase
  • Intermediate Tagatose-6-Phosphate (T6P) Intermediate Tagatose-6-Phosphate (T6P) is removed from the phosphate group by 6-Phosphate Tagatose phosphatase to produce Tagatose.
  • Bacillus subtilis strains known in the art can be used as starting strains in the present invention, such as Bacillus subtilis 168, DB104, WB800, WB600, SCK6, 1A751, ATCC6051a, ATCC6051 and the like.
  • the Bacillus subtilis starting strain is a protease knockout Bacillus subtilis strain, such as WB800, WB600, SCK6, 1A751 and the like. More preferably, the starting strain of Bacillus subtilis is SCK6.
  • the Bacillus subtilis genetically engineered bacteria include co-expression of ⁇ -glucan phosphorylase, glucose phosphate mutase, glucose phosphate isomerase, and 6-phosphate tagatose epimerase and the expression vector of 6-phosphate tagatose phosphatase, or the genetically engineered bacteria containing the expression vector of ⁇ -glucan phosphorylase, the genetically engineered bacteria of the expression vector of glucose phosphomutase, glucose phosphate isomerase A mixture of genetically engineered bacteria for the expression vector, genetically engineered bacteria for the expression vector of 6-phosphate tagatose epimerase, and genetically engineered bacteria for the expression vector of 6-phosphate tagatose phosphatase.
  • the various enzymes are thermostable, namely thermostable ⁇ -glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphate isomerase, thermostable 6-phosphate taggase Glycoepimerase and thermostable 6-phosphate tagatose phosphatase.
  • thermostable enzymes Compared with thermolabile normal temperature enzymes, the use of thermostable enzymes has advantages in strain inactivation, that is, the latter can inactivate strains by heat treatment after fermentation, while at the same time tagatose synthesis-related The enzyme remains active so that inactivated strains can be mixed for tagatose production, which is more suitable for industrial applications. If the related enzymes at room temperature are used, after the fermentation production is completed, the pure enzymes need to be obtained by breaking the cells and enzyme purification steps before the related enzymes can be obtained for the production of tagatose.
  • thermostable ⁇ -glucan phosphorylase refers to a temperature of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or An enzyme with the function of phosphorylating starch to glucose-1-phosphate (G1P) above 80°C.
  • thermostable ⁇ -glucan phosphorylase is derived from a thermophilic microorganism, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermomophilum, Caldicellulosirensis, Clostridium thermocellum, Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, etc.; or the amino acid sequence of the thermostable ⁇ -glucan phosphorylase and the thermostable ⁇ -glucan derived from the thermophilic microorganism
  • the sugar phosphorylases are at least 70%, preferably at
  • thermostable glucose phosphomutase refers to a temperature of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher.
  • Glucose-1-phosphate G1P is mutated into an enzyme that functions as glucose-6-phosphate (G6P).
  • thermostable glucose phosphomutase is derived from thermophilic microorganisms, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor kronotsky cell, Clostridium ensis Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, etc.; or the amino acid sequence of the thermostable glucose phosphomutase and the thermostable glucose phosphomutase derived from the thermophilic microorganism have at least 70%, Preferably at least 80%, more
  • thermostable glucose phosphate isomerase refers to a temperature of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C or higher.
  • thermostable glucose phosphate isomerase is derived from thermophilic microorganisms, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor kronotskyensis Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, etc.; or the amino acid sequence of said thermostable glucose phosphate isomerase has at least 70% of the thermostable glucose phosphate isomerase derived from said thermophilic microorganism, Preferably at least 80%, more preferably at least 90%, most
  • the heat-stable 6-phosphate tagatose epimerase is 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or An enzyme with the function of isomerizing fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) above 80°C.
  • F6P fructose-6-phosphate
  • T6P tagatose-6-phosphate
  • thermostable 6-phosphate tagatose epimerase is derived from thermophilic microorganisms, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga themarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermophilum, Caldicellulosiruptor kronotskyensis, Clostridium thermocellum, Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, etc; Thermostable 6-phosphate tagatose epimerases are at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identical. More preferably, the 6-
  • the tagatose 6-phosphate phosphatase refers to a temperature above 40°C, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, or 80°C
  • the above enzymes have the function of tagatose-6-phosphate (T6P) removing the phosphate group as the product Tagatose.
  • the tagatose 6-phosphate phosphatase is derived from thermophilic microorganisms, such as Geobacillus kaustophilus, Geobacillus stearothermophilus, Thermotoga maritima, Pseudothermotoga thermarum, Thermococcus kodakarensis, Archaeoglobus fulgidus, Thermoanaerobacter indiensis, Dictyoglomus thermomophilum, Caldicellulosiruptor kroostium thermocells , Caldilinea aerophila, Pyrococcus furiosus, Thermus thermophilus, Methanothermobacter marburgensis, Archaeoglobus profundus, etc.; or the amino acid sequence of the 6-phosphate tagatose phosphatase and the 6-phosphate tagatose phosphatase derived from the thermophilic microorganism have at least 70%, preferably at least
  • the present invention accordingly provides an expression vector for the above-mentioned genetically engineered bacteria, which comprises ⁇ -glucan phosphorylase, glucose phosphate mutase, glucose phosphate isomerase, and 6-phosphate tagatose epimerization enzyme and 6-phosphate tagatose phosphatase genes, and can achieve co-expression of these genes.
  • the vectors and genetically engineered bacteria involved in the present invention can be prepared by conventional methods known in the art, for example, by recombinant DNA technology construction to obtain ⁇ -glucan phosphorylase gene, glucose phosphorylase locase gene, glucose phosphate isomerase gene, 6-phosphate tagatose epimerase gene, 6-phosphate tagatose phosphatase gene, construct a recombinant expression vector, and then transfer it into Bacillus subtilis by known methods Obtain genetically engineered bacteria.
  • the vector includes a promoter, a thermostable ⁇ -glucan phosphorylase gene, a thermostable glucose phosphate mutase gene, a thermostable glucose phosphate isomerase gene, a thermostable 6-phosphate tagatose gene.
  • a promoter a promoter, a thermostable ⁇ -glucan phosphorylase gene, a thermostable glucose phosphate mutase gene, a thermostable glucose phosphate isomerase gene, a thermostable 6-phosphate tagatose gene.
  • thermostable ⁇ -glucan phosphorylase respectively include a promoter, a thermostable ⁇ -glucan phosphorylase gene, and a terminator;
  • the vectors for expressing thermostable glucose phosphomutase include Promoter, thermostable glucose phosphomutase gene, terminator;
  • the vector expressing thermostable 6-phosphate tagatose epimerase includes promoter, thermostable 6-phosphate tagatose epimerase Gene, terminator;
  • the vector expressing thermostable 6-phosphate tagatose epimerase includes promoter, thermostable 6-phosphate tagatose epimerase gene, terminator;
  • the expression thermostable 6-phosphate epimerase The vector of 6-phosphate tagatose phosphatase includes a promoter, a thermostable 6-phosphate tagatose phosphatase gene, and a terminator.
  • promoters known in the art can be used as promoters in the present invention, including but not limited to P43 promoter, Pylb promoter, PamyL promoter, Plaps promoter, PhpaII promoter, PamyE promoter Promoter, Pgrac promoter, PsacB promoter, PsigX promoter, PaprE promoter, PgroES promoter, etc.
  • the promoter of the present invention is selected in tandem between the PhpaII promoter and the Pylb promoter.
  • terminators known in the art can also be used as terminators in the present invention.
  • the endogenous uracil phosphoribosyltransferase gene, and/or the ⁇ -amylase gene, and/or the sporulation RNA polymerase ⁇ F factor gene in the genetically engineered bacteria, and /or the surfactin-active peptide synthase subunit 3 gene is inactivated or knocked out.
  • the endogenous uracil phosphoribosyltransferase gene, ⁇ -amylase gene, spore-forming RNA polymerase ⁇ F factor gene, and surface active peptide synthase subunit 3 gene in the genetically engineered bacteria are all inactivated or knocked out.
  • the inactivation or knockout of the above-mentioned genes can further improve the efficiency of tagatose production by genetically engineered bacteria.
  • knockout gene i.e. knockout gene
  • inactivation or knockout of ⁇ -amylase gene can cut off the way that strains utilize exogenous starch and prevent the production of tagatose.
  • Starch is metabolized by the strain as carbon source; inactivating or knocking out the spore-forming RNA polymerase ⁇ F factor gene can flow to the direction of heterologous protein synthesis and expression related to tagatose synthesis for the bacterial metabolism, and can also make the strain fermented Control does not form spores; inactivation or knockout of the surfactant peptide synthase subunit 3 gene can make it easier to control the fermentation production of bacteria without producing too much foam. Therefore, inactivation or knockout of the above-mentioned four genes can be more beneficial to the production of tagatose by genetically engineered bacteria, so it is a preferred embodiment.
  • Methods known in the art are used to achieve inactivation or knockout of endogenous genes, preferably using gene editing methods.
  • the present invention further provides a method for preparing and producing tagatose using the above-mentioned genetically engineered bacteria whole cell catalyzed starch, which comprises the following steps:
  • step (2) performing cell membrane permeability treatment on the Bacillus subtilis whole cells obtained in step (1) to obtain permeable whole cells;
  • step (3) using the permeable whole-cell catalytic starch obtained in step (2) to prepare tagatose, wherein the co-expressed Bacillus subtilis engineering bacteria are directly used for catalysis, and the Bacillus subtilis engineering bacteria expressing various enzymes respectively are used for catalysis. Mixed for catalysis.
  • the method also includes immobilizing the permeable whole cells of Bacillus subtilis obtained in step (2) to obtain immobilized whole cells, or a mixture of immobilized whole cells, which is then used for catalysis.
  • the preparation of the whole cells in step (1) is performed using methods known in the art. Fermentation can use any medium suitable for exogenous protein expression, including but not limited to LB medium, SR medium, TB medium, and the like.
  • the cell membrane permeability treatment in step (2) can adopt known methods, including but not limited to heat treatment, addition of organic solvents and/or addition of surfactants.
  • the organic solvent includes, but is not limited to, acetone, acetonitrile, and the like.
  • Surfactants include, but are not limited to, cetyltrimethylammonium bromide (CTAB), Tween-80, and the like.
  • CTAB cetyltrimethylammonium bromide
  • Tween-80 Tween-80
  • the cell membrane permeability treatment is heat treatment. The purpose of permeabilizing the cell membrane is to enable the extracellular starch to enter the cell through the cell membrane.
  • the heat treatment temperature is 45-100°C; more preferably, the heat treatment temperature is 70-80°C.
  • the heat treatment time is 10-100 min; more preferably, the heat treatment time is 50-70 min.
  • the heat treatment can be carried out in a buffer-free system or a buffer system; preferably, the heat treatment is carried out in a buffer system, and the buffer can be HEPES buffer, phosphate buffer, Tris buffer, Acetate buffer, etc. Among them, phosphate buffer such as sodium phosphate buffer, potassium phosphate buffer and the like.
  • the concentration of the substrate starch is 50-300 g/L; more preferably, the concentration of the substrate starch is 100-200 g/L.
  • the reaction conditions are: at pH 5.0-8.0, 40-80°C for 0.5-96h; more preferably at pH 6.5-7.5, 45-75°C for 12-60h; most preferably at pH 7.5, React at 60-70°C for 12-96h.
  • the catalysis can be carried out in a buffer-free system or a buffer system; preferably, in a buffer system, the buffer can be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, etc. Among them, phosphate buffer, such as sodium phosphate buffer, potassium phosphate buffer, etc.
  • the mixture is a mixture of the permeable whole cells expressing thermostable ⁇ -glucan phosphorylase, the permeable whole cells expressing thermostable glucose phosphate mutase Permeable whole cells, permeable whole cells expressing thermostable glucose phosphate isomerase, permeable whole cells expressing thermostable tagatose 6-phosphate epimerase, permeable whole cells expressing thermostable tagatose 6-phosphate phosphatase
  • the ratio of permeable whole cells is (0.1-10):(0.1-10):(0.1-10):(0.1-10); more preferably (0.5-5):(0.5 -5): (0.5-5): (0.5-5): (0.5-5), most preferably 1:1:1:1:1.
  • the permeable whole cell immobilization treatment method is: resuspending with sodium phosphate or potassium phosphate buffer to co-express thermostable ⁇ -glucan phosphorylase, thermostable glucose phosphomutase, Permeable whole cells of thermostable glucose phosphate isomerase, thermostable 6-phosphate tagatose epimerase and thermostable 6-phosphate tagatose phosphatase, add appropriate amount of inorganic soil, and stir well. Add polyethylenimine aqueous solution to flocculate at room temperature, and then add crosslinking agent for crosslinking. Then suction filtration to obtain a filter cake layer, the filter cake is washed with deionized water, and then extruded into particles, and dried to obtain immobilized whole cells.
  • the processing method for the mixture of immobilized permeable whole cells is as follows: resuspend the permeable whole cells expressing thermostable ⁇ -glucan phosphorylase with sodium phosphate or potassium phosphate buffer, respectively; Phosphomutase permeable whole cells, permeable whole cells expressing thermostable glucose phosphate isomerase, permeable whole cells expressing thermostable 6-phosphate tagatose epimerase, permeable whole cells expressing thermostable 6-phosphate To permeabilize whole cells with tagatose phosphatase, add an appropriate amount of inorganic soil and stir well. Add polyethylenimine aqueous solution to flocculate at room temperature, and then add crosslinking agent for crosslinking. Then suction filtration to obtain a filter cake layer, the filter cake is washed with deionized water, and then extruded into particles, and dried to obtain immobilized whole cells.
  • the inorganic earth includes but is not limited to montmorillonite, diatomite, kaolin, bentonite, etc.
  • the inorganic earth is diatomite;
  • the cross-linking agent includes but is not limited to glutaraldehyde, Hydroxymethylphosphorus, N,N-methylenebisacrylamide, epichlorohydrin, genistein, etc.
  • the crosslinking agent is glutaraldehyde.
  • the present invention has the following beneficial effects:
  • thermostable ⁇ -glucan phosphorylase thermostable glucose phosphate mutase
  • thermostable glucose phosphate isomerase thermostable 6-phosphate tagatose epimerase
  • thermostable glucose phosphate isomerase thermostable glucose phosphate isomerase
  • Bacillus subtilis is a food-grade microorganism generally recognized as safe (Generally Recognized As Safe, GRAS) and does not produce endotoxin. Furthermore, knocking out the gene encoding ⁇ -amylase is beneficial to the catalytic application of subsequent substrate starch; knocking out the encoding gene of spore-forming RNA polymerase ⁇ F factor is beneficial to the subsequent fermentation and production of genetically engineered strains in substrate transformation. Application of tagatose production; Knockout of the gene encoding surface active peptide synthase subunit 3 is beneficial to the application of subsequent fermentation production of genetically engineered strains in the production of tagatose by substrate conversion;
  • the preparation of tagatose can be carried out at a higher temperature, thereby increasing the solubility of the substrate starch.
  • the present invention can be used in The preparation of tagatose is realized at a higher substrate concentration, which is beneficial to improve the production efficiency and further reduce the production cost.
  • the conversion reaction of tagatose can be carried out in a buffer-free system or a buffer system, and it is not necessary to use a medium containing carbon sources, nitrogen sources, inorganic salts and antibiotics.
  • a medium containing carbon sources, nitrogen sources, inorganic salts and antibiotics On the one hand, it is beneficial to reduce the The production cost, on the other hand, is beneficial to the separation and purification of the product tagatose.
  • FIG. 1 is a schematic diagram of the whole cell catalyzed starch production of tagatose of the present invention.
  • Figure 2 is a map of the recombinant expression vector pMA5-Pylb-aGP.
  • Figure 3 is a map of the recombinant expression vector pMA5-Pylb-PGM.
  • FIG. 4 Map of the recombinant expression vector pMA5-Pylb-PGI.
  • Figure 5 is a map of the recombinant expression vector pMA5-Pylb-TPE.
  • Figure 6 is a map of the recombinant expression vector pMA5-Pylb-TPP.
  • Figure 7 is a map of the recombinant expression vector pMA5-Pylb-aGP-PGM-PGI-TPE-TPP.
  • Figure 8 is a graph showing the change process of the yield of tagatose with reaction time.
  • Figure 9 is a trend diagram of the production of tagatose produced by immobilized co-expression engineered strains.
  • Figure 10 is a trend diagram of the immobilized production of tagatose by sub-expression engineered strains.
  • upp gene sequence (NCBI-ProteinID: NP_391570) of the uracil phosphoribosyltransferase encoding gene from Bacillus subtilis 168 in the KEGG database
  • primers were designed, and a 500bp homologous fragment upstream of the upp gene was obtained by PCR amplification and The downstream 500bp homologous fragment is connected by simple cloning (You, C., Zhang, X.Z., & Zhang, Y.H. (2012). Simple cloning via direct transformation of PCR product(DNA Multimer) to Escherichia coli and Bacillus subtilis.Appl. Environ.Microbiol., 78(5), 1593-1595.doi:10.1128/AEM.07105-11) was constructed into the integration vector pSS to obtain the recombinant integration vector pSS-upp-FR.
  • chloramphenicol 5 ⁇ g/mL
  • the positive single-crossover transformant colonies grown on chloramphenicol-resistant plates were picked for colony PCR verification, and two bands of 1000bp DNA fragment and 2000bp DNA fragment were obtained by PCR amplification (wherein the size of the 1000bp DNA fragment was the vector pSS-upp).
  • the fragment size of the upstream and downstream homology arms of the upp-encoding gene in the vector in the FR, the 2000bp DNA fragment size is the size of the genome including the upstream homology arm of the upp-encoding gene, the upp-encoding gene and the downstream homology arm of the upp-encoding gene) positive clone.
  • amyG gene sequence (NCBI-ProteinID: NP_388186) of the ⁇ -amylase encoding gene of Bacillus subtilis 168 in the KEGG database, primers were designed, and the upstream 500bp homologous fragment and the downstream 500bp homologous fragment of the amyG gene were obtained by PCR amplification
  • the homologous fragment of using simple cloning connection (You, C., Zhang, X.Z., & Zhang, Y.H. (2012). Simple cloning via direct transformation of PCR product(DNA Multimer) to Escherichia coli and Bacillus subtilis.Appl.Environ. Microbiol., 78(5), 1593-1595.doi:10.1128/AEM.07105-11) was constructed into the integration vector pSS to obtain the recombinant integration vector pSS-amyE-FR.
  • Bacillus subtilis strain SCK8 super-competent cells 200 ⁇ l
  • put it into a shaker at 37°C Resuscitate for 90min spread the bacterial solution on solid LB medium (5g/L yeast extract, 10g/L peptone, 10g/L sodium chloride) containing chloramphenicol (5 ⁇ g/mL), and place at 37°C Culture in the incubator for 14-16h.
  • solid LB medium 5g/L yeast extract, 10g/L peptone, 10g/L sodium chloride
  • the positive single-crossover transformant colonies grown on chloramphenicol-resistant plates were picked for colony PCR verification, and two bands of 1000bp DNA fragment and 2000bp DNA fragment were obtained by PCR amplification (wherein the size of the 1000bp DNA fragment was the vector pSS-amyE -The fragment size of the upstream and downstream homology arms of the amyE-encoding gene in the vector in the FR, the 2000bp DNA fragment size is the size of the genome including the upstream homology arms of the amyE-encoding gene, the amyE-encoding gene and the downstream homology arms of the amyE-encoding gene) positive clone.
  • spoIIAC gene sequence NCBI-ProteinID: NP_390226
  • NP_390226 the gene encoding sporulation RNA polymerase ⁇ F factor of Bacillus subtilis 168 in the KEGG database
  • primers were designed, and the 500 bp upstream of the sporulation spoIIAC gene was obtained by PCR amplification
  • the homologous fragment and the downstream 500bp homologous fragment were connected by simple cloning (You, C., Zhang, XZ, & Zhang, YH (2012).
  • Bacillus subtilis strain SCK8-ST1 super competent cells 200 ⁇ l
  • put into 37 Resuscitate in a shaker for 90 min spread the bacterial solution in solid LB medium (5 g/L yeast extract, 10 g/L peptone, 10 g/L sodium chloride) containing chloramphenicol (5 ⁇ g/mL)
  • the positive single-crossover transformant colonies grown on chloramphenicol-resistant plates were picked for colony PCR verification, and two bands of 1000bp DNA fragment and 2000bp DNA fragment were obtained by PCR amplification (wherein the size of the 1000bp DNA fragment was the vector pSS-spoIIAC).
  • the fragment size of the upstream and downstream homology arms of the spoIIAC-encoding gene in the vector in the FR, the 2000bp DNA fragment size is the size of the genome including the upstream homology arms of the spoIIAC-encoding gene, the spoIIAC-encoding gene and the downstream homology arms of the spoIIAC-encoding gene) positive clone.
  • the purpose of this step is to promote the intramolecular homologous recombination of the positive transformants by culturing in the LB medium without adding antibiotics, and then screen and culture in the 5-FU basic salt medium to obtain the target transformants of spoIIAC knockout.
  • srfAC gene sequence (NCBI-ProteinID: NP_388233) of the surface-active peptide synthase subunit 3 encoding gene of Bacillus subtilis 168 in the KEGG database
  • primers were designed, and a 500bp homologous fragment upstream of the srfAC gene was obtained by PCR amplification and the downstream 500bp homologous fragment, using simple cloning connection (You, C., Zhang, X.Z., & Zhang, Y.H. (2012).
  • Bacillus subtilis strain SCK8-ST2 super competent cells 200 ⁇ l
  • the positive single-crossover transformant colonies grown on chloramphenicol-resistant plates were picked for colony PCR verification, and two bands of 1000bp DNA fragment and 2000bp DNA fragment were obtained by PCR amplification (wherein the size of the 1000bp DNA fragment was the vector pSS-srfAC).
  • the fragment size of the upstream and downstream homology arms of the srfAC encoding gene in the vector in the FR, the 2000bp DNA fragment size is the size of the genome including the upstream homology arms of the srfAC encoding gene, the srfAC encoding gene and the downstream homology arms of the srfAC encoding gene) positive clone.
  • thermostable ⁇ -glucan phosphorylase in this example is from Thermococcus kodakarensis.
  • Thermostable ⁇ -glucan phosphorylase encoding gene agp sequence (NCBI-ProteinID: BAD85595) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and linked to a common plasmid.
  • the thermostable ⁇ -glucan phosphorylase-encoding gene agp gene was obtained by PCR using a pair of primers.
  • the linear backbone of pMA5-Pylb was obtained by PCR using a pair of primers using primers 299-F: 5'-AGAAACAACAAAGGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3' and 300-R: 5'-gcttgagctcgactctagaggatcctcagtcaagtcccttccacttgacca-3'.
  • primers 301-F 5'-tggtcaagtggaagggacttgactgaggatcctctagagtcgagctcaagc-3' and 302-R: 5'-caacggcattggaaacgttcaccatACAAATCTCCCCCTTTGTTGTTTCT-3';
  • All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the PCR conditions for the gene were denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.
  • the products obtained by PCR reaction were analyzed by 0.8% agarose gel electrophoresis. After the correct size of the fragment was confirmed by the gel imaging system, the target fragment was recovered using a DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd., China) for the construction of a recombinant expression vector.
  • thermostable ⁇ -glucan phosphorylase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR.
  • the POE-PCR system is as follows: purified pMA5-Pylb linear backbone, 200ng; purified heat-resistant ⁇ -glucan phosphorylase gene fragment, 131ng; 2 ⁇ PrimeSTAR MAX DNA Polymerase (Dalian Bao Bio, China), 25 ⁇ L, Add water to make up 50 ⁇ L.
  • the POE-PCR conditions were denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 3.5 min, and final extension at 72°C for 5 min.
  • the ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformants were selected for colony PCR and double-enzyme digestion identification, and 2-3 positive transformants were selected for further verification by sequencing.
  • the sequencing results showed that pMA5-Pylb- aGP recombinant co-expression vector, the plasmid map is shown in Figure 2.
  • thermostable glucose phosphomutase was from Thermococcus kodakarensis.
  • Thermostable glucose phosphomutase encoding gene pgm sequence (NCBI-ProteinID: BAD85297) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and connected to a common plasmid.
  • the thermostable glucose phosphomutase-encoding gene pgm was obtained by PCR from genomic DNA using a pair of primers.
  • Primers 327-F 5'-AGAAACAACAAAGGGGGAGATTTGTatgggcaaactgtttggtaccttcg-3' and 328-R: 5'-agcttgagctcgactctagaggatccTTAacctttcagtgcttcttccagc-3'; pMA5-Pylb linear backbone was obtained by PCR using a pair of primers.
  • primers 329-F 5'-gctggaagaagcactgaaaggtTAAggatcctctagagtcgagctcaagct-3' and 330-R: 5'-cgaaggtaccaaacagtttgcccatACAAATCTCCCCCTTTGTTGTTTCT-3';
  • All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the PCR conditions for the gene were denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.
  • the products obtained by PCR reaction were analyzed by 0.8% agarose gel electrophoresis. After the correct size of the fragment was confirmed by the gel imaging system, the target fragment was recovered using a DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd., China) for the construction of a recombinant expression vector.
  • thermostable glucose phosphomutase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR.
  • the POE-PCR system is as follows: purified pMA5-Pylb linear backbone, 200 ng; purified thermostable glucose phosphate mutase gene fragment 131 ng; 2 ⁇ PrimeSTAR MAX DNA Polymerase (Dalian Bao Bio, China), 25 ⁇ L, add water to make up 50 ⁇ L .
  • the POE-PCR conditions were denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 3.5 min, and final extension at 72°C for 5 min.
  • the ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformants were selected for colony PCR and double-enzyme digestion identification, and 2-3 positive transformants were selected for further verification by sequencing.
  • the sequencing results showed that pMA5-Pylb- PGM recombinant co-expression vector, the plasmid map is shown in Figure 3.
  • thermostable glucose phosphate isomerase in this example is from Thermus thermophilus.
  • Thermostable glucose phosphate isomerase encoding gene pgi sequence (NCBI-ProteinID: AAS82052) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and connected to a common plasmid.
  • the thermostable glucose phosphate isomerase-encoding gene pgi was obtained by PCR from genomic DNA using a pair of primers.
  • Primers 331-F 5'-AGAAACAACAAAGGGGGAGATTTGTATGCTGCGTCTGGATACTCGCTTTC-3' and 332-R: 5'-agcttgagctcgactctagaggatccTTAACCAGCCAGGCGTTTTACGAGTC-3'; pMA5-Pylb linear backbone was obtained by PCR using a pair of primers. Using primers 333-F: 5'-GACTCGTAAACGCCTGGCTGGTTAAggatcctctagagtcgagctcaagct-3' and 334-R: 5'-GAAAGCGAGTATCCAGACGCAGCATACAAATCTCCCCCTTTGTTGTTTCT-3';
  • All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the PCR conditions for the gene were denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.
  • the products obtained by PCR reaction were analyzed by 0.8% agarose gel electrophoresis. After the correct size of the fragment was confirmed by the gel imaging system, the target fragment was recovered using a DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd., China) for the construction of a recombinant expression vector.
  • thermostable glucose phosphate isomerase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR.
  • the POE-PCR system is as follows: purified pMA5-Pylb linear backbone, 200 ng; purified thermostable glucose phosphate isomerase gene fragment 131 ng; 2 ⁇ PrimeSTAR MAX DNA Polymerase (Dalian Bao Bio, China), 25 ⁇ L, add water to make up 50 ⁇ L .
  • the POE-PCR conditions were denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 3.5 min, and final extension at 72°C for 5 min.
  • the ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformants were selected for colony PCR and double-enzyme digestion identification, and 2-3 positive transformants were selected for further verification by sequencing.
  • the sequencing results showed that pMA5-Pylb- PGI recombinant co-expression vector, the plasmid map is shown in Figure 4.
  • thermostable 6-phosphate tagatose epimerase in this example is from Thermoanaerobacter indiensis.
  • the thermostable 6-phosphate tagatose epimerase-encoding gene tpe sequence (NCBI-ProteinID: B044_RS0101530) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and linked to a common plasmid.
  • the thermostable tagatose 6-phosphate epimerase-encoding gene tpe was obtained by PCR from genomic DNA using a pair of primers.
  • the linear backbone was obtained by PCR using primers 335-F: 5'-AGAAACAACAAAGGGGGAGATTTGTatgaaagtttggctggttggtgcct-3' and 324-R: 5'-agcttgagctcgactctagaggatccTTAtttcaggttgctataccattct-3'; pMA5-Pylb linear backbone using a pair of primers.
  • primers 325-F 5'-agaatggtatagcaacctgaaaTAAggatcctctagagtcgagctcaagct-3' and 326-R: 5'-aggcaccaaccagccaaactttcatACAAATCTCCCCCTTTGTTGTTTCT-3';
  • All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the PCR conditions for the gene were denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.
  • the products obtained by PCR reaction were analyzed by 0.8% agarose gel electrophoresis. After the correct size of the fragment was confirmed by the gel imaging system, the target fragment was recovered using a DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd., China) for the construction of a recombinant expression vector.
  • thermostable tagatose 6-phosphate epimerase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR.
  • the POE-PCR system is as follows: purified pMA5-Pylb linear backbone, 200ng; purified heat-resistant 6-phosphate tagatose epimerase gene fragment 131ng; 2 ⁇ PrimeSTAR MAX DNA Polymerase (Dalian Bao Bio, China) , 25 ⁇ L, add water to make up 50 ⁇ L.
  • the POE-PCR conditions were denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 3.5 min, and final extension at 72°C for 5 min.
  • the ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformants were selected for colony PCR and double-enzyme digestion identification, and 2-3 positive transformants were selected for further verification by sequencing.
  • the sequencing results showed that pMA5-Pylb- TPE recombinant co-expression vector, the plasmid map is shown in Figure 5.
  • thermostable 6-phosphate tagatose phosphatase in this example was from Archaeoglobus fulgidus.
  • the thermostable 6-phosphate tagatose phosphatase encoding gene tpp sequence (NCBI-ProteinID: AAB90791) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and connected to a common plasmid.
  • the thermostable 6-phosphate tagatose phosphatase-encoding gene tpp was obtained by PCR from genomic DNA using a pair of primers.
  • the linear backbone was obtained by PCR using primers 339-F: 5'-AGAAACAACAAAGGGGGAGATTTGTATGTTCAAGCCGAAAGCGATCGCGG-3' and 340-R: 5'-agcttgagctcgactctagaggatccTTAACGCAGCAGGCCCAGAAACTG-3'; pMA5-Pylb using a pair of primers. Primers were used; 341-F: 5'-CAGTTTCTGGGCCTGCTGCGTTAAggatcctctagagtcgagctcaagct-3' and 342-R: 5'-CCGCGATCGCTTTCGGCTTGAACATACAAATCTCCCCCTTTGTTGTTTCT-3'.
  • All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the PCR conditions for the gene were denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.
  • the products obtained by PCR reaction were analyzed by 0.8% agarose gel electrophoresis. After the correct size of the fragment was confirmed by the gel imaging system, the target fragment was recovered by DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd., China) for the construction of recombinant expression vector.
  • thermostable tagatose 6-phosphate phosphatase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR.
  • the POE-PCR system is as follows: purified pMA5-Pylb linear backbone, 200 ng; purified heat-resistant 6-phosphate tagatose phosphatase gene fragment 131 ng; 2 ⁇ PrimeSTAR MAX DNA Polymerase (Dalian Bao Bio, China), 25 ⁇ L, Add water to make up 50 ⁇ L.
  • the POE-PCR conditions were denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 3.5 min, and final extension at 72°C for 5 min.
  • the ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformants were selected for colony PCR and double-enzyme digestion identification, and 2-3 positive transformants were selected for further verification by sequencing.
  • the sequencing results showed that pMA5-Pylb- TPP recombinant co-expression vector, the plasmid map is shown in Figure 6.
  • thermostable ⁇ -glucan phosphorylase is from Thermococcus kodakarensis; thermostable glucose phosphate mutase is from Thermococcus kodakarensis; thermostable glucose phosphate isomerase is from Thermus thermophilus; thermostable 6-phosphate tagatose epidermase Isomerase was from Thermoanaerobacter indiensis; and thermostable 6-phosphate tagatose phosphatase was from Archaeoglobus fulgidus.
  • Thermostable ⁇ -glucan phosphorylase encoding gene agp (NCBI-ProteinID: BAD85595) was obtained by PCR using primers 350-F: 5′-AGAAACAACAAAGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3′ and 351-R: 5′-cgaaggtaccaaacagtttgcccatTTTGAATTCCTCCTTTtcagtcaagtcccttccacttgacc-3′;
  • Thermostable glucose phosphomutase encoding gene pgm (NCBI-ProteinID: BAD85297) was obtained by PCR using primers 352-F: 5'-ggtcaagtggaagggacttgactgaAAAGGAGGAATTCAAAatgggcaaactgtttggtaccttcg-3' and 353-R: 5'-GAAAGCGAGTATCCAGACG
  • All primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the PCR conditions for the gene were denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.
  • the products obtained by PCR reaction were analyzed by 0.8% agarose gel electrophoresis. After the correct size of the fragment was confirmed by the gel imaging system, the target fragment was recovered using a DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd., China) for the construction of a recombinant expression vector.
  • thermostable ⁇ -glucan phosphorylase gene fragment thermostable glucose phosphomutase gene fragment, thermostable 6-phosphate tagatose epimerase gene fragment, thermostable 6-phosphate Tagatose phosphatase gene fragment and pMA5-Pylb vector backbone.
  • the POE-PCR system is as follows: purified pMA5-Pylb linear backbone, 200ng; purified thermostable ⁇ -glucan phosphorylase gene fragment 131ng, thermostable glucose phosphomutase gene fragment 131ng, thermostable glucose phosphoisotrope 131ng Constructing enzyme gene fragment 131ng, thermostable 6-phosphate tagatose epimerase gene fragment 131ng, thermostable 6-phosphate tagatose phosphatase gene fragment 131ng; 2 ⁇ PrimeSTAR MAX DNA Polymerase (Dalian Bao Bio, China) , 25 ⁇ L, add water to make up 50 ⁇ L.
  • the POE-PCR conditions were denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 3.5 min, and final extension at 72°C for 5 min.
  • the ligation product was transformed into competent E.coli Top10 by the calcium chloride method, and the transformants were selected for colony PCR and double-enzyme digestion identification, and 2-3 positive transformants were selected for further verification by sequencing.
  • the sequencing results showed that pMA5-Pylb- aGP-PGM-PGI-TPE-TPP recombinant co-expression vector, the plasmid map is shown in Figure 7.
  • the constructed recombinant expression vectors pMA5-Pylb-aGP, pMA5-Pylb-PGM, pMA5-Pylb-PGI, pMA5-Pylb-TPE, pMA5-Pylb-TPP, pMA5-Pylb-aGP-PGM-PGI-TPE-TPP were respectively Transform into Bacillus subtilis engineering bacteria SCK8-ST3, culture in LB test tube overnight, extract plasmid with plasmid extraction kit, and extract the correct clones SCK8-ST3/pMA5-Pylb-aGP, SCK8-ST3/pMA5-Pylb-PGM, SCK8-ST3/pMA5-Pylb-PGI, SCK8-ST3/pMA5-Pylb-TPE, SCK8-ST3/pMA5-Pylb-TPP and SCK8-ST3/pMA5-Pylb-aGP-P
  • the culture was transferred to fresh LB medium containing spectinomycin at an inoculum of 1%, incubated overnight at 37°C with shaking, centrifuged at 5500 rpm for 10 min, and discarded the supernatant to obtain the expression of thermostable ⁇ -glucan phosphorylation.
  • thermostable glucose phosphomutase whole cells expressing thermostable glucose phosphate isomerase
  • thermostable 6-phosphate tagatose epimerase whole cells expressing thermostable glucose phosphate isomerase
  • Whole cells and co-expression of thermostable ⁇ -glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphoisomerase, thermostable 6-phosphate tagatose phosphatase in whole cells Whole cells to isomerase and thermostable 6-phosphate tagatose phosphatase.
  • thermostable ⁇ -glucan phosphorylase thermostable glucose phosphomutase
  • thermostable glucose phosphate isomerase thermostable 6-phosphate tagatose epimerase
  • Figure 8 shows the graph of the change in the yield of tagatose with the reaction time. After 46 hours of reaction in a water bath shaker at 70°C, the HPLC test results showed that the yield of tagatose reached 50 g/L, and the yield was 50 g/L. up to 50%.
  • Example 9 Whole cell mixture catalyzes the preparation of tagatose from starch
  • thermostable ⁇ -glucan phosphorylase whole cells expressing thermostable glucose phosphomutase
  • whole cells expressing thermostable glucose phosphoisomerase whole cells expressing thermostable 6-phosphate tagatose epimerase
  • thermostable 6 - Whole cells were added at a ratio of 1:1:1:1 for tagatose phosphate phosphatase.
  • the reaction was carried out at 70°C in a water bath and shaker for 46 hours, and samples were taken for HPLC analysis.
  • thermostable ⁇ -glucan phosphorylase Co-express thermostable ⁇ -glucan phosphorylase, thermostable glucose phosphomutase, thermostable glucose phosphate isomerase, thermostable 6-phosphate tagatose difference by resuspending in 1L sodium phosphate buffer (pH 7.0).
  • 1 g of montmorillonite was added, and stirred evenly.
  • 40ml of 5% (w/v) polyethyleneimine aqueous solution was added for flocculation at room temperature, and 20ml of 50% glutaraldehyde aqueous solution was added for crosslinking at room temperature for 3h. Then suction filtration to obtain a filter cake layer, the filter cake is washed with deionized water, and then extruded into particles, and dried to obtain immobilized whole cells.
  • Example 11 Immobilized whole cell mixture catalyzed the preparation of tagatose from starch
  • Permeabilized whole cells expressing thermostable ⁇ -glucan phosphorylase and permeabilized whole cells expressing thermostable glucose phosphate mutase were resuspended in 1L sodium phosphate buffer (pH 7.0) at a ratio of 1:1:1:1:1.

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Abstract

提供了一种产塔格糖的枯草芽孢杆菌基因工程菌及制备塔格糖的方法，所述基因工程菌包括构建单独表达或共表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶。利用所述基因工程菌可有效将淀粉转化为塔格糖。与现有生产塔格糖的方法相比，该方法可适用于全细胞循环使用，具有安全性能高、产率高、生产工艺简单、成本低且易于规模化制备等优点。

Description

产塔格糖的枯草芽孢杆菌基因工程菌及制备塔格糖的方法 技术领域
本发明涉及生物工程技术领域，尤其涉及产塔格糖的基因工程菌及其制备塔格糖的方法。
背景技术
塔格糖是天然存在的一种稀有单糖，是半乳糖的酮糖形式，果糖的差向异构体。塔格糖的甜味特性与蔗糖相似，热量只有蔗糖的三分之一，被称为低热量甜味剂。天然的塔格糖主要存在于酸乳、奶粉等乳制品中。塔格糖提供非常新鲜和纯净的甜度，其味道的特点与果糖相似。研究表明塔格糖具有低热量、低升糖指数、抗龋齿、抗氧化、益生元、改善肠道功能、免疫调节、药物前体等重要生理功能特性，可以广泛应用于食品、饮料、医药、保健等领域，具有巨大的经济价值(Oh D-K:Tagatose:properties,applications,and biotechnological processes.App.Microbiol.Biotechnol.2007,76:1-8)。
2001年，美国食品及药物管理局(FDA)对塔格糖安全性进行了确认，批准其为GRAS(Generally Regarded As Safe)产品。FDA在2002年12月批准塔格糖作为牙齿友好的成分，以及2003年10月批准其作为食品添加剂，可作为甜味剂用于食品饮料业以及医药领域。2001年，联合国粮农组织和世界卫生组织联合食品添加剂委员会(JECFA)推荐塔格糖为一种新的低热量甜味剂，可以作为食品添加剂使用。2004年第63次会议中声明不需要限制塔格糖的允许日摄取量(ADI)，并将“未指定的”ADI分配在JECFA可安排食品组分的最安全的目录中。韩国，澳洲(澳大利亚和新西兰)和欧盟分别于2003年，2004年和2005年批准塔格糖在本地区上市，其中2005年12月，塔格糖在欧盟被正式批准为无任何使用限制的新型食品组分。我国也于2014年5月批准塔格糖为新食品原料。目前，塔格糖已获全球30多个国家以及WHO/FAO和国际食品法典委员会的核准，并对其每天允许摄入量和使用用途不作限制。
塔格糖的生产方法主要有化学合成法和生物转化法。一般都以半乳糖为原料，通过化学方法或生物转化进行异构化反应而成。化学合成法是利用可溶性碱金属盐或碱土金属盐为催化剂，促使半乳糖在碱性条件下生成塔格糖，并形成金属氢氧化物-塔格糖复合物，再用酸中和获得塔格糖。化学法能耗高，产物复杂，纯化困难，副反应多，产生化学污染。生物法是利用半乳糖醇或半乳糖，在酶或者微生物的催化作用下将相应的底物转化为塔格糖。半乳 糖醇价格较高且来源受限，不适宜用作工业化生产原料。目前塔格糖主流生产方法是经过半乳糖异构化、脱盐、脱色、分离、浓缩、结晶等步骤制成塔格糖纯品。然而，这种方法也存在缺陷，半乳糖不能完全转化为塔格糖，最终产物是半乳糖和塔格糖的混合物，导致塔格糖分离工艺复杂，转化率低，分离成本高昂，同时原料半乳糖的价格并不低廉，最终塔格糖的生产成本高昂。(Rhimi M,Aghajari N,Juy M,Chouayekh H,Maguin E,Haser R,Bejar S:Rational design of Bacillus stearothermophilus US100l-arabinose isomerase:Potential applications for d-tagatose production.Biochim.2009,91:650-653.Oh H-J,Kim H-J,Oh D-K:Increase in d-tagatose production rate by site-directed mutagenesis of l-arabinose isomerase from Geobacillus thermodenitrificans.Biotechnol.Lett.2006,28:145-149.Bosshart A,Hee CS,Bechtold M,Schirmer T,Panke S:Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.ChemBioChem 2015,16:592-601.)
韩国CJ公司发明多酶催化果糖转化为塔格糖技术(Oh DK,HONG SH,Lee SH:Aldolase,aldolase mutants and tagatose using the same production methods and compositions for production.WO 2015016544A1)，包括果糖激酶、醛缩酶、植酸酶，但是从果糖生产6-磷酸果糖需要ATP对果糖进行底物磷酸化，添加昂贵的ATP导致塔格糖生产成本高，不具备工业化生产价值(TW107111500，US20160186162A1)。韩国CJ公司挖掘和改造己糖醛酸酯4-差向异构酶将果糖转化为塔格糖(CN105431541B，CN109415715A)，经过改造的己糖醛酸酯4-差向异构酶酶活性极低，不具备工业化应用价值。韩国CJ公司还挖掘新型来源的6-磷酸塔格糖磷酸酶并将其应用到淀粉、麦芽糊精和蔗糖等底物转化生产塔格糖(WO2018004310A1，CN 109790524A)，但是该6-磷酸塔格糖磷酸酶酶活性极低，现阶段不具备工业化应用价值。
中国科学院天津工业生物技术研究所以廉价玉米淀粉、麦芽糊精、蔗糖等为原料，建立了塔格糖体外多酶合成新路线，从根本上改变了现有塔格糖的生产工艺(CN106399427A)。在此基础上，中国科学院天津工业生物技术研究所以廉价玉米淀粉、纤维素、麦芽糊精、蔗糖等为原料，利用全细胞催化制备塔格糖的方法(CN107988286A)，该工艺减少了多酶纯化工艺步骤，降低了生产成本低和环境污染，提高了塔格糖产率。但是该方法中关键酶的发酵生产宿主是大肠杆菌BL21(DE3)，大肠杆菌不适合作为食品制剂生产相关酶的生产菌株进行工业化生产，除此之外体外酶分离纯化步骤繁琐，酶回收利用率低，循环使用困难，无法进一步降低生产成本，无法实现工业化高浓度产物大批量生产应用。
因此，亟待开发一种全细胞循环使用、安全性能高、产率高、生产工艺简单、成本低且且适用于高底物浓度投放和高产物塔格糖产出，易于规模化制备塔格糖的新方法。
发明内容
针对现有的多酶催化制备塔格糖的方法所存在的问题如大肠杆菌不利于食品制剂的工业 化生产，纯化步骤繁琐，酶回收利用利用率低和循环使用困难，以及低浓度底物淀粉的投料问题，本发明的主要目的是提供一种利用枯草芽孢杆菌全细胞催化高浓度淀粉制备生产高浓度塔格糖的方法。
本发明首先提供一种产塔格糖的枯草芽孢杆菌基因工程菌，其是共表达α-葡聚糖磷酸化酶基因、葡萄糖磷酸变位酶基因、葡萄糖磷酸异构酶基因、6-磷酸塔格糖差向异构酶基因和6-磷酸塔格糖磷酸酶基因的枯草芽孢杆菌基因工程菌、或分别表达α-葡聚糖磷酸化酶基因、葡萄糖磷酸变位酶基因、葡萄糖磷酸异构酶基因、6-磷酸塔格糖差向异构酶基因和6-磷酸塔格糖磷酸酶基因的枯草芽孢杆菌基因工程菌的混合物。
上述技术方案的原理在于充分利用相关催化途径而活体细胞水平表达并实现有效催化反应，其包括由α-葡聚糖磷酸化酶在无机磷存在下将底物淀粉转化为中间体葡萄糖-1-磷酸(G1P)；由葡萄糖磷酸变位酶将中间体葡萄糖-1-磷酸(G1P)变位为另一中间体葡萄糖-6-磷酸(G6P)；由葡萄糖磷酸异构酶将中间体葡萄糖-6-磷酸(G6P)变位为另一中间体果糖-6-磷酸(F6P)；由6-磷酸塔格糖差向异构酶将中间体果糖-6-磷酸(F6P)异构化为另一中间体塔格糖-6-磷酸(T6P)；由6-磷酸塔格糖磷酸酶将中间体塔格糖-6-磷酸(T6P)脱掉磷酸基团为产物塔格糖(Tagatose)。
本领域已知的各种枯草芽孢杆菌菌株均可用作本发明的出发菌株，如Bacillus subtilis 168、DB104、WB800、WB600、SCK6、1A751、ATCC6051a、ATCC6051等。优选地，所述枯草芽孢杆菌出发菌株为敲除蛋白酶的枯草芽孢杆菌菌株，例如WB800、WB600、SCK6、1A751等。更优选地，所述枯草芽孢杆菌出发菌株为SCK6。
在具体实施方式中，所述枯草芽孢杆菌基因工程菌中包括共表达α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶的表达载体，或者为包含α-葡聚糖磷酸化酶的表达载体的基因工程菌、葡萄糖磷酸变位酶的表达载体的基因工程菌、葡萄糖磷酸异构酶的表达载体的基因工程菌、6-磷酸塔格糖差向异构酶的表达载体的基因工程菌和6-磷酸塔格糖磷酸酶的表达载体的基因工程菌的混合物。
优选地，所述的各种酶是耐热型的，即耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶。相对于不耐热的常温酶来说，采用耐热的酶在菌株灭活方面具有优势，即后者在发酵结束后可以通过加热处理的方式将菌株灭活，而同时塔格糖合成相关的酶仍保持活性，从而能够将灭活的菌株混合用来生产塔格糖，更适于工业应用。如果使用常温的相关酶，那么在发酵生产结束后，则需要通过破碎菌体和酶纯化步骤获取纯酶，才能够获得相关酶用来塔格糖的生产。
具体地，所述耐热α-葡聚糖磷酸化酶指在40℃以上、45℃以上、50℃以上、55℃以上、 60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有将淀粉磷酸化为葡萄糖-1-磷酸(G1P)功能的酶。进一步优选地，所述耐热α-葡聚糖磷酸化酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热α-葡聚糖磷酸化酶的氨基酸序列与来源于所述嗜热微生物的耐热α-葡聚糖磷酸化酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐热α-葡聚糖磷酸化酶来源于Thermococcus kodakarensis。
具体地，耐热葡萄糖磷酸变位酶指在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有将葡萄糖-1-磷酸(G1P)变位为葡萄糖-6-磷酸(G6P)功能的酶。进一步优选地，所述耐热葡萄糖磷酸变位酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热葡萄糖磷酸变位酶的氨基酸序列与来源于所述嗜热微生物的耐热葡萄糖磷酸变位酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐热葡萄糖磷酸变位酶来源于Thermococcus kodakarensis。
具体地，耐热葡萄糖磷酸异构酶指在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有将葡萄糖-6-磷酸(G6P)变位为果糖-6-磷酸(F6P)功能的酶。进一步优选地，所述耐热葡萄糖磷酸异构酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热葡萄糖磷酸异构酶的氨基酸序列与来源于所述嗜热微生物的耐热葡萄糖磷酸异构酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐热葡萄糖磷酸异构酶 来源于Thermus thermophilus。
具体地，耐热6-磷酸塔格糖差向异构酶在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有果糖-6-磷酸(F6P)异构为塔格糖-6-磷酸(T6P)功能的酶。进一步优选地，所述耐热6-磷酸塔格糖差向异构酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述耐热6-磷酸塔格糖差向异构酶的氨基酸序列与来源于所述嗜热微生物的耐热6-磷酸塔格糖差向异构酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述耐6-磷酸塔格糖差向异构酶来源于Thermoanaerobacter indiensis。
具体地，所述6-磷酸塔格糖磷酸酶指在40℃以上、45℃以上、50℃以上、55℃以上、60℃以上、65℃以上、70℃以上、75℃以上、或80℃以上具有塔格糖-6-磷酸(T6P)脱掉磷酸基团为产物塔格糖(Tagatose)功能的酶。进一步优选地，所述6-磷酸塔格糖磷酸酶来源于嗜热微生物，例如Geobacillus kaustophilus、Geobacillus stearothermophilus、Thermotoga maritima、Pseudothermotoga thermarum、Thermococcus kodakarensis、Archaeoglobus fulgidus、Thermoanaerobacter indiensis、Dictyoglomus thermophilum、Caldicellulosiruptor kronotskyensis、Clostridium thermocellum、Caldilinea aerophila、Pyrococcus furiosus、Thermus thermophilus、Methanothermobacter marburgensis、Archaeoglobus profundus等；或所述6-磷酸塔格糖磷酸酶的氨基酸序列与来源于所述嗜热微生物的6-磷酸塔格糖磷酸酶具有至少70％，优选至少80％，更优选至少90％，最优选至少95％的同一性。更优选地，所述6-磷酸塔格糖磷酸酶来源于Archaeoglobus fulgidus。
本发明相应地提供一种用于上述基因工程菌的表达载体，其包含α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶基因，并能够实现这些基因的共表达。
本领域技术人员可以理解，本发明所涉及的载体和基因工程菌可以通过本领域已知的常规方法制备，例如，通过重组DNA技术构建，获取α-葡聚糖磷酸化酶基因、葡萄糖磷酸变位酶基因、葡萄糖磷酸异构酶基因、6-磷酸塔格糖差向异构酶基因、6-磷酸塔格糖磷酸酶基因，构建重组表达载体，再通过已知的方法转入枯草芽孢杆菌获得基因工程菌。
更优选地，所述载体包括启动子、耐热α-葡聚糖磷酸化酶基因、耐热葡萄糖磷酸变位 酶基因、耐热葡萄糖磷酸异构酶基因、耐热6-磷酸塔格糖差向异构酶基因、耐热6-磷酸塔格糖磷酸酶基因和终止子。而分别各所述表达耐热α-葡聚糖磷酸化酶的载体包括启动子、耐热α-葡聚糖磷酸化酶基因、终止子；所述表达耐热葡萄糖磷酸变位酶的载体包括启动子、耐热葡萄糖磷酸变位酶基因、终止子；所述表达耐热6-磷酸塔格糖差向异构酶的载体包括启动子、耐热6-磷酸塔格糖差向异构酶基因、终止子；所述表达耐热6-磷酸塔格糖差向异构酶的载体包括启动子、耐热6-磷酸塔格糖差向异构酶基因、终止子；所述表达耐热6-磷酸塔格糖磷酸酶的载体包括启动子、耐热6-磷酸塔格糖磷酸酶基因、终止子。
本领域技术人员可以理解，本领域已知的各种启动子均可用作本发明的启动子包括但不限于P43启动子、Pylb启动子、PamyL启动子、Plaps启动子、PhpaII启动子、PamyE启动子、Pgrac启动子、PsacB启动子、PsigX启动子、PaprE启动子、PgroES启动子等。优选地，本发明的启动子选择PhpaII启动子和Pylb启动子串联。本领域已知的各种终止子也可用作本发明中的终止子。
在优选的实施方式中，所述基因工程菌中的内源性的尿嘧啶磷酸核糖基转移酶基因，和/或α-淀粉酶基因、和/或孢子形成RNA聚合酶σ F因子基因，和/或表面活性肽合成酶亚基3基因被失活或敲除。最优选地，所述基因工程菌中内源性的尿嘧啶磷酸核糖基转移酶基因，α-淀粉酶基因，孢子形成RNA聚合酶σ F因子基因，和表面活性肽合成酶亚基3基因均被失活或敲除。其中失活或敲除上述基因能够进一步提高基因工程菌生产塔格糖的效率，具体理由是，失活或敲除尿嘧啶磷酸核糖基转移酶基因可以构建无痕遗传操作系统，后续的遗传操作(即敲除基因)可以做到无痕操作，不引入抗性基因等外源基因；失活或敲除α-淀粉酶基因可以切除菌株利用外源淀粉的途径，防止生产塔格糖的底物淀粉被菌株作为碳源代谢利用；失活或敲除孢子形成RNA聚合酶σ F因子基因，可以为菌种代谢流向塔格糖合成相关的异源蛋白合成表达方向，同时也可以使菌株发酵控制不形成芽孢；失活或敲除表面活性肽合成酶亚基3基因可以在发酵生产菌体的时候更容易控制，不产生太多的泡沫。因此，失活或敲除上述4个基因能够更有利于基因工程菌产生塔格糖，因此是优选的实施方式。
本领域已知的方法来实现内源性基因的失活或敲除，优选采用基因编辑的方法实现。
本发明进一步提供利用上述基因工程菌全细胞催化淀粉制备生产塔格糖的方法，其包括下述步骤：
(1)将所述枯草芽孢杆菌工程菌进行发酵获得全细胞；
(2)将步骤(1)获得的枯草芽孢杆菌全细胞进行细胞膜通透性处理，获得透性全细胞；
(3)利用步骤(2)获得的透性全细胞催化淀粉制备塔格糖，其中对于共表达的枯草芽孢杆菌工程菌直接用于催化，对于分别表达各种酶的枯草芽孢杆菌工程菌则进行混合用于催化。
优选地，还包括将步骤(2)中获得的枯草芽孢杆菌透性全细胞进行固定化处理，获得固定化全细胞，或者固定化全细胞混合物，然后再用于催化。
在具体实施方式中，步骤(1)所述全细胞的制备使用本领域已知的方法进行。发酵可使用任何适合外源蛋白表达的培养基，包括但不限于LB培养基、SR培养基、TB培养基等。
在优选实施方式中地，步骤(2)中所述细胞膜通透性处理可以采用已知方法，包括但不限于热处理、添加有机溶剂和/或添加表面活性剂等。其中，有机溶剂包括但不限于丙酮、乙腈等。表面活性剂包括但不限于十六烷基三甲基溴化铵(CTAB)、Tween-80等。优选地，细胞膜通透性处理为热处理。对细胞膜进行通透性处理的目的是为了使细胞外的淀粉能够通过细胞膜进入到细胞内。
其中优选地，所述热处理温度为45-100℃；更优选地，热处理温度为70-80℃。优选地，热处理时间为10-100min；更优选地，热处理时间为50-70min。优选地，热处理时的细胞浓度为OD 600＝10-300；更优选地，细胞浓度为OD 600＝30-150。同时，所述热处理可在无缓冲液体系或缓冲液体系中进行；优选地，所述热处理在缓冲液体系中进行，所述缓冲液可为HEPES缓冲液、磷酸盐缓冲液、Tris缓冲液、醋酸盐缓冲液等。其中，磷酸盐缓冲液例如磷酸钠缓冲液、磷酸钾缓冲液等。
在具体实施方式中，催化的反应体系中，底物淀粉的浓度为50-300g/L；更优选地，底物淀粉的浓度为100-200g/L。优选地，其反应条件为：在pH 5.0-8.0、40-80℃下反应0.5-96h；更优选地在pH 6.5-7.5、45-75℃下反应12-60h；最优选地在pH 7.5、60-70℃下反应12-96h。所述的催化可在无缓冲液体系或缓冲液体系中进行；优选地，是在缓冲液体系中进行，所述缓冲液可为HEPES缓冲液、磷酸盐缓冲液、Tris缓冲液、醋酸盐缓冲液等。其中，磷酸盐缓冲液，例如磷酸钠缓冲液、磷酸钾缓冲液等。
优选地，对于分别表达各种酶的枯草芽孢杆菌工程菌透性全细胞，其混合物是将表达耐热α-葡聚糖磷酸化酶的透性全细胞、表达耐热葡萄糖磷酸变位酶的透性全细胞、表达耐热葡萄糖磷酸异构酶的透性全细胞、表达耐热6-磷酸塔格糖差向异构酶的透性全细胞、表达耐热6-磷酸塔格糖磷酸酶的透性全细胞的比例为(0.1-10)：(0.1-10)：(0.1-10)：(0.1-10)：(0.1-10)；更优选地为(0.5-5)：(0.5-5)：(0.5-5)：(0.5-5)：(0.5-5)，最优选为1:1:1:1:1。
在具体实施方式中，所述透性全细胞固定化处理方法为：用磷酸钠或磷酸钾缓冲液重悬共表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶的透性全细胞，加入适量无机土，搅拌均匀。加入聚乙烯亚胺水溶液于室温条件下絮凝，在加入交联剂进行交联。然后抽滤得到滤饼层，滤饼用去离子水洗涤后挤压制备成颗粒，干燥后得到固定化全细胞。
而对于所述固定化透性全细胞的混合物的处理方法为：分别用磷酸钠或磷酸钾缓冲液重悬表达耐热α--葡聚糖磷酸化酶的透性全细胞、表达耐热葡萄糖磷酸变位酶的透性全细胞、表达耐热葡萄糖磷酸异构酶的透性全细胞、表达耐热6-磷酸塔格糖差向异构酶的透性全细胞、表达耐热6-磷酸塔格糖磷酸酶的透性全细胞，加入适量无机土，搅拌均匀。加入聚乙烯亚胺水溶液于室温条件下絮凝，在加入交联剂进行交联。然后抽滤得到滤饼层，滤饼用去离子水洗涤后挤压制备成颗粒，干燥后得到固定化全细胞。
其中，所述无机土包括但并不限于蒙脱土、硅藻土、高岭土和膨润土等，优选地，所述无机土为硅藻土；所述交联剂包括但不限于戊二醛、三羟甲基磷、N,N-亚甲基双丙烯酰胺、环氧氯丙烷、京尼平等，优选地，所述交联剂为戊二醛。
与现有技术相比，本发明具有如下有益效果：
(1)本发明首次利用表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶的全细胞催化淀粉生产塔格糖，开发了一种简单且易于规模化制备塔格糖的新方法。
(2)枯草芽孢杆菌是一般认为安全(Generally Recognized As Safe,GRAS)的食品级微生物，不产生内毒素。更进一步地是，敲除α-淀粉酶编码基因，有利于后续底物淀粉的催化应用；敲除孢子形成RNA聚合酶σ F因子的编码基因，有利于后续发酵生产基因工程菌株在底物转化生产塔格糖的应用；敲除表面活性肽合成酶亚基3的编码基因，有利于后续发酵生产基因工程菌株在底物转化生产塔格糖的应用；
(3)在优选实施方式中，采用耐热的各种酶，则塔格糖的制备可以在较高温度下进行，从而可以增加底物淀粉的溶解度，相对于现有技术，本发明可在较高底物浓度下实现塔格糖的制备，有利于提高生产效率，进一步降低生产成本。
(4)本发明的方法中塔格糖的转化反应可在无缓冲液体系或缓冲液体系中进行，不需要使用含有碳源、氮源、无机盐及抗生素的培养基，一方面有利于降低生产成本，另一方面有利于产物塔格糖的分离纯化。
附图说明
图1为本发明的全细胞催化淀粉生产塔格糖的示意图。
图2为重组表达载体pMA5-Pylb-aGP的图谱。
图3为重组表达载体pMA5-Pylb-PGM的图谱。
图4重组表达载体pMA5-Pylb-PGI的图谱。
图5为重组表达载体pMA5-Pylb-TPE的图谱。
图6为重组表达载体pMA5-Pylb-TPP的图谱。
图7为重组表达载体pMA5-Pylb-aGP-PGM-PGI-TPE-TPP的图谱。
图8为塔格糖的产量随反应时间变化过程曲线图。
图9为固定化共表达工程菌株生产塔格糖产量趋势图。
图10为分表达工程菌株固定化生产塔格糖产量趋势图。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下通过具体实施例来进一步说明本发明的技术方案。但是应理解所述实施例仅是范例性的而且是优选的实施例，但不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
实施例1：枯草芽孢杆菌重组菌株SCK8的构建
(1)构建重组整合载体pSS-upp-FR
根据KEGG数据库中来源于枯草芽孢杆菌Bacillus subtilis 168的尿嘧啶磷酸核糖基转移酶编码基因upp基因序列(NCBI-ProteinID:NP_391570)，设计引物，并通过PCR扩增获得upp基因上游500bp同源片段和下游500bp的同源片段，采用simple cloning连接方式(You,C.,Zhang,X.Z.,&Zhang,Y.H.(2012).Simple cloning via direct transformation of PCR product(DNA Multimer)to Escherichia coli and Bacillus subtilis.Appl.Environ.Microbiol.,78(5),1593-1595.doi:10.1128/AEM.07105-11)构建至整合载体pSS中，得到重组整合载体pSS-upp-FR。
(2)构建枯草芽孢杆菌重组菌株SCK8
制备枯草芽孢杆菌菌株SCK6超级感受态细胞(200μl)(Zhang,X.Z.,&Zhang,Y.H.P.(2011).Simple,fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis.Microb.Biotechnol.,4(1),98-105.doi:10.1111/j.1751-7915.2010.00230.x)，将重组整合载体pSS-upp-FR(1μg)与枯草芽孢杆菌菌株SCK6超级感受态细胞(200μl)混合均匀，随后放入37℃摇床中复苏90min，将菌液涂布于含有氯霉素(5μg/mL)的固体LB培养基(酵母抽提物5g/L，蛋白胨10g/L，氯化钠10g/L)中，放置于37℃培养箱中培养14-16h。
挑取在氯霉素抗性平板上生长的阳性单交换转化子菌落进行菌落PCR验证，经PCR扩增获得1000bp DNA片段和2000bp DNA片段两条条带(其中1000bp DNA片段大小是载体pSS-upp-FR中载体中的upp编码基因上下游同源臂的片段大小，2000bp DNA片段大小是基因组上包括upp编码基因上游同源臂、upp编码基因和upp编码基因下游同源臂的片段大小)的为阳性克隆。
挑取阳性克隆转接到不添加抗生素的LB培养基中培养8-12h，然后取200μl菌液离心 去除上清，再用无菌水重悬涂布于5-氟尿嘧啶(5-FU)基本盐培养基固体平板(40％葡萄糖20.0ml/L,4％谷氨酰胺50.0mL/L,0.5％色氨酸10.0mL/L,1％维他命B1 1.0mL/L,20mM 5-氟尿嘧啶500μL/L,10×基本盐100.0mL/L,1000×微量元素1.0mL/L)中，放置37℃培养箱中培养24h。此步骤的目的是在于通过不添加抗生素的LB培养基培养，促使阳性转化子发生分子内同源重组，再通过5-FU基本盐培养基筛选培养获得upp敲除的目的转化子。
从5-FU基本盐培养基固体平板上挑取若干菌落，再次进行菌落PCR验证，经PCR扩增获得仅有1000bp的DNA片段的转化子为阳性克隆。将转化子PCR送测序验证正确并保存正确的菌株，即upp基因敲除的枯草芽孢杆菌重组工程菌株，即无尿嘧啶磷酸核糖基转移酶酶活性的枯草芽孢杆菌重组工程菌株，命名为SCK8。
实施例2 枯草芽孢杆菌重组菌株SCK8-ST1的构建
(1)构建重组整合载体pSS-amyE-FR
根据KEGG数据库中来源于枯草芽孢杆菌Bacillus subtilis 168的α-淀粉酶的编码基因amyG基因序列(NCBI-ProteinID:NP_388186)，设计引物，并通过PCR扩增获得amyG基因上游500bp同源片段和下游500bp的同源片段，采用simple cloning连接方式(You,C.,Zhang,X.Z.,&Zhang,Y.H.(2012).Simple cloning via direct transformation of PCR product(DNA Multimer)to Escherichia coli and Bacillus subtilis.Appl.Environ.Microbiol.,78(5),1593-1595.doi:10.1128/AEM.07105-11)构建至整合载体pSS中，得到重组整合载体pSS-amyE-FR。
(2)构建枯草芽孢杆菌重组菌株SCK8-ST1
制备枯草芽孢杆菌菌株SCK8超级感受态细胞(200μl)，将重组整合载体pSS-amyE-FR(1μg)与枯草芽孢杆菌菌株SCK8超级感受态细胞(200μl)混合均匀，随后放入37℃摇床中复苏90min，将菌液涂布于含有氯霉素(5μg/mL)的固体LB培养基(酵母抽提物5g/L，蛋白胨10g/L，氯化钠10g/L)中，放置于37℃培养箱中培养14-16h。
挑取在氯霉素抗性平板上生长的阳性单交换转化子菌落进行菌落PCR验证，经PCR扩增获得1000bp DNA片段和2000bp DNA片段两条条带(其中1000bp DNA片段大小是载体pSS-amyE-FR中载体中的amyE编码基因上下游同源臂的片段大小，2000bp DNA片段大小是基因组上包括amyE编码基因上游同源臂、amyE编码基因和amyE编码基因下游同源臂的片段大小)的为阳性克隆。
挑取阳性克隆转接到不添加抗生素的LB培养基中培养8-12h，然后取200μl菌液离心去除上清，再用无菌水重悬涂布于5-FU基本盐培养基固体平板(40％葡萄糖20.0ml/L,4％谷氨酰胺50.0mL/L,0.5％色氨酸10.0mL/L,1％维他命B1 1.0mL/L,20mM 5-氟尿嘧啶 500μL/L,10×基本盐100.0mL/L,1000×微量元素1.0mL/L)中，放置37℃培养箱中培养24h。此步骤的目的是在于通过不添加抗生素的LB培养基培养，促使阳性转化子发生分子内同源重组，再通过5-FU基本盐培养基筛选培养获得amyE敲除的目的转化子。
从5-FU基本盐培养基固体平板上挑取若干菌落，再次进行菌落PCR验证，经PCR扩增获得仅有1000bp的DNA片段的转化子为阳性克隆。将转化子PCR送测序验证正确并保存正确的菌株，即amyG基因敲除的枯草芽孢杆菌重组工程菌株，即无α-淀粉酶酶活性的枯草芽孢杆菌重组工程菌株，命名为SCK8-ST1。
实施例3 枯草芽孢杆菌重组菌株SCK8-ST2的构建
(1)构建重组整合载体pSS-spoIIAC-FR
根据KEGG数据库中来源于枯草芽孢杆菌Bacillus subtilis 168的孢子形成RNA聚合酶σ F因子的编码基因spoIIAC基因序列(NCBI-ProteinID:NP_390226)，设计引物，并通过PCR扩增获得孢子形成spoIIAC基因上游500bp同源片段和下游500bp的同源片段，采用simple cloning连接方式(You,C.,Zhang,X.Z.,&Zhang,Y.H.(2012).Simple cloning via direct transformation of PCR product(DNA Multimer)to Escherichia coli and Bacillus subtilis.Appl.Environ.Microbiol.,78(5),1593-1595.doi:10.1128/AEM.07105-11)构建至整合载体pSS中，得到重组整合载体pSS-spoIIAC-FR。
(2)构建枯草芽孢杆菌重组菌株SCK8-ST2
制备枯草芽孢杆菌菌株SCK8-ST1超级感受态细胞(200μl)，将重组整合载体pSS-spoIIAC-FR(1μg)与枯草芽孢杆菌菌株SCK8-ST1超级感受态细胞(200μl)混合均匀，随后放入37℃摇床中复苏90min，将菌液涂布于含有氯霉素(5μg/mL)的固体LB培养基(酵母抽提物5g/L，蛋白胨10g/L，氯化钠10g/L)中，放置于37℃培养箱中培养14-16h。
挑取在氯霉素抗性平板上生长的阳性单交换转化子菌落进行菌落PCR验证，经PCR扩增获得1000bp DNA片段和2000bp DNA片段两条条带(其中1000bp DNA片段大小是载体pSS-spoIIAC-FR中载体中的spoIIAC编码基因上下游同源臂的片段大小，2000bp DNA片段大小是基因组上包括spoIIAC编码基因上游同源臂、spoIIAC编码基因和spoIIAC编码基因下游同源臂的片段大小)的为阳性克隆。
挑取阳性克隆转接到不添加抗生素的LB培养基中培养8-12h，然后取200μl菌液离心去除上清，再用无菌水重悬涂布于5-FU基本盐培养基固体平板(40％葡萄糖20.0ml/L,4％谷氨酰胺50.0mL/L,0.5％色氨酸10.0mL/L,1％维他命B1 1.0mL/L,20mM 5-氟尿嘧啶500μL/L,10×基本盐100.0mL/L,1000×微量元素1.0mL/L)中，放置37℃培养箱中培养24h。此步骤的目的是在于通过不添加抗生素的LB培养基培养，促使阳性转化子发生分子 内同源重组，再通过5-FU基本盐培养基筛选培养获得spoIIAC敲除的目的转化子。
从5-FU基本盐培养基固体平板上挑取若干菌落，再次进行菌落PCR验证，经PCR扩增获得仅有1000bp的DNA片段的转化子为阳性克隆。将转化子PCR送测序验证正确并保存正确的菌株，即孢子形成spoIIAC基因敲除的枯草芽孢杆菌重组工程菌株，即无孢子形成RNA聚合酶σ F因子活性的枯草芽孢杆菌重组工程菌株，命名为SCK8-ST2。
实施例4 枯草芽孢杆菌重组菌株SCK8-ST3的构建
(1)构建重组整合载体pSS-srfAC-FR
根据KEGG数据库中来源于枯草芽孢杆菌Bacillus subtilis 168的表面活性肽合成酶亚基3编码基因srfAC基因序列(NCBI-ProteinID:NP_388233)，设计引物，并通过PCR扩增获得srfAC基因上游500bp同源片段和下游500bp的同源片段，采用simple cloning连接方式(You,C.,Zhang,X.Z.,&Zhang,Y.H.(2012).Simple cloning via direct transformation of PCR product(DNA Multimer)to Escherichia coli and Bacillus subtilis.Appl.Environ.Microbiol.,78(5),1593-1595.doi:10.1128/AEM.07105-11)构建至整合载体pSS中，得到重组整合载体pSS-srfAC-FR。
(2)构建枯草芽孢杆菌重组菌株SCK8-ST3
制备枯草芽孢杆菌菌株SCK8-ST2超级感受态细胞(200μl)，将重组整合载体pSS-srfAC-FR(1μg)与枯草芽孢杆菌菌株SCK8-ST2超级感受态细胞(200μl)混合均匀，随后放入37℃摇床中复苏90min，将菌液涂布于含有氯霉素(5μg/mL)的固体LB培养基(酵母抽提物5g/L，蛋白胨10g/L，氯化钠10g/L)中，放置于37℃培养箱中培养14-16h。
挑取在氯霉素抗性平板上生长的阳性单交换转化子菌落进行菌落PCR验证，经PCR扩增获得1000bp DNA片段和2000bp DNA片段两条条带(其中1000bp DNA片段大小是载体pSS-srfAC-FR中载体中的srfAC编码基因上下游同源臂的片段大小，2000bp DNA片段大小是基因组上包括srfAC编码基因上游同源臂、srfAC编码基因和srfAC编码基因下游同源臂的片段大小)的为阳性克隆。
挑取阳性克隆转接到不添加抗生素的LB培养基中培养8-12h，然后取200μl菌液离心去除上清，再用无菌水重悬涂布于5-FU基本盐培养基固体平板(40％葡萄糖20.0ml/L,4％谷氨酰胺50.0mL/L,0.5％色氨酸10.0mL/L,1％维他命B1 1.0mL/L,20mM 5-氟尿嘧啶500μL/L,10×基本盐100.0mL/L,1000×微量元素1.0mL/L)中，放置37℃培养箱中培养24h。此步骤的目的是在于通过不添加抗生素的LB培养基培养，促使阳性转化子发生分子内同源重组，再通过5-FU基本盐培养基筛选培养获得srfAC敲除的目的转化子。
从5-FU基本盐培养基固体平板上挑取若干菌落，再次进行菌落PCR验证，经PCR扩增 获得仅有1000bp的DNA片段的转化子为阳性克隆。将转化子PCR送测序验证正确并保存正确的菌株，即srfAC基因敲除的枯草芽孢杆菌重组工程菌株，即无表面活性肽合成酶亚基3酶活性的枯草芽孢杆菌重组工程菌株，命名为SCK8-ST3。
实施例5：重组载体的构建
(1)pMA5-Pylb-aGP的构建
本实施例中耐热α-葡聚糖磷酸化酶来自Thermococcus kodakarensis。耐热α-葡聚糖磷酸化酶编码基因agp序列(NCBI-ProteinID:BAD85595)通过苏州金唯智生物科技有限公司合成并连接到普通质粒上。耐热α-葡聚糖磷酸化酶编码基因agp基因使用一对引物通过PCR获取。使用引物299-F：5′-AGAAACAACAAAGGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3′和300-R：5′-gcttgagctcgactctagaggatcctcagtcaagtcccttccacttgacca-3′；pMA5-Pylb线性骨架使用一对引物通过PCR获取。使用引物301-F：5′-tggtcaagtggaagggacttgactgaggatcctctagagtcgagctcaagc-3′和302-R：5′-caacggcattggaaacgttcaccatACAAATCTCCCCCTTTGTTGTTTCT-3′；
所有引物均由苏州金唯智生物科技有限公司合成。基因的PCR条件为94℃变性5min，按如下参数循环30次：94℃变性15s，58℃退火15s，72℃延伸1min，最后72℃延伸10min。PCR反应所得到的产物分别用0.8％的琼脂糖凝胶电泳分析结果。经凝胶成像系统成像确认片段大小正确后，采用DNA纯化回收试剂盒(天根生化科技有限公司，中国)回收目的片段用于重组表达载体的构建。
然后使用POE-PCR组装耐热α-葡聚糖磷酸化酶基因片段和pMA5-Pylb载体骨架。POE-PCR体系如下：纯化后的pMA5-Pylb线性骨架，200ng；纯化后的耐热α-葡聚糖磷酸化酶基因片段131ng；2×PrimeSTAR MAX DNA Polymerase(大连宝生物，中国)，25μL，加水补足50μL。POE-PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸3.5min，最后72℃延伸5min。连接产物用氯化钙法转化入感受态E.coli Top10，挑选转化子进行菌落PCR及双酶切鉴定，选择2-3个阳性转化子送测序进一步验证，测序结果显示成功获得pMA5-Pylb-aGP重组共表达载体，质粒图谱如图2所示。
(2)pMA5-Pylb-PGM的构建
本实施例中耐热葡萄糖磷酸变位酶来自Thermococcus kodakarensis。耐热葡萄糖磷酸变位酶编码基因pgm序列(NCBI-ProteinID:BAD85297)通过苏州金唯智生物科技有限公司合成并连接到普通质粒上。耐热葡萄糖磷酸变位酶编码基因pgm使用一对引物从基因组DNA中通过PCR获取。使用引物327-F：5′-AGAAACAACAAAGGGGGAGATTTGTatgggcaaactgtttggtaccttcg-3′和328-R：5′- agcttgagctcgactctagaggatccTTAacctttcagtgcttcttccagc-3′；pMA5-Pylb线性骨架使用一对引物通过PCR获取。使用引物329-F：5′-gctggaagaagcactgaaaggtTAAggatcctctagagtcgagctcaagct-3′和330-R：5′-cgaaggtaccaaacagtttgcccatACAAATCTCCCCCTTTGTTGTTTCT-3′；
所有引物均由苏州金唯智生物科技有限公司合成。基因的PCR条件为94℃变性5min，按如下参数循环30次：94℃变性15s，58℃退火15s，72℃延伸1min，最后72℃延伸10min。PCR反应所得到的产物分别用0.8％的琼脂糖凝胶电泳分析结果。经凝胶成像系统成像确认片段大小正确后，采用DNA纯化回收试剂盒(天根生化科技有限公司，中国)回收目的片段用于重组表达载体的构建。
然后使用POE-PCR组装耐热葡萄糖磷酸变位酶基因片段和pMA5-Pylb载体骨架。POE-PCR体系如下：纯化后的pMA5-Pylb线性骨架，200ng；纯化后的耐热葡萄糖磷酸变位酶基因片段131ng；2×PrimeSTAR MAX DNA Polymerase(大连宝生物，中国)，25μL，加水补足50μL。POE-PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸3.5min，最后72℃延伸5min。连接产物用氯化钙法转化入感受态E.coli Top10，挑选转化子进行菌落PCR及双酶切鉴定，选择2-3个阳性转化子送测序进一步验证，测序结果显示成功获得pMA5-Pylb-PGM重组共表达载体，质粒图谱如图3所示。
(3)pMA5-Pylb-PGI的构建
本实施例中耐热葡萄糖磷酸异构酶来自Thermus thermophilus。耐热葡萄糖磷酸异构酶编码基因pgi序列(NCBI-ProteinID:AAS82052)通过苏州金唯智生物科技有限公司合成并连接到普通质粒上。耐热葡萄糖磷酸异构酶编码基因pgi使用一对引物从基因组DNA中通过PCR获取。使用引物331-F：5′-AGAAACAACAAAGGGGGAGATTTGTATGCTGCGTCTGGATACTCGCTTTC-3′和332-R：5′-agcttgagctcgactctagaggatccTTAACCAGCCAGGCGTTTACGAGTC-3′；pMA5-Pylb线性骨架使用一对引物通过PCR获取。使用引物333-F：5′-GACTCGTAAACGCCTGGCTGGTTAAggatcctctagagtcgagctcaagct-3′和334-R：5′-GAAAGCGAGTATCCAGACGCAGCATACAAATCTCCCCCTTTGTTGTTTCT-3′；
所有引物均由苏州金唯智生物科技有限公司合成。基因的PCR条件为94℃变性5min，按如下参数循环30次：94℃变性15s，58℃退火15s，72℃延伸1min，最后72℃延伸10min。PCR反应所得到的产物分别用0.8％的琼脂糖凝胶电泳分析结果。经凝胶成像系统成像确认片段大小正确后，采用DNA纯化回收试剂盒(天根生化科技有限公司，中国)回收目的片段用于重组表达载体的构建。
然后使用POE-PCR组装耐热葡萄糖磷酸异构酶基因片段和pMA5-Pylb载体骨架。POE- PCR体系如下：纯化后的pMA5-Pylb线性骨架，200ng；纯化后的耐热葡萄糖磷酸异构酶基因片段131ng；2×PrimeSTAR MAX DNA Polymerase(大连宝生物，中国)，25μL，加水补足50μL。POE-PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸3.5min，最后72℃延伸5min。连接产物用氯化钙法转化入感受态E.coli Top10，挑选转化子进行菌落PCR及双酶切鉴定，选择2-3个阳性转化子送测序进一步验证，测序结果显示成功获得pMA5-Pylb-PGI重组共表达载体，质粒图谱如图4所示。
(4)pMA5-Pylb-TPE的构建
本实施例中耐热6-磷酸塔格糖差向异构酶来自Thermoanaerobacter indiensis。耐热6-磷酸塔格糖差向异构酶编码基因tpe序列(NCBI-ProteinID:B044_RS0101530)通过苏州金唯智生物科技有限公司合成并连接到普通质粒上。耐热6-磷酸塔格糖差向异构酶编码基因tpe使用一对引物从基因组DNA中通过PCR获取。使用引物335-F：5′-AGAAACAACAAAGGGGGAGATTTGTatgaaagtttggctggttggtgcct-3′和324-R：5′-agcttgagctcgactctagaggatccTTAtttcaggttgctataccattct-3′；pMA5-Pylb线性骨架使用一对引物通过PCR获取。使用引物325-F：5′-agaatggtatagcaacctgaaaTAAggatcctctagagtcgagctcaagct-3′和326-R：5′-aggcaccaaccagccaaactttcatACAAATCTCCCCCTTTGTTGTTTCT-3′；
所有引物均由苏州金唯智生物科技有限公司合成。基因的PCR条件为94℃变性5min，按如下参数循环30次：94℃变性15s，58℃退火15s，72℃延伸1min，最后72℃延伸10min。PCR反应所得到的产物分别用0.8％的琼脂糖凝胶电泳分析结果。经凝胶成像系统成像确认片段大小正确后，采用DNA纯化回收试剂盒(天根生化科技有限公司，中国)回收目的片段用于重组表达载体的构建。
然后使用POE-PCR组装耐热6-磷酸塔格糖差向异构酶基因片段和pMA5-Pylb载体骨架。POE-PCR体系如下：纯化后的pMA5-Pylb线性骨架，200ng；纯化后的耐热6-磷酸塔格糖差向异构酶基因片段131ng；2×PrimeSTAR MAX DNA Polymerase(大连宝生物，中国)，25μL，加水补足50μL。POE-PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸3.5min，最后72℃延伸5min。连接产物用氯化钙法转化入感受态E.coli Top10，挑选转化子进行菌落PCR及双酶切鉴定，选择2-3个阳性转化子送测序进一步验证，测序结果显示成功获得pMA5-Pylb-TPE重组共表达载体，质粒图谱如图5所示。
(5)pMA5-Pylb-TPP的构建
本实施例中耐热6-磷酸塔格糖磷酸酶来自Archaeoglobus fulgidus。耐热6-磷酸塔格糖磷酸酶编码基因tpp序列(NCBI-ProteinID:AAB90791)通过苏州金唯智生物科技有限公司合 成并连接到普通质粒上。耐热6-磷酸塔格糖磷酸酶编码基因tpp使用一对引物从基因组DNA中通过PCR获取。使用引物339-F：5′-AGAAACAACAAAGGGGGAGATTTGTATGTTCAAGCCGAAAGCGATCGCGG-3′和340-R：5′-agcttgagctcgactctagaggatccTTAACGCAGCAGGCCCAGAAACTG-3′；pMA5-Pylb线性骨架使用一对引物通过PCR获取。使用引物；341-F：5′-CAGTTTCTGGGCCTGCTGCGTTAAggatcctctagagtcgagctcaagct-3′和342-R：5′-CCGCGATCGCTTTCGGCTTGAACATACAAATCTCCCCCTTTGTTGTTTCT-3′。
所有引物均由苏州金唯智生物科技有限公司合成。基因的PCR条件为94℃变性5min，按如下参数循环30次：94℃变性15s，58℃退火15s，72℃延伸1min，最后72℃延伸10min。PCR反应所得到的产物分别用0.8％的琼脂糖凝胶电泳分析结果。经凝胶成像系统成像确认片段大小正确后，采用DNA纯化回收试剂盒(天根生化科技有限公司，中国)回收目的片段用于重组表达载体的构建。
然后使用POE-PCR组装耐热6-磷酸塔格糖磷酸酶基因片段和pMA5-Pylb载体骨架。POE-PCR体系如下：纯化后的pMA5-Pylb线性骨架，200ng；纯化后的耐热6-磷酸塔格糖磷酸酶基因片段131ng；2×PrimeSTAR MAX DNA Polymerase(大连宝生物，中国)，25μL，加水补足50μL。POE-PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸3.5min，最后72℃延伸5min。连接产物用氯化钙法转化入感受态E.coli Top10，挑选转化子进行菌落PCR及双酶切鉴定，选择2-3个阳性转化子送测序进一步验证，测序结果显示成功获得pMA5-Pylb-TPP重组共表达载体，质粒图谱如图6所示。
(6)pMA5-Pylb-aGP-PGM-PGI-TPE-TPP的构建
本实施例中耐热α-葡聚糖磷酸化酶来自Thermococcus kodakarensis；耐热葡萄糖磷酸变位酶来自Thermococcus kodakarensis；耐热葡萄糖磷酸异构酶来自Thermus thermophilus；耐热6-磷酸塔格糖差向异构酶来自Thermoanaerobacter indiensis；和耐热6-磷酸塔格糖磷酸酶来自Archaeoglobus fulgidus。耐热α-葡聚糖磷酸化酶编码基因agp(NCBI-ProteinID:BAD85595)通过PCR获取，使用引物350-F：5′-AGAAACAACAAAGGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3′和351-R：5′-cgaaggtaccaaacagtttgcccatTTTGAATTCCTCCTTTtcagtcaagtcccttccacttgacc-3′；耐热葡萄糖磷酸变位酶编码基因pgm(NCBI-ProteinID:BAD85297)通过PCR获取，使用引物352-F：5′-ggtcaagtggaagggacttgactgaAAAGGAGGAATTCAAAatgggcaaactgtttggtaccttcg-3′和353-R：5′-GAAAGCGAGTATCCAGACGCAGCATTTTGAATTCCTCCTTTTTAacctttcagtgcttcttccagc-3′； 耐热葡萄糖磷酸异构酶编码基因pgi(NCBI-ProteinID:AAS82052)通过PCR获取，使用引物354-F：5′-gctggaagaagcactgaaaggtTAAAAAGGAGGAATTCAAAATGCTGCGTCTGGATACTCGCTTTC-3′和355-R：5′-TTTTCAGCGGATGTTCGGTGTTCATTTTGAATTCCTCCTTTTCAACCAGCCAGGCGTTTACGAGTC-3′；耐热6-磷酸塔格糖差向异构酶编码基因tpe(NCBI-ProteinID:B044_RS0101530)通过PCR获取，使用引物356-F：5′-GACTCGTAAACGCCTGGCTGGTTGAAAAGGAGGAATTCAAAATGAACACCGAACATCCGCTGAAAA-3′和357-R：5′-ACCGCGATCGCTTTCGGCTTGAACATTTTGAATTCCTCCTTTttaAATCAGTTTGAATTCACCGCTG-3′；耐热6-磷酸塔格糖磷酸酶编码基因tpp(NCBI-ProteinID:AAB90791)通过PCR获取，使用引物358-F：5′-CAGCGGTGAATTCAAACTGATTtaaAAAGGAGGAATTCAAAATGTTCAAGCCGAAAGCGATCGCGGT-3′和359-R：5′-gcttgagctcgactctagaggatccTTAACGCAGCAGGCCCAGAAACTGCA-3′；pMA5-Pylb线性骨架使用通过PCR获取，使用引物；360-F：5′-TGCAGTTTCTGGGCCTGCTGCGTTAAggatcctctagagtcgagctcaagc-3′和361-R：5′-caacggcattggaaacgttcaccatACAAATCTCCCCCTTTGTTGTTTCT-3′。
所有引物均由苏州金唯智生物科技有限公司合成。基因的PCR条件为94℃变性5min，按如下参数循环30次：94℃变性15s，58℃退火15s，72℃延伸1min，最后72℃延伸10min。PCR反应所得到的产物分别用0.8％的琼脂糖凝胶电泳分析结果。经凝胶成像系统成像确认片段大小正确后，采用DNA纯化回收试剂盒(天根生化科技有限公司，中国)回收目的片段用于重组表达载体的构建。
然后使用POE-PCR组装耐热α-葡聚糖磷酸化酶基因片段、耐热葡萄糖磷酸变位酶基因片段、耐热6-磷酸塔格糖差向异构酶基因片段、耐热6-磷酸塔格糖磷酸酶基因片段和pMA5-Pylb载体骨架。POE-PCR体系如下：纯化后的pMA5-Pylb线性骨架，200ng；纯化后的耐热α-葡聚糖磷酸化酶基因片段131ng、耐热葡萄糖磷酸变位酶基因片段131ng、耐热葡萄糖磷酸异构酶基因片段131ng、耐热6-磷酸塔格糖差向异构酶基因片段131ng、耐热6-磷酸塔格糖磷酸酶基因片段131ng；2×PrimeSTAR MAX DNA Polymerase(大连宝生物，中国)，25μL，加水补足50μL。POE-PCR条件为98℃变性2min，按如下参数循环30次：98℃变性15s，58℃退火15s，72℃延伸3.5min，最后72℃延伸5min。连接产物用氯化钙法转化入感受态E.coli Top10，挑选转化子进行菌落PCR及双酶切鉴定，选择2-3个阳性转化子送 测序进一步验证，测序结果显示成功获得pMA5-Pylb-aGP-PGM-PGI-TPE-TPP重组共表达载体，质粒图谱如图7所示。
实施例6 重组工程菌的构建
将构建的重组表达载体pMA5-Pylb-aGP、pMA5-Pylb-PGM、pMA5-Pylb-PGI、pMA5-Pylb-TPE、pMA5-Pylb-TPP、pMA5-Pylb-aGP-PGM-PGI-TPE-TPP分别转化入枯草芽孢杆菌工程菌SCK8-ST3，LB试管培养过夜，质粒抽提试剂盒抽提质粒，将正确的克隆子SCK8-ST3/pMA5-Pylb-aGP、SCK8-ST3/pMA5-Pylb-PGM、SCK8-ST3/pMA5-Pylb-PGI、SCK8-ST3/pMA5-Pylb-TPE、SCK8-ST3/pMA5-Pylb-TPP和SCK8-ST3/pMA5-Pylb-aGP-PGM-PGI-TPE-TPP保存。
实施例7 重组工程菌全细胞的制备
分别挑取重组工程菌SCK8-ST3/pMA5-Pylb-aGP、SCK8-ST3/pMA5-Pylb-PGM、SCK8-ST3/pMA5-Pylb-PGI、SCK8-ST3/pMA5-Pylb-TPE、SCK8-ST3/pMA5-Pylb-TPP和SCK8-ST3/pMA5-Pylb-aGP-PGM-PGI-TPE-TPP接种于含有奇霉素的LB培养基中，37℃振荡过夜培养。将培养物以1％的接种量转接于新鲜的含有奇霉素的LB培养基中，37℃震荡培养过夜，5500rpm离心10min，弃去上清，获得表达耐热α-葡聚糖磷酸化酶的全细胞、表达耐热葡萄糖磷酸变位酶的全细胞、表达耐热葡萄糖磷酸异构酶的全细胞、表达耐热6-磷酸塔格糖差向异构酶的全细胞、表达耐热6-磷酸塔格糖磷酸酶的全细胞以及共表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶的全细胞。
实施例8 共表达全细胞催化淀粉制备塔格糖
将实施例7中制备的共表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶的全细胞使用50mM Tris-HCl缓冲液(pH 7.5)洗涤1次，5500rpm离心10min，弃去上清，向沉淀中加入50mM Tris-HCl(pH 7.5)缓冲液，重悬菌体至OD 600＝300左右。将重悬的菌体在75℃热处理90min。
在1L反应体系中，分别加入终浓度为100g/L淀粉、50mM磷酸钠缓冲液(pH 7.5)以及热处理的全细胞，使OD 600＝20左右。在70℃水浴摇床反应46h，取样进行高效液相色谱(HPLC)分析。HPLC检测条件如下：色谱柱为Bio-Rad HPX-87H；流速为0.6mL/min；柱温为60℃；检测器为示差折光检测器；进样量为20μL。
共计进行了三次平行重复试验，图8显示出了塔格糖的产量随反应时间变化过程曲线图，70℃水浴摇床反应46h后，HPLC测试结果显示塔格糖产量达到50g/L，产率可达50％。
实施例9 全细胞混合物催化淀粉制备塔格糖
将实施例7中制备的表达耐热α-葡聚糖磷酸化酶的全细胞、表达耐热葡萄糖磷酸变位酶的全细胞、表达耐热葡萄糖磷酸异构酶的全细胞、表达耐热6-磷酸塔格糖差向异构酶的全细胞、表达耐热6-磷酸塔格糖磷酸酶的全细胞分别使用50mM Tris-HCl缓冲液(pH 7.5)洗涤1次，5500rpm离心10min，弃去上清，分别向沉淀中加入50mM磷酸钠缓冲液(pH 7.5)，重悬菌体至OD 600＝300左右。将重悬的菌体75℃热处理90min。
在1L反应体系中，加入终浓度为100g/L淀粉、50mM磷酸钠缓冲液(pH 7.5)以及以上四种热处理的全细胞使OD 600＝20左右，表达耐热α-葡聚糖磷酸化酶的全细胞、表达耐热葡萄糖磷酸变位酶的全细胞、表达耐热葡萄糖磷酸异构酶的全细胞、表达耐热6-磷酸塔格糖差向异构酶的全细胞、表达耐热6-磷酸塔格糖磷酸酶的全细胞的加入比例为1:1:1:1。在70℃水浴摇床反应46h，取样进行HPLC分析。共计进行了三次平行重复试验，HPLC检测条件同实施例8。70℃水浴摇床反应46h后，HPLC测试结果显示塔格糖产量达到73g/L，产率可达73％。
实施例10 固定化全细胞催化淀粉制备塔格糖
用1L磷酸钠缓冲液(pH 7.0)重悬共表达耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶的透性全细胞，使得OD600＝400左右，加入1g蒙脱土，搅拌均匀。加入40ml 5％(w/v)聚乙烯亚胺水溶液于室温条件下絮凝，在加入20ml 50％戊二醛水溶液于室温交联3h。然后抽滤得到滤饼层，滤饼用去离子水洗涤后挤压制备成颗粒，干燥后得到固定化全细胞。
在1L反应体系中，分别加入终浓度为100g/L淀粉、50mM磷酸钠缓冲液(pH 7.5)以及固定化全细胞，使OD600＝20左右，在70℃水浴摇床反应。反应结束后反应液于4℃离心，进行高效液相色谱(HPLC)分析塔格糖含量；收集固定化颗粒，经缓冲液洗涤进行下一批次反应。实验结果如图9所示，结果表明，固定化全细胞连续催化淀粉生产塔格糖过程中，初始产物得率最高可达50％，随着连续催化反应批次的增加，产物得率逐渐降低，连续催化50批次后，产物得率可维持36％左右。
实施例11 固定化全细胞混合物催化淀粉制备塔格糖
用1L磷酸钠缓冲液(pH 7.0)按照比例1:1:1:1:1重悬表达耐热α-葡聚糖磷酸化酶的透性全细胞、表达耐热葡萄糖磷酸变位酶的透性全细胞、表达耐热葡萄糖磷酸异构酶的透性全细胞、表达耐热6-磷酸塔格糖差向异构酶的透性全细胞、表达耐热6-磷酸塔格糖磷酸酶的透性全细胞，使得OD600＝300左右，加入0.8g蒙脱土，搅拌均匀。加入35ml 5％(w/v)聚乙烯亚胺水溶液于室温条件下絮凝，在加入18ml 50％戊二醛水溶液于室温交联3h。然后抽滤 得到滤饼层，滤饼用去离子水洗涤后挤压制备成颗粒，干燥后得到固定化全细胞混合物。
在1L反应体系中，分别加入终浓度为100g/L淀粉、50mM磷酸钠缓冲液(pH 7.5)以及固定化全细胞，使OD 600＝20左右，在70℃水浴摇床反应。反应结束后反应液于4℃离心，进行高效液相色谱(HPLC)分析塔格糖含量；收集固定化颗粒，经缓冲液洗涤进行下一批次反应。实验结果如图10所示，结果表明，连续催化60批次，产物得率均大于50％，最高可达73％以上。实验结果如图10所示，结果表明，固定化全细胞混合物连续催化反应时，初始产物得率最高可达73％以上，随着连续催化反应的进行，产物得率逐渐降低，连续催化60批次后，产物得率仍可维持在52％。

Claims (21)

  1. 一种产塔格糖的枯草芽孢杆菌基因工程菌，其特征在于，所述基因工程菌是共表达α-葡聚糖磷酸化酶基因、葡萄糖磷酸变位酶基因、葡萄糖磷酸异构酶基因、6-磷酸塔格糖差向异构酶基因和6-磷酸塔格糖磷酸酶基因的枯草芽孢杆菌基因工程菌、或者是分别表达α-葡聚糖磷酸化酶基因、葡萄糖磷酸变位酶基因、葡萄糖磷酸异构酶基因、6-磷酸塔格糖差向异构酶基因和6-磷酸塔格糖磷酸酶基因的枯草芽孢杆菌基因工程菌的混合物。
  2. 如权利要求1所述的基因工程菌，其特征在于，所述枯草芽孢杆菌的出发菌株为敲除蛋白酶的枯草芽孢杆菌菌株。
  3. 如权利要求1所述的基因工程菌，其特征在于，所述基因工程菌中包括共表达α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶的表达载体，或者为包含α-葡聚糖磷酸化酶的表达载体的基因工程菌、包含葡萄糖磷酸变位酶的表达载体的基因工程菌、包含葡萄糖磷酸异构酶的表达载体的基因工程菌、包含6-磷酸塔格糖差向异构酶的表达载体的基因工程菌和包含6-磷酸塔格糖磷酸酶的表达载体的基因工程菌的混合物。
  4. 如权利要求1所述的基因工程菌，其特征在于，所述的α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶分别为耐热α-葡聚糖磷酸化酶、耐热葡萄糖磷酸变位酶、耐热葡萄糖磷酸异构酶、耐热6-磷酸塔格糖差向异构酶和耐热6-磷酸塔格糖磷酸酶。
  5. 如权利要求4所述的基因工程菌，其特征在于，所述耐热是指在40℃以上具有酶的活性。
  6. 如权利要求1至5任一项所述的基因工程菌，其特征在于，所述基因工程菌中内源性的尿嘧啶磷酸核糖基转移酶基因，α-淀粉酶基因，孢子形成RNA聚合酶σ F因子基因，和表面活性肽合成酶亚基3基因均被失活或敲除。
  7. 一种表达载体，其特征在于，其包含α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、6-磷酸塔格糖差向异构酶和6-磷酸塔格糖磷酸酶基因，并能够实现这些基因的共表达。
  8. 利用如权利要求1至6任一项所述的基因工程菌的全细胞催化淀粉制备生产塔格糖的方法，其包括下述步骤：
    (1)将所述枯草芽孢杆菌工程菌进行发酵获得全细胞；
    (2)将步骤(1)获得的枯草芽孢杆菌全细胞进行细胞膜通透性处理，获得透性全细胞；
    (3)利用步骤(2)获得的透性全细胞催化淀粉制备塔格糖，其中对于共表达的枯草芽孢杆菌工程菌全细胞直接用于催化，对于分别表达各种酶的枯草芽孢杆菌工程菌全细胞，则进行混合用于催化。
  9. 如权利要求8所述的方法，其特征在于，还包括将步骤(2)中获得的枯草芽孢杆菌透性全细胞进行固定化处理，获得固定化全细胞，或者固定化全细胞混合物，然后再用于催化。
  10. 如权利要求8所述的方法，其特征在于，所述步骤(1)的全细胞的制备是通过适于表达外源蛋白的发酵方法获得的。
  11. 如权利要求8所述的方法，其特征在于，所述步骤(2)的细胞膜通透性处理是通过热处理、添加有机溶剂和/或添加表面活性剂处理获得。
  12. 如权利要求11所述的方法，其特征在于，所述有机溶剂选自丙酮、乙腈；所述表面活性剂选自十六烷基三甲基溴化铵、Tween-80。
  13. 如权利要求11所述的方法，其特征在于，所述热处理的温度为45-100℃，热处理时间为10-100min；处理时细胞浓度为OD 600＝10-300。
  14. 如权利要求13所述的方法，其特征在于，所述热处理的温度为70-80℃，热处理时间为50-70min；处理时细胞浓度为OD 600＝30-150。
  15. 如权利要求11所述的方法，其特征在于，所述热处理是在选自HEPES缓冲液、磷酸盐缓冲液、Tris缓冲液、醋酸盐缓冲液的缓冲液中进行。
  16. 如权利要求8所述的方法，其特征在于，所述步骤(3)中催化的反应体系中，底物淀粉的浓度为50-300g/L；反应条件为：在pH 5.0-8.0、40-80℃下反应0.5-96h。
  17. 如权利要求17所述的方法，其特征在于，所述步骤(3)中催化的反应体系中，底物淀粉的浓度为100-200g/L；反应条件为：pH 6.5-7.5、45-75℃下反应12-60h。
  18. 如权利要求8所述的方法，其特征在于，所述步骤(3)中催化的反应是在选自HEPES缓冲液、磷酸盐缓冲液、Tris缓冲液、醋酸盐缓冲液的缓冲液中进行的。
  19. 如权利要求8所述的方法，其特征在于，对于分别表达各种酶的枯草芽孢杆菌工程菌透性全细胞，其混合物是将表达α-葡聚糖磷酸化酶的透性全细胞、表达葡萄糖磷酸变位酶的透性全细胞、表达葡萄糖磷酸异构酶的透性全细胞、表达6-磷酸塔格糖差向异构酶的透性全细胞、表达6-磷酸塔格糖磷酸酶的透性全细胞按比例为(0.1-10)：(0.1-10)：(0.1-10)：(0.1-10)进行混合。
  20. 如权利要求9所述的方法，其特征在于，所述透性全细胞固定化处理方法为：用磷酸钠或磷酸钾缓冲液重悬所述透性全细胞，加入无机土，搅拌均匀；随后加入聚乙烯亚胺水溶液进行絮凝，再加入交联剂进行交联；然后抽滤得到滤饼层，滤饼用去离子水洗涤后挤压制备 成颗粒，干燥后得到固定化全细胞。
  21. 如权利要求20所述的方法，其特征在于，所述无机土选自蒙脱土、硅藻土、高岭土和膨润土；所述交联剂选自戊二醛、三羟甲基磷、N,N-亚甲基双丙烯酰胺、环氧氯丙烷和京尼平。
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