CN112980754A - Method for preparing inositol by catalyzing starch with bacillus subtilis whole cells - Google Patents
Method for preparing inositol by catalyzing starch with bacillus subtilis whole cells Download PDFInfo
- Publication number
- CN112980754A CN112980754A CN201911282987.XA CN201911282987A CN112980754A CN 112980754 A CN112980754 A CN 112980754A CN 201911282987 A CN201911282987 A CN 201911282987A CN 112980754 A CN112980754 A CN 112980754A
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- Prior art keywords
- inositol
- promoter
- thermotolerant
- phosphate
- thermostable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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Abstract
The invention discloses a method for preparing high-concentration inositol by catalyzing high-concentration starch through whole cells of bacillus subtilis, which comprises the steps of constructing engineering bacteria and/or engineering bacteria mixture which co-expresses or independently expresses heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphoglucomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylase, carrying out cell membrane permeability treatment on the engineering bacteria and/or the engineering bacteria mixture, and converting starch into inositol by utilizing permeable engineering bacteria and/or the permeable engineering bacteria mixture. Compared with the existing method for producing inositol, the method provided by the invention has the advantages of whole cell recycling, high safety performance, high yield, simple production process, low cost, easiness in large-scale preparation and the like.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to a method for preparing and producing high-concentration inositol by catalyzing high-concentration starch with bacillus subtilis whole cells, belonging to the field of preparation of inositol.
Background
Inositol, also known as inositol, is one of water-soluble vitamin B group and is widely applied to industries such as feed, food, medicine and the like. The addition of 0.2-0.5% of inositol into the feed can effectively promote the growth of livestock and prevent death. Inositol is added in the goldfish culture process to obviously improve the swimming capacity of goldfishes, and a certain amount of inositol is added in the feed for fish and shrimp to promote the growth of fish and shrimp and increase the growth speed of fish and shrimp by more than 10%. Inositol is an essential substance for growth of human, animals and microorganisms, so the inositol is often used as a nutrition enhancer in health care products, beverages (red cattle functional beverage) and milk products, and the infant food is recommended to be added with a certain amount of inositol in 1987 in the United states. A health food and nutritional health product containing inositol for reducing weight and blood lipid are called as European style. Inositol has good medicinal value, can promote the metabolism of liver and fat, treat various vitamin deficiencies, is widely used as a raw material for preparing a compound vitamin preparation, can treat various diseases such as liver cirrhosis, fatty liver, vascular sclerosis, high cholesterol, obesity, diabetes, carbon tetrachloride poisoning and the like, and has the medical effects of promoting cell growth and preventing aging. Inositol is used as pharmaceutical intermediate for synthesizing inositol nicotinate, inositol selenate, etc. to treat hypercholesterolemia, arteriosclerosis, diabetes, cancer, etc. Wherein, inositol selenate is called 'anticancer king' in microelement, and can be used for preparing selenium-rich anticancer drugs, foods and beverages. The new product fluoroinositol developed in recent years has the functions of resisting cancer, treating cancer and improving immunity. Inositol and its derivatives can also treat melancholia and obsessive-compulsive disorder by a compound amine reuptake inhibitor different from blood.
The chemical production of inositol is mainly the traditional high-temperature pressurized hydrolysis of phytic acid (inositol hexaphosphate). The process has strict requirements on the material of equipment, large one-time investment, and high production cost, can only control the operating pressure within a certain range, limits the improvement of the utilization rate of raw materials, and has the disadvantages of complicated crude product refining process, more loss and high production cost.
There are documents and patents reporting that inositol is produced using glucose as a substrate by a microbial fermentation method. The inositol-1-phosphate synthase derived from Saccharomyces cerevisiae and myo-inositol monophosphorylase over-expressed in Escherichia coli are introduced into Escherichia coli to convert glucose into myo-inositol. But glucose in microbial cells is used as a substrate to participate in multiple metabolic reactions simultaneously for maintaining cell metabolism; in addition, glucose-6-phosphate is obtained by phosphorylation of glucose, which requires the consumption of energy (ATP or phosphoenolpyruvate), and the conversion rate of glucose into inositol is low, and in addition, Escherichia coli contains endotoxin, and the application of products produced by Escherichia coli as a host in the food preparation industry is limited.
Zhang Heng and Yongchun invented a method for the enzymatic conversion of inositol (CN106148425A, method for preparing inositol), which provides a method for the enzymatic conversion of inositol, and a method for producing inositol by in vitro multi-enzyme catalysis of starch or cellulose and their derivatives and glucose, and has the advantages of high yield and conversion rate of inositol, etc. However, the fermentation production host of the key enzyme in the method is escherichia coli BL21(DE3), the escherichia coli contains endotoxin which is not suitable for industrial production of production strains of relevant enzymes for production of food preparations, and in addition, the steps of in vitro enzyme separation and purification are complicated, the enzyme recovery rate is low, the recycling is difficult, and the production cost cannot be further reduced. In addition, the concentration of the substrate starch is 10g/L, the substrate concentration is very low, the yield of the inositol product is lower, and the industrial high-concentration product mass production application cannot be realized.
Therefore, a new method which is capable of recycling whole cells, high in safety performance, high in yield, simple in production process, low in cost, suitable for high-substrate-concentration input and high-yield inositol output and easy for large-scale inositol preparation is urgently needed to be developed.
Disclosure of Invention
Aiming at the problems of the existing method for preparing inositol by multi-enzyme catalysis, such as the disadvantages that escherichia coli contains endotoxin, which is not beneficial to the industrial production of food preparations, the purification steps are complicated, the recycling utilization rate of enzyme is low, the recycling is difficult, and the feeding problem of low-concentration substrate starch, the invention mainly aims to provide a method for preparing high-concentration inositol by catalyzing high-concentration starch by using bacillus subtilis whole cells.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention relates to a method for preparing inositol by using bacillus subtilis whole cell catalysis high-concentration starch, which is characterized by comprising the following steps:
(1) constructing bacillus subtilis host bacteria for knocking out alpha-amylase;
(2) constructing engineering bacteria for co-expressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphate mutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylase and/or engineering bacteria of bacillus subtilis for respectively expressing the heat-resistant alpha-glucan phosphorylase, the heat-resistant glucose phosphate mutase, the heat-resistant inositol-3-phosphate synthase and the heat-resistant inositol monophosphorylase on the basis of the host bacteria constructed in the step (1);
(3) fermenting the bacillus subtilis engineering bacteria constructed in the step (2) to obtain whole cells;
(4) performing cell membrane permeability treatment on the bacillus subtilis whole cell obtained in the step (3) to obtain a permeable whole cell;
(5) catalyzing starch to prepare inositol by using the mixture of the permeable whole cells co-expressing the thermostable alpha-glucan phosphorylase, the thermostable glucose phosphate mutase, the thermostable inositol-3-phosphate synthase and the thermostable inositol monophosphorylase obtained in the step (4), the permeable whole cells expressing the thermostable alpha-glucan phosphorylase, the permeable whole cells expressing the thermostable glucose phosphate mutase, the permeable whole cells expressing the thermostable inositol-3-phosphate synthase and the permeable whole cells expressing the thermostable inositol monophosphorylase.
According to the invention, amyE gene in bacillus subtilis encodes alpha-amylase, which can be secreted to extracellular degraded substrate starch in the fermentation process, in order to avoid degrading the substrate starch in the amylase produced by fermenting the genetically engineered bacteria so as to influence the application of substrate conversion to produce inositol, the invention knocks out the alpha-amylase protein encoding gene amyE in a bacillus subtilis host, and constructs the bacillus subtilis host bacteria with alpha-amylase knocked out.
It will be appreciated by those skilled in the art that various strains of Bacillus subtilis known in the art may be used as starting strains for the present invention. Preferably, the bacillus subtilis starting strain is a protease-knock-out bacillus subtilis strain, such as WB800, WB600, SCK6, 1a751 and the like. More preferably, the starting strain of bacillus subtilis is SCK 6.
According to the invention, the catalytic pathway comprises: converting the substrate starch to an intermediate glucose-1-phosphate (G1P) by thermostable α -glucan phosphorylase in the presence of inorganic phosphorus; (ii) converting the intermediate glucose-1-phosphate (G1P) to another intermediate glucose-6-phosphate (G6P) by thermostable phosphoglucomutase; isomerizing intermediate glucose-6-phosphate (G6P) to another intermediate inositol-1-phosphate (I1P) by a thermostable inositol-3-phosphate synthase; the intermediate Inositol-1-phosphate (I1P) was dephosphorylated by thermotolerant Inositol monophosphatase to the product Inositol (Inositol).
Preferably, according to the present invention, in the step (2), the engineered bacterium comprises a vector co-expressing thermotolerant α -glucan phosphorylase, thermotolerant phosphoglucomutase, thermotolerant inositol-3-phosphate synthase, and thermotolerant inositol monophosphorylase, or a vector expressing thermotolerant α -glucan phosphorylase, or a vector expressing thermotolerant phosphoglucomutase, or a vector expressing thermotolerant inositol-3-phosphate synthase, or a vector expressing thermotolerant inositol monophosphorylase. As will be understood by those skilled in the art, the vectors and engineered bacteria of the present invention can be prepared by conventional methods known in the art, for example, by recombinant DNA technology construction, obtaining the gene α gp encoding α -glucan phosphorylase, pgm encoding glucose phosphoglucomutase, ips encoding inositol-3-phosphate synthase, and imp encoding inositol monophosphorylase, constructing recombinant expression vectors, and transferring into host bacteria to obtain genetically engineered bacteria.
Preferably, according to the present invention, in step (2), "thermostable α -glucan phosphorylase" refers to an enzyme having a function of phosphorylating starch to glucose-1-phosphate (G1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher.
Further preferably, the thermostable α -glucan phosphorylase is derived from a thermophilic microorganism, such as Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Geobacillus maritima (thermooga maritima), thermoascus kodakarensis and the like; or the amino acid sequence of said thermotolerant alpha-glucan phosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant alpha-glucan phosphorylase derived from said thermophilic microorganism.
More preferably, the thermostable a-glucan phosphorylase is derived from Thermococcus kodakarensis; or the amino acid sequence of said thermotolerant α -glucan phosphorylase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with a thermotolerant α -glucan phosphorylase derived from Thermococcus kodakarensis.
Preferably, according to the present invention, in step (2), "thermostable glucose phosphate mutase" refers to an enzyme having a function of changing glucose-1-phosphate (G1P) into glucose-6-phosphate (G6P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher.
Further preferably, the thermostable phosphoglucomutase is derived from a thermophilic microorganism, such as Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Thermococcus kodakarensis and the like; or the amino acid sequence of said thermotolerant phosphoglucomutase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with a thermotolerant phosphoglucomutase derived from said thermophilic microorganism.
More preferably, the thermostable phosphoglucomutase is derived from Thermococcus kodakarensis; or the amino acid sequence of said thermotolerant phosphoglucomutase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity to a thermotolerant phosphoglucomutase derived from Thermococcus kodakarensis.
Preferably, according to the present invention, in step (2), "thermostable inositol-3-phosphate synthase" refers to an enzyme having a function of converting glucose-6-phosphate (G1P) into inositol-1-phosphate (I1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher.
Further preferably, the thermostable inositol-3-phosphate synthase is derived from a thermophilic microorganism, such as Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Thermococcus kodakarensis, Archaeoglobus fulgidus, etc.; or the amino acid sequence of said thermotolerant inositol-3-phosphate synthase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with a thermotolerant inositol-3-phosphate synthase derived from said thermophilic microorganism.
More preferably, the thermotolerant inositol-3-phosphate synthase is derived from Archaeoglobubus fulgidus; or said thermotolerant inositol-3-phosphate synthase has an amino acid sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant inositol-3-phosphate synthase derived from Archaeoglobus fulgidus.
Preferably, according to the present invention, in the step (2), "Inositol monophosphorylase" refers to an enzyme having a function of dephosphorylating Inositol-1-phosphate (I1P) to Inositol (Inositol) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher.
Further preferably, the inositol monophosphorylase is derived from a thermophilic microorganism, such as Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Thermococcus kodakarensis and the like; or the amino acid sequence of said myo-inositol monophosphorylase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the myo-inositol monophosphorylase derived from said thermophilic microorganism.
More preferably, the myo-inositol monophosphorylase is derived from Thermotoga maritima; or the amino acid sequence of said myo-inositol monophosphorylase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the myo-inositol monophosphorylase derived from Thermotoga maritima.
Preferably, according to the present invention, the co-expressed vector comprises a promoter, a thermostable α -glucan phosphorylase gene, a thermostable glucose phosphate mutase gene, a thermostable myo-inositol-3-phosphate synthase gene, a thermostable myo-inositol monophosphorylate gene and a terminator; the vector for expressing the heat-resistant alpha-glucan phosphorylase comprises a promoter, a heat-resistant alpha-glucan phosphorylase gene and a terminator; the vector for expressing the heat-resistant glucose phosphoglucomutase comprises a promoter, a heat-resistant glucose phosphoglucomutase gene and a terminator; the vector for expressing the heat-resistant inositol-3-phosphate synthetase comprises a promoter, a heat-resistant inositol-3-phosphate synthetase gene and a terminator; the vector for expressing the heat-resistant inositol monophosphorylase comprises a promoter, a heat-resistant inositol monophosphorylase gene and a terminator.
It will be appreciated by those skilled in the art that a variety of promoters known in the art may be used as the promoter of the present invention including, but not limited to, the P43 promoter, the Pylb promoter, the Pamyl promoter, the Plaps promoter, the PhpaII promoter, the PamyE promoter, the Pgrac promoter, and the like. Preferably, the promoter of the invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
According to the present invention, in step (3), the preparation of the whole cells is performed using a method known in the art. The fermentation may use any medium suitable for the expression of the foreign protein, including, but not limited to, LB medium, TB medium, and the like.
According to the present invention, in step (4), the cell membrane permeability treatment includes, but is not limited to, heat treatment, addition of an organic solvent and/or addition of a surfactant, and the like. Among them, the organic solvent includes, but is not limited to, acetone, acetonitrile, etc. Surfactants include, but are not limited to, cetyltrimethylammonium bromide (CTAB), Tween-80, and the like. Preferably, the cell membrane permeability treatment is a heat treatment. The purpose of the permeability treatment of the cell membrane is to allow the extracellular starch to enter the cell through the cell membrane.
Preferably, the heat treatment temperature is 45-95 ℃; more preferably, the heat treatment temperature is 60 to 80 ℃.
Preferably, the heat treatment time is 1-60 min; more preferably, the heat treatment time is 10-50 min.
Preferably, the cell concentration at the time of heat treatment is OD60010-300; more preferably, the cell concentration is OD600＝30-150。
According to the invention, the heat treatment can be carried out in a buffer-free system or in a buffer system; preferably, the heat treatment is performed in a buffer system, which may be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, or the like. Among them, phosphate buffer solutions such as sodium phosphate buffer solution, potassium phosphate buffer solution, and the like.
Preferably, in step (5), the concentration of the substrate starch in the reaction system for preparing the inositol by catalyzing starch with the permeable whole cells or the mixture of the permeable whole cells is 10-300 g/L; more preferably, the concentration of the substrate starch is 20-200 g/L.
Preferably, according to the present invention, in step (4), the reaction conditions for catalyzing the preparation of inositol from starch by using permeable whole cells or a mixture of permeable whole cells are as follows: reacting at pH 6.0-8.0 and 50-80 deg.C for 0.5-96 hr; more preferably at pH 6.5-7.5, at 55-75 deg.C for 12-60 h; most preferably at a pH of 7.0 at 60-65 ℃ for 12-96 h.
According to the invention, the reaction catalyzed by permeable whole cells or a mixture of permeable whole cells can be carried out in a buffer-free system or a buffer system; preferably, the catalytic reaction is performed in a buffer system using permeable whole cells or a mixture of permeable whole cells, and the buffer may be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, or the like. Among them, phosphate buffer solutions such as sodium phosphate buffer solution, potassium phosphate buffer solution, and the like.
Preferably, according to the present invention, in step (5), the ratio of permeable whole cells expressing thermostable α -glucan phosphorylase, permeable whole cells expressing thermostable glucose phosphate mutase, permeable whole cells expressing thermostable inositol-3-phosphate synthase, permeable whole cells expressing thermostable inositol monophosphorylatese in the mixture of permeable whole cells is 0.1:1:1:0.1-10:1:1: 10; more preferably 0.5:1:1:0.5 to 5:1:1: 5.
According to the invention, it is also possible to use a mixture of permeable whole cells for the catalytic preparation of inositol from starch, under the following reaction conditions: reacting at pH 6.0-8.0 and 50-80 deg.C for 0.5-96 hr; more preferably at a pH of 6.5-7.5 at 55-75 deg.C for 12-96 h.
According to another aspect of the present invention, the present invention relates to the above-mentioned vector co-expressing thermotolerant α -glucan phosphorylase, thermotolerant phosphoglucomutase, thermotolerant inositol-3-phosphate synthase and thermotolerant inositol monophosphorylase, vector expressing thermotolerant α -glucan phosphorylase, vector expressing thermotolerant inositol-3-phosphate synthase, and vector expressing thermotolerant inositol monophosphorylase.
According to another aspect of the present invention, the present invention relates to the above-mentioned engineered bacteria coexpressing thermotolerant α -glucan phosphorylase, thermotolerant phosphoglucomutase, thermotolerant inositol-3-phosphate synthase and thermotolerant inositol monophosphorylase, engineered bacteria expressing thermotolerant α -glucan phosphorylase, engineered bacteria expressing thermotolerant glucose phosphoglucomutase, engineered bacteria expressing thermotolerant inositol-3-phosphate synthase and engineered bacteria expressing thermotolerant inositol monophosphorylase. According to the present invention, an engineered bacterium co-expressing a thermotolerant α -glucan phosphorylase, a thermotolerant glucose phosphate mutase, a thermotolerant inositol-3-phosphate synthase and a thermotolerant inositol monophosphorylase comprises a vector co-expressing the thermotolerant α -glucan phosphorylase, the thermotolerant glucose phosphate mutase, the thermotolerant inositol-3-phosphate synthase and the thermotolerant inositol monophosphorylase, or comprises a vector expressing the thermotolerant α -glucan phosphorylase, a vector expressing the thermotolerant glucose phosphate mutase, a vector expressing the thermotolerant inositol-3-phosphate synthase and a vector expressing the thermotolerant inositol monophosphorylase together; the engineering bacteria for expressing the heat-resistant alpha-glucan phosphorylase comprise a carrier for expressing the heat-resistant alpha-glucan phosphorylase; the engineering bacteria for expressing the heat-resistant glucose phosphoglucomutase comprise a carrier for expressing the heat-resistant glucose phosphoglucomutase; the engineering bacteria for expressing the heat-resistant inositol-3-phosphate synthetase comprise a vector for expressing the heat-resistant inositol-3-phosphate synthetase; the engineering bacteria for expressing the thermotolerant myo-inositol monophosphorylase comprise a vector for expressing the thermotolerant myo-inositol monophosphorylase.
Compared with the prior art, the invention has the following beneficial effects:
(1) the bacillus subtilis is a food-grade microorganism which is Generally regarded As Safe (Generally Recognized As Safe, GRAS), does not produce endotoxin, knocks out an alpha-amylase coding gene, and is beneficial to the catalytic application of subsequent substrate starch;
(2) the invention firstly utilizes the whole-cell catalytic starch expressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphoglucomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylase to produce inositol, and develops a novel method for simply preparing inositol on a large scale.
(3) No additional NAD is required to be added into the reaction system+Is beneficial to reducing the production cost and has important industrial application value.
(4) The preparation of the inositol in the method can be carried out at higher temperature, so that the solubility of the substrate starch can be increased.
(5) The conversion reaction of the inositol in the method can be carried out in a buffer solution-free system or a buffer solution system, and a culture medium containing a carbon source, a nitrogen source, inorganic salts and antibiotics is not needed, so that the method is favorable for reducing the production cost on one hand, and is favorable for separating and purifying the inositol product on the other hand.
Drawings
FIG. 1 is a schematic diagram of the permeable whole-cell catalyzed starch-catalyzed inositol production of the present invention.
FIGS. 2A-E are maps of recombinant expression vectors pMA5-Pylb-aGP, pMA5-Pylb-PGM, pMA5-Pylb-IPS, pMA5-Pylb-IMP and pMA5-Pylb-aGP-PGM-IPS-IMP, respectively.
FIG. 3 is a graph showing the course of inositol production as a function of reaction time.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following embodiments are provided to further illustrate the technical solutions of the present invention. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1: construction of bacillus subtilis engineering bacteria with alpha-amylase knockout function
And (3) competent preparation: the activated SCK6 strain was streaked on LB solid plate containing 0.3. mu.g/mL of erythromycin and cultured overnight at 37 ℃. The next day, a single clone was picked and inoculated into 5mL of LB liquid medium containing 0.3. mu.g/mL of erythromycin, and cultured at 37 ℃ for 8-12 hours at 200 rpm. The absorbance at 600nm was measured, and then the culture was diluted to a600 ═ 1.0 with fresh LB liquid medium containing 0.3 μ g/mL erythromycin, preheated at 37 ℃. D-xylose was added to a final concentration of 1% (w/v), and the culture was continued at 37 ℃ and 200rpm for 2 hours, and was ready for transformation.
And (3) transformation: 50ng of PDG1730 plasmid was mixed with 200. mu.L of competent cells, incubated at 37 ℃ for 1.5h at 200rpm, plated with LB solid plates containing 100. mu.g/mL spectinomycin, and incubated overnight at 37 ℃.
Colony PCR identification: a small amount of transformant cells were picked from the plate and diluted in 30. mu.L of sterile water, subjected to boiling water bath for 5min, frozen at-20 ℃ for 5min, dissolved at room temperature, centrifuged at 12000rpm for 1min, and 2. mu.L of the supernatant was taken as a template, and the primer amyE-F: 5'-ACCTCTTTACTGCCGTTATTCG-3' and amyE-R: 5'-TAGCACGTAATCAAAGCCAGG-3' PCR amplification was performed according to the following conditions: denaturation at 98 ℃ for 2min, cycling 30 times with the following parameters: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 30s, and final extension at 72 ℃ for 5 min. And (3) analyzing the PCR result by electrophoresis, only amplifying a single band with the size of-2.6 kb, further sequencing and verifying that amyE is successfully knocked out, and marking the bacillus subtilis strain with the amyE gene successfully knocked out as SCK 6/amyE.
Example 2: construction of recombinant vectors
(1) Construction of pMA5-Pylb-aGP
In the embodiment, the heat-resistant alpha-glucan phosphorylase is from Thermotoga maritima, and the KEGG has the accession number of TM 1168; thermotoga maritima is purchased from China general microbiological culture Collection center (CGMCC). The thermostable α -glucan phosphorylase-encoding gene agp was obtained from genomic DNA by PCR using a pair of primers. Primers 299-F were used: 5'-AGAAACAACAAAGGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3' and 300-R: 5'-gcttgagctcgactctagaggatcctcagtcaagtcccttccacttgacca-3', respectively; the pMA5-Pylb linear scaffold was obtained by PCR using a pair of primers. Primers 301-F were used: 5'-tggtcaagtggaagggacttgactgaggatcctctagagtcgagctcaagc-3' and 302-R: 5'-caacggcattggaaacgttcaccatACAAATCTCCCCCTTTGTTGTTTCT-3', respectively;
all primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc. The PCR conditions of the gene are 94 ℃ denaturation for 5min, and the cycle is 30 times according to the following parameters: denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, and final extension at 72 ℃ for 10 min. The products obtained from the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After confirming that the size of the fragment is correct by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., Ltd., China) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable α -glucan phosphorylase gene fragment and the pMA5-Pylb vector backbone were then assembled using POE-PCR. The POE-PCR system is as follows: purified pMA5-Pylb linear skeleton, 200 ng; 131ng of purified heat-resistant alpha-glucan phosphorylase gene fragment; 2 × PrimeSTAR MAX DNA Polymerase (Dalianbao bio, China), 25 μ L, and water to make up 50 μ L. The POE-PCR condition is that the denaturation is carried out for 2min at 98 ℃, and the cycle is carried out for 30 times according to the following parameters: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 3.5min, and final extension at 72 ℃ for 5 min. The ligation product was transformed into competent E.coli Top10 by calcium chloride method, transformants were selected for colony PCR and double enzyme digestion identification, 2-3 positive transformants were selected for further validation by sequencing, the sequencing result showed successful acquisition of pMA5-Pylb-aGP recombinant co-expression vector, the plasmid map is shown in FIG. 2A.
(2) Construction of pMA5-Pylb-PGM
In the embodiment, the heat-resistant phosphoglucomutase is from Thermococcus kodakarensis, and the KEGG has a registration number of TK 1108; thermococcus kodakarensis is purchased from China general microbiological culture Collection center (CGMCC). The thermostable phosphoglucomutase-encoding gene pgm was obtained from genomic DNA by PCR using a pair of primers. Primers 327-F were used: 5'-AGAAACAACAAAGGGGGAGATTTGTatgggcaaactgtttggtaccttcg-3' and 328-R: 5'-agcttgagctcgactctagaggatccTTAacctttcagtgcttcttccagc-3', respectively; the pMA5-Pylb linear scaffold was obtained by PCR using a pair of primers. Primers 329-F were used: 5'-gctggaagaagcactgaaaggtTAAggatcctctagagtcgagctcaagct-3' and 330-R: 5'-cgaaggtaccaaacagtttgcccatACAAATCTCCCCCTTTGTTGTTTCT-3', respectively;
all primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc. The PCR conditions of the gene are 94 ℃ denaturation for 5min, and the cycle is 30 times according to the following parameters: denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, and final extension at 72 ℃ for 10 min. The products obtained from the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After confirming that the size of the fragment is correct by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., Ltd., China) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable phosphoglucomutase gene fragment and the pMA5-Pylb vector backbone were then assembled using POE-PCR. The POE-PCR system is as follows: purified pMA5-Pylb linear skeleton, 200 ng; 131ng of purified heat-resistant glucose phosphate mutase gene fragment; 2 × PrimeSTAR MAX DNA Polymerase (Dalianbao bio, China), 25 μ L, and water to make up 50 μ L. The POE-PCR condition is that the denaturation is carried out for 2min at 98 ℃, and the cycle is carried out for 30 times according to the following parameters: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 3.5min, and final extension at 72 ℃ for 5 min. The ligation product was transformed into competent E.coli Top10 by calcium chloride method, transformants were selected for colony PCR and double enzyme digestion identification, 2-3 positive transformants were selected for further validation by sequencing, the sequencing result showed successful acquisition of pMA5-Pylb-PGM recombinant co-expression vector, the plasmid map is shown in FIG. 2B.
(3) Construction of pMA5-Pylb-IPS
In this example, the thermostable inositol-3-phosphate synthase is from Archaeoglobus fulgidus, KEGG accession number is AF 1794; archaeoglobus fulgidus is purchased from China general microbiological culture Collection center (CGMCC). The gene ips encoding thermostable inositol-3-phosphate synthase is obtained by PCR from genomic DNA using a pair of primers. Using primers 323-F: 5'-AGAAACAACAAAGGGGGAGATTTGTatgaaagtttggctggttggtgcct-3' and 324-R: 5'-agcttgagctcgactctagaggatccTTAtttcaggttgctataccattct-3', respectively; the pMA5-Pylb linear scaffold was obtained by PCR using a pair of primers. Primers 325-F were used: 5'-agaatggtatagcaacctgaaaTAAggatcctctagagtcgagctcaagct-3' and 326-R: 5'-aggcaccaaccagccaaactttcatACAAATCTCCCCCTTTGTTGTTTCT-3', respectively;
all primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc. The PCR conditions of the gene are 94 ℃ denaturation for 5min, and the cycle is 30 times according to the following parameters: denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, and final extension at 72 ℃ for 10 min. The products obtained from the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After confirming that the size of the fragment is correct by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., Ltd., China) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable myo-inositol-3-phosphate synthase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR. The POE-PCR system is as follows: purified pMA5-Pylb linear skeleton, 200 ng; 131ng of purified heat-resistant inositol-3-phosphate synthetase gene fragment; 2 × PrimeSTAR MAX DNApolymerase (Dalianbao bio, China), 25 μ L, and water to make up to 50 μ L. The POE-PCR condition is that the denaturation is carried out for 2min at 98 ℃, and the cycle is carried out for 30 times according to the following parameters: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 3.5min, and final extension at 72 ℃ for 5 min. The ligation product is transformed into competent E.coli Top10 by calcium chloride method, transformants are selected for colony PCR and double enzyme digestion identification, 2-3 positive transformants are selected for further verification by sequencing, the sequencing result shows that the pMA5-Pylb-IPS recombinant co-expression vector is successfully obtained, and the plasmid map is shown in figure 2C.
(4) Construction of pMA5-Pylb-IMP
Thermotolerant inositol monophosphatase in this example is from Thermotoga maritima, KEGG accession No. TM 1415; thermotoga maritima is purchased from China general microbiological culture Collection center (CGMCC). The thermotolerant myo-inositol monophosphorylase encoding gene ips is obtained by PCR from genomic DNA using a pair of primers. Using primers 311-F: 5'-AGAAACAACAAAGGGGGAGATTTGTatgctggatcgcctggatttctcta-3' and 312-R: 5'-gcttgagctcgactctagaggatccTCAtttaccgccgatttcttcaaca-3', respectively; the pMA5-Pylb linear scaffold was obtained by PCR using a pair of primers. Using a primer; 313-F: 5'-tgttgaagaaatcggcggtaaaTGAggatcctctagagtcgagctcaagc-3' and 314-R: 5'-tagagaaatccaggcgatccagcatACAAATCTCCCCCTTTGTTGTTTCT-3' are provided.
All primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc. The PCR conditions of the gene are 94 ℃ denaturation for 5min, and the cycle is 30 times according to the following parameters: denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, and final extension at 72 ℃ for 10 min. The products obtained from the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After confirming that the size of the fragment is correct by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., Ltd., China) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable myo-inositol monophosphatase gene fragment and the pMA5-Pylb vector backbone were then assembled using POE-PCR. The POE-PCR system is as follows: purified pMA5-Pylb linear skeleton, 200 ng; 131ng of purified heat-resistant inositol monophosphatase gene fragment; 2 × PrimeSTAR MAX DNA Polymerase (Dalianbao bio, China), 25 μ L, and water to make up 50 μ L. The POE-PCR condition is that the denaturation is carried out for 2min at 98 ℃, and the cycle is carried out for 30 times according to the following parameters: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 3.5min, and final extension at 72 ℃ for 5 min. The ligation product was transformed into competent E.coli Top10 by calcium chloride method, transformants were selected for colony PCR and double enzyme digestion identification, 2-3 positive transformants were selected for further validation by sequencing, the sequencing result showed successful acquisition of pMA5-Pylb-IMP recombinant co-expression vector, the plasmid map is shown in FIG. 2D.
(5) Construction of pMA5-Pylb-aGP-PGM-IPS-IMP
In the embodiment, the heat-resistant alpha-glucan phosphorylase is from Thermotoga maritima, and the KEGG has the accession number of TM 1168; the heat-resistant glucose phosphate mutase is from Thermococcus kodakarensis, and the accession number of KEGG is TK 1108; the thermotolerant inositol-3-phosphate synthase is from Archaeoglobus fulgidus, KEGG accession AF 1794; and thermotolerant myo-inositol monophosphatase from Thermotoga maritima, KEGG accession No. TM 1415; thermococcus kodakarensis, Archaeoglobus fulgidus and Thermotoga maritima are all purchased from China general microbiological culture Collection center (CGMCC). Thermostable a-glucan phosphorylase; the thermostable a-glucan phosphorylase encoding gene agp was obtained by PCR using primers 330-F: 5'-AGAAACAACAAAGGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3' and 331-R: 5'-cgaaggtaccaaacagtttgcccattcagtcaagtcccttccacttgacc-3', respectively; thermostable a-glucan phosphorylase; the thermostable phosphoglucomutase-encoding gene pgm was obtained by PCR using primers 332-F: 5'-ggtcaagtggaagggacttgactgaatgggcaaactgtttggtaccttcg-3' and 333-R: 5'-aggcaccaaccagccaaactttcatTTAacctttcagtgcttcttccagc-3', respectively; the gene ips encoding thermostable inositol-3-phosphate synthase was obtained by PCR using primers 334-F: 5'-gctggaagaagcactgaaaggtTAAatgaaagtttggctggttggtgcct-3' and 335-R: 5'-tagagaaatccaggcgatccagcatTTAtttcaggttgctataccattct-3', respectively; the thermotolerant myo-inositol monophosphorylase encoding gene imp was obtained by PCR using primers 335-F: 5'-agaatggtatagcaacctgaaaTAAatgctggatcgcctggatttctcta-3' and 336-R: 5'-gcttgagctcgactctagaggatccTCAtttaccgccgatttcttcaaca-3', respectively; the pMA5-Pylb linear scaffold was obtained by PCR using primers; 337-F: 5'-tgttgaagaaatcggcggtaaaTGAggatcctctagagtcgagctcaagc-3' and 338-R: 5'-caacggcattggaaacgttcaccatACAAATCTCCCCCTTTGTTGTTTCT-3' are provided.
All primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc. The PCR conditions of the gene are 94 ℃ denaturation for 5min, and the cycle is 30 times according to the following parameters: denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, and final extension at 72 ℃ for 10 min. The products obtained from the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After confirming that the size of the fragment is correct by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., Ltd., China) is adopted to recover the target fragment for constructing the recombinant expression vector.
Then, using POE-PCR to assemble a heat-resistant alpha-glucan phosphorylase gene fragment, a heat-resistant glucose phosphate mutase gene fragment, a heat-resistant inositol-3-phosphate synthase gene fragment, a heat-resistant inositol monophosphorylate gene fragment and a pMA5-Pylb vector skeleton. The POE-PCR system is as follows: purified pMA5-Pylb linear skeleton, 200 ng; 131ng of purified heat-resistant alpha-glucan phosphorylase gene fragment, 131ng of heat-resistant glucose phosphate mutase gene fragment, 131ng of heat-resistant inositol-3-phosphate synthase gene fragment and 131ng of heat-resistant inositol monophosphorylate gene fragment; 2 × PrimeSTAR MAX DNA Polymerase (Dalianbao bio, China), 25 μ L, and water to make up 50 μ L. The POE-PCR condition is that the denaturation is carried out for 2min at 98 ℃, and the cycle is carried out for 30 times according to the following parameters: denaturation at 98 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 3.5min, and final extension at 72 ℃ for 5 min. The ligation product was transformed into competent E.coli Top10 by calcium chloride method, transformants were selected for colony PCR and double enzyme digestion identification, 2-3 positive transformants were selected for further validation by sequencing, the sequencing result showed successful acquisition of pMA5-Pylb-aGP-PGM-IPS-IMP recombinant co-expression vector, and the plasmid map is shown in FIG. 2E.
Example 3 construction of recombinant engineering bacteria
The constructed recombinant expression vectors pMA5-Pylb-aGP, pMA5-Pylb-PGM, pMA5-Pylb-IPS, pMA5-Pylb-IMP and pMA5-Pylb-aGP-PGM-IPS-IMP are respectively transformed into Bacillus subtilis host bacteria SCK6/amyE by a calcium chloride method, LB test tube culture is carried out overnight, a plasmid is extracted by a plasmid extraction kit, and correct clones SCK6/amyE/pMA5-Pylb-aGP, SCK6/amyE/pMA5-Pylb-PGM, SCK6/amyE/pMA5-Pylb-IPS, SCK6/amyE/pMA5-Pylb-IMP and SCK6/amyE/pMA 5-PyP-IMP are preserved.
EXAMPLE 4 preparation of Whole cells of recombinant engineered bacteria
The recombinant engineering bacteria SCK6/amyE/pMA5-Pylb-aGP, SCK6/amyE/pMA5-Pylb-PGM, SCK6/amyE/pMA5-Pylb-IPS, SCK6/amyE/pMA5-Pylb-IMP and SCK6/amyE/pMA5-Pylb-aGP-PGM-IPS-IMP are respectively picked and inoculated in an LB culture medium containing spectinomycin, and are shaken at 37 ℃ for overnight culture. Transferring the culture into a fresh LB culture medium containing spectinomycin with the inoculation amount of 1%, shaking and culturing at 37 ℃ overnight, centrifuging at 5500rpm for 10min, and discarding the supernatant to obtain whole cells expressing heat-resistant alpha-glucan phosphorylase, whole cells expressing heat-resistant glucose phosphoglucomutase, whole cells expressing heat-resistant inositol-3-phosphate synthase, whole cells expressing heat-resistant inositol monophosphorylate and whole cells co-expressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphoglucomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylate.
EXAMPLE 5 Whole-cell catalysis of starch to inositol
The whole cells co-expressing thermostable α -glucan phosphorylase, thermostable phosphoglucomutase, thermostable myo-inositol 3-phosphate synthase and thermostable myo-inositol monophosphorylase prepared in example 4 were washed 1 time with 50mM Tris-HCl buffer (pH 7.5), centrifuged at 5500rpm for 10min, the supernatant was discarded, 50mM Tris-HCl buffer (pH 7.5) was added to the pellet, and the cells were resuspended to OD600About 150. The resuspended cells were heat treated at 75 ℃ for 90 min.
In a 1L reaction system, starch at a final concentration of 200g/L, 50mM sodium phosphate buffer (pH 7.0), and heat-treated whole cells were added to make OD600About 20. Shaking table reaction at 55 deg.C for 63h, sampling, and analyzing by High Performance Liquid Chromatography (HPLC). The HPLC detection conditions were as follows: the chromatographic column is Bio-Rad HPX-87H; the flow rate is 0.6 mL/min; the column temperature is 60 ℃; the detector is a differential refraction detector; the amount of sample was 20. mu.L.
FIG. 3 shows a graph of the production of inositol as a function of reaction time, showing that the yield of inositol can reach 70%.
Example 6 Whole-cell catalysis of starch to inositol
The whole cells expressing thermostable α -glucan phosphorylase, the whole cells expressing thermostable phosphoglucomutase, the whole cells expressing thermostable myo-inositol-3-phosphate synthase, and the whole cells expressing thermostable myo-inositol monophosphorylase prepared in example 4 were washed with 0.9% NaCl for 1 time, centrifuged at 5500rpm for 10min, the supernatant was discarded, and 50mM sodium phosphate was added to the precipitateBuffer (pH 7.0), resuspend the cells to OD600About 150. The resuspended cells were heat treated at 75 ℃ for 90 min.
In a 1L reaction system, starch at a final concentration of 200g/L, 50mM sodium phosphate buffer (pH 7.0), and the above four heat-treated whole cells were added to make OD600Approximately 20, the ratio of whole cells expressing thermostable α -glucan phosphorylase, whole cells expressing thermostable phosphoglucomutase, whole cells expressing thermostable inositol-3-phosphate synthase, and whole cells expressing thermostable inositol monophosphorylase was 1:1:1: 1. Shaking table reaction at 55 deg.C water bath for 63h, sampling and HPLC analysis. HPLC detection conditions were the same as in example 5. The results show that the inositol yield can reach 73%.
Claims (11)
1. A method for preparing high-concentration inositol by catalyzing high-concentration starch through whole cells of bacillus subtilis, which is characterized by comprising the following steps:
(1) constructing bacillus subtilis host bacteria for knocking out alpha-amylase;
(2) constructing engineering bacteria for co-expressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphate mutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylase and/or engineering bacteria of bacillus subtilis for respectively expressing the heat-resistant alpha-glucan phosphorylase, the heat-resistant glucose phosphate mutase, the heat-resistant inositol-3-phosphate synthase and the heat-resistant inositol monophosphorylase on the basis of the host bacteria constructed in the step (1);
(3) fermenting the bacillus subtilis engineering bacteria constructed in the step (2) to obtain whole cells;
(4) performing cell membrane permeability treatment on the bacillus subtilis whole cell obtained in the step (3) to obtain a permeable whole cell;
(5) utilizing the permeable whole cells co-expressing the thermotolerant α -glucan phosphorylase, the thermotolerant glucose phosphate mutase, the thermotolerant inositol-3-phosphate synthase and the thermotolerant inositol monophosphorylase obtained in step (4), the permeable whole cells expressing the thermotolerant α -glucan phosphorylase, the permeable whole cells expressing the thermotolerant glucose phosphate mutase, a mixture of the permeable whole cells expressing the thermotolerant inositol-3-phosphate synthase and the permeable whole cells expressing the thermotolerant inositol monophosphorylate, the permeable whole cells co-expressing the thermotolerant α -glucan phosphorylase, the thermotolerant inositol-3-phosphate synthase and the thermotolerant inositol monophosphorylate, the permeable whole cells expressing the thermotolerant glucose phosphate mutase, the permeable whole cells expressing the thermotole, The mixture of permeable whole cells expressing thermostable myo-inositol-3-phosphate synthase and permeable whole cells expressing thermostable myo-inositol monophosphorylase catalyzes the production of high-concentration myo-inositol from high-concentration starch.
2. The method of claim 1, wherein the engineered bacteria comprise a vector co-expressing a thermotolerant α -glucan phosphorylase, a thermotolerant phosphoglucomutase, a thermotolerant inositol-3-phosphate synthase, and a thermotolerant inositol monophosphorylase; or a vector expressing a thermotolerant alpha-glucan phosphorylase, a vector expressing a thermotolerant phosphoglucomutase, a vector expressing a thermotolerant inositol-3-phosphate synthase, or a vector expressing a thermotolerant inositol monophosphorylase.
Preferably, the thermostable α -glucan phosphorylase is an enzyme having a function of converting starch into glucose-1-phosphate (G1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermostable a-glucan phosphorylase is derived from a thermophilic microorganism or the amino acid sequence of the thermostable a-glucan phosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the thermostable a-glucan phosphorylase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis.
Preferably, the thermostable phosphoglucomutase is an enzyme having a function of converting glucose-1-phosphate (G1P) into glucose-6-phosphate (G6P) as another intermediate at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermotolerant phosphoglucomutase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant phosphoglucomutase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant phosphoglucomutase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis.
Preferably, the thermostable inositol-3-phosphate synthase is an enzyme having a function of isomerizing glucose-6-phosphate (G6P) to inositol-1-phosphate (I1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, said thermotolerant inositol-3-phosphate synthase is derived from a thermophilic microorganism or said thermotolerant inositol-3-phosphate synthase has an amino acid sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant inositol-3-phosphate synthase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis, Archaeoglobus fulgidus.
Preferably, the thermostable myo-Inositol monophosphorylase is an enzyme having a function of removing phosphate groups from Inositol-1-phosphate (I1P) to produce myo-Inositol (Inositol) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermotolerant myo-inositol monophosphorylase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant myo-inositol monophosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the thermotolerant myo-inositol monophosphorylase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis, Archaeoglobus fulgidus.
Preferably, the co-expressed vector comprises a promoter, a thermostable α -glucan phosphorylase gene, a thermostable phosphoglucomutase gene, a thermostable myo-inositol-3-phosphate synthase gene, a thermostable myo-inositol monophosphorylate gene, and a terminator; the vector for expressing the heat-resistant alpha-glucan phosphorylase comprises a promoter, a heat-resistant alpha-glucan phosphorylase gene and a terminator; the vector for expressing the heat-resistant glucose phosphoglucomutase comprises a promoter, a heat-resistant glucose phosphoglucomutase gene and a terminator; the vector for expressing the heat-resistant inositol-3-phosphate synthetase comprises a promoter, a heat-resistant inositol-3-phosphate synthetase gene and a terminator; the vector for expressing the heat-resistant inositol monophosphorylase comprises a promoter, a heat-resistant inositol monophosphorylase gene and a terminator.
Preferably, the promoter includes, but is not limited to, the P43 promoter, the Pylb promoter, the Pamyl promoter, the plats promoter, the PhpaII promoter, the PamyE promoter, the Pgrac promoter, and the like. More preferably, the promoter of the invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
3. The method according to claim 1 or 2, wherein the cell membrane permeability treatment comprises but is not limited to heat treatment, addition of organic solvents and/or addition of surfactants.
Preferably, the heat treatment temperature is 45-95 ℃; more preferably, the heat treatment temperature is 50 to 80 ℃.
Preferably, the heat treatment time is 1-100 min; more preferably, the heat treatment time is 10-60 min.
Preferably, the cell concentration at the time of the heat treatment is OD60010-300; more preferably, the cell concentration is OD600＝30-200。
Preferably, the heat treatment can be carried out in a buffer-free system or a buffer system; more preferably, the heat treatment is performed in a buffer system, and the buffer may be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, or the like. Among them, phosphate buffer solutions such as sodium phosphate buffer solution, potassium phosphate buffer solution, and the like.
Preferably, the organic solvent includes, but is not limited to, acetone, acetonitrile, and the like.
Preferably, the surfactant includes, but is not limited to, cetyltrimethylammonium bromide (CTAB), Tween-80, and the like.
4. The method according to any one of claims 1 to 3, wherein in the reaction system for preparing the inositol by catalyzing starch with permeable whole cells or a mixture of permeable whole cells, the concentration of the fed substrate starch is 10 to 300 g/L; more preferably, the concentration of the substrate starch fed is 20-200 g/L.
Preferably, the ratio of permeable whole cells expressing thermostable α -glucan phosphorylase, permeable whole cells expressing thermostable glucose phosphate mutase, permeable whole cells expressing thermostable myo-inositol-3-phosphate synthase, permeable whole cells expressing thermostable myo-inositol monophosphorylase in the mixture of permeable whole cells is 0.1:1:1:0.1-10:1:1: 10; more preferably 0.5:1:1:0.5 to 5:1:1: 5.
5. The process according to any one of claims 1 to 5, characterized in that the conditions of the catalytic reaction are: reacting at pH 6.0-8.0 and 50-80 deg.C for 0.5-96 hr; more preferably at a pH of 6.5-7.5 at 55-75 deg.C for 12-96 h.
Preferably, the catalytic reaction can be carried out in a buffer-free system or a buffer system; preferably in a buffer system, which may be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer, etc. Among them, phosphate buffer solutions such as sodium phosphate buffer solution, potassium phosphate buffer solution, and the like.
6. A vector co-expressing thermostable alpha-glucan phosphorylase, thermostable phosphoglucomutase, thermostable inositol-3-phosphate synthase and thermostable inositol monophosphorylase.
Preferably, the thermostable α -glucan phosphorylase is an enzyme having a function of converting starch into glucose-1-phosphate (G1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermostable a-glucan phosphorylase is derived from a thermophilic microorganism or the amino acid sequence of the thermostable a-glucan phosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the thermostable a-glucan phosphorylase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis.
Preferably, the thermostable phosphoglucomutase is an enzyme having a function of converting glucose-1-phosphate (G1P) into glucose-6-phosphate (G6P) as another intermediate at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermotolerant phosphoglucomutase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant phosphoglucomutase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant phosphoglucomutase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis.
Preferably, the thermostable inositol-3-phosphate synthase is an enzyme having a function of isomerizing glucose-6-phosphate (G6P) to inositol-1-phosphate (I1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, said thermotolerant inositol-3-phosphate synthase is derived from a thermophilic microorganism or said thermotolerant inositol-3-phosphate synthase has an amino acid sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant inositol-3-phosphate synthase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis, Archaeoglobus fulgidus.
Preferably, the thermostable myo-Inositol monophosphorylase is an enzyme having a function of removing phosphate groups from Inositol-1-phosphate (I1P) to produce myo-Inositol (Inositol) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermotolerant myo-inositol monophosphorylase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant myo-inositol monophosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the thermotolerant myo-inositol monophosphorylase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis, Archaeoglobus fulgidus.
Preferably, the co-expressed vector comprises a promoter, a thermostable α -glucan phosphorylase gene, a thermostable phosphoglucomutase gene, a thermostable myo-inositol-3-phosphate synthase gene, a thermostable myo-inositol monophosphorylate gene, and a terminator;
preferably, the promoter includes, but is not limited to, the P43 promoter, the Pylb promoter, the Pamyl promoter, the plats promoter, the PhpaII promoter, the PamyE promoter, the Pgrac promoter, and the like. More preferably, the promoter of the invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
7. A vector for expressing thermostable a-glucan phosphorylase.
Preferably, the thermostable α -glucan phosphorylase is an enzyme having a function of converting starch into glucose-1-phosphate (G1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermostable a-glucan phosphorylase is derived from a thermophilic microorganism or the amino acid sequence of the thermostable a-glucan phosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the thermostable a-glucan phosphorylase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis.
Preferably, the vector for expressing the thermostable α -glucan phosphorylase comprises a promoter, a thermostable α -glucan phosphorylase gene, and a terminator.
Preferably, the promoter includes, but is not limited to, the P43 promoter, the Pylb promoter, the Pamyl promoter, the plats promoter, the PhpaII promoter, the PamyE promoter, the Pgrac promoter, and the like. More preferably, the promoter of the invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
8. A vector for expressing a thermostable phosphoglucomutase.
Preferably, the thermostable phosphoglucomutase is an enzyme having a function of converting glucose-1-phosphate (G1P) into glucose-6-phosphate (G6P) as another intermediate at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermotolerant phosphoglucomutase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant phosphoglucomutase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant phosphoglucomutase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis.
Preferably, the vector for expressing the thermostable phosphoglucomutase comprises a promoter, a thermostable phosphoglucomutase gene and a terminator.
Preferably, the promoter includes, but is not limited to, the P43 promoter, the Pylb promoter, the Pamyl promoter, the plats promoter, the PhpaII promoter, the PamyE promoter, the Pgrac promoter, and the like. More preferably, the promoter of the invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
9. A vector for expressing a thermotolerant inositol-3-phosphate synthase.
Preferably, the thermostable inositol-3-phosphate synthase is an enzyme having a function of isomerizing glucose-6-phosphate (G6P) to inositol-1-phosphate (I1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, said thermotolerant inositol-3-phosphate synthase is derived from a thermophilic microorganism or said thermotolerant inositol-3-phosphate synthase has an amino acid sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant inositol-3-phosphate synthase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis, Archaeoglobus fulgidus.
Preferably, the vector for expressing a thermotolerant inositol-3-phosphate synthase comprises a promoter, a thermotolerant inositol-3-phosphate synthase gene, and a terminator.
Preferably, the promoter includes, but is not limited to, the P43 promoter, the Pylb promoter, the Pamyl promoter, the plats promoter, the PhpaII promoter, the PamyE promoter, the Pgrac promoter, and the like. More preferably, the promoter of the invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
10. A vector for expressing thermotolerant myo-inositol monophosphorylase.
Preferably, the thermostable myo-Inositol monophosphorylase is an enzyme having a function of removing phosphate groups from Inositol-1-phosphate (I1P) to produce myo-Inositol (Inositol) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher. Further preferably, the thermotolerant myo-inositol monophosphorylase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant myo-inositol monophosphorylase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the thermotolerant myo-inositol monophosphorylase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermomarum, Thermococcus kodakarensis, Archaeoglobus fulgidus.
Preferably, the vector for expressing the thermotolerant myo-inositol monophosphorylase comprises a promoter, a thermotolerant myo-inositol monophosphorylase gene and a terminator.
Preferably, the promoter includes, but is not limited to, the P43 promoter, the Pylb promoter, the Pamyl promoter, the plats promoter, the PhpaII promoter, the PamyE promoter, the Pgrac promoter, and the like. More preferably, the promoter of the invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
11. Engineering bacteria for coexpressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphate mutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylase, engineering bacteria for expressing heat-resistant alpha-glucan phosphorylase, engineering bacteria for expressing heat-resistant glucose phosphate mutase, engineering bacteria for expressing heat-resistant inositol-3-phosphate synthase and engineering bacteria for expressing heat-resistant inositol monophosphorylase.
Preferably, the engineered bacterium comprises the vector of claim 6, 7, 8, 9 or 10.
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Inventor after: Zhang Yiheng Inventor after: You Chun Inventor after: Shi Ting Inventor before: You Chun Inventor before: Shi Ting |