阅读说明：本技术 一种鲨肌醇的制备方法 (Preparation method of scyllo-inositol ) 是由 游淳 李元 刘珊 于 2018-08-24 设计创作，主要内容包括：本发明公开了一种利用微生物全细胞和/或其裂解液催化肌肉肌醇制备鲨肌醇的方法,包括构建表达耐热肌肉肌醇脱氢酶和/或耐热鲨肌醇脱氢酶基因的工程菌,并将工程菌进行细胞膜通透性处理和/或破碎,然后利用透性工程菌或其裂解液将肌肉肌醇转化为鲨肌醇。本发明的方法与现有生产鲨肌醇的方法相比,具有生产工艺简单、生产成本低、产率高等优点。(The invention discloses a method for preparing scyllo-inositol by catalyzing myoinositol with whole cells of microorganisms and/or lysate thereof, which comprises the steps of constructing engineering bacteria for expressing thermotolerant myoinositol dehydrogenase and/or thermotolerant scyllo-inositol dehydrogenase genes, carrying out cell membrane permeability treatment and/or disruption on the engineering bacteria, and then converting the myoinositol into the scyllo-inositol by utilizing the permeable engineering bacteria or the lysate thereof. Compared with the existing method for producing scyllo-inositol, the method of the invention has the advantages of simple production process, low production cost, high yield and the like.)

1. A method for preparing scyllo-inositol, comprising the steps of:

(1) constructing engineering bacteria for co-expressing thermotolerant myo-inositol dehydrogenase and thermotolerant scyllo-inositol dehydrogenase and/or engineering bacteria for respectively expressing the thermotolerant myo-inositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase;

(2) fermenting the engineering bacteria constructed in the step (1) to obtain whole cells;

(3) performing cell membrane permeability treatment and/or disruption on the whole cells obtained in the step (2) to obtain permeable whole cells and/or lysate;

(4) utilizing the whole cell obtained in step (3) to co-express a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a mixture of whole cells expressing a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a lysate of whole cells co-expressing a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a mixture of a lysate of whole cells expressing a thermomycent myo-inositol dehydrogenase and a lysate of whole cells expressing a thermovent scyllo-inositol dehydrogenase, and/or a mixed lysate of whole cells co-expressing a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a lysate of whole cells expressing a thermomycent myo-inositol dehydrogenase and a lysate of whole cells expressing a thermovent scyllo-inositol dehydrogenase The compounds catalyze myo-inositol to produce scyllo-inositol.

2. The method of claim 1, wherein the engineered bacterium comprises a vector co-expressing a thermotolerant myo-inositol dehydrogenase and a thermotolerant scyllo-inositol dehydrogenase, or comprises both a vector expressing a thermotolerant myo-inositol dehydrogenase and a vector expressing a thermotolerant scyllo-inositol dehydrogenase, or comprises a vector expressing a thermotolerant myo-inositol dehydrogenase, or comprises a vector expressing a thermotolerant scyllo-inositol dehydrogenase.

Preferably, the thermotolerant myo-inositol dehydrogenase is an enzyme having a function of oxidizing myo-inositol to myo-inositol monoketone 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 dehydrogenase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant myo-inositol dehydrogenase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant myo-inositol dehydrogenase 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.

Preferably, the thermostable scyllo-inositol dehydrogenase is an enzyme having a function of reducing myoinositol monoketone to scyllo-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 thermostable scyllo-inositol dehydrogenase is derived from a thermophilic microorganism or the amino acid sequence of the thermostable scyllo-inositol dehydrogenase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermostable scyllo-inositol dehydrogenase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Geobacillus thermophiliovorans, Thermotoga maritima (Thermotoga maritima).

Preferably, the vector co-expressing the thermotolerant myoinositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase comprises a first promoter, a thermotolerant myoinositol dehydrogenase gene, a first terminator, a second promoter, a thermotolerant scyllo-inositol dehydrogenase gene and a second terminator, or comprises a first promoter, a thermotolerant myoinositol dehydrogenase gene, a first terminator, a second promoter, a thermotolerant myoinositol dehydrogenase gene and a second terminator, or comprises a promoter, a thermotolerant scyllo-inositol dehydrogenase gene, a thermotolerant myoinositol dehydrogenase gene and a terminator, or comprises a promoter, a thermotolerant myoinositol dehydrogenase gene, a thermotolerant scyllo-inositol dehydrogenase gene and a terminator; the vector for expressing the heat-resistant myoinositol dehydrogenase comprises a promoter, a heat-resistant myoinositol dehydrogenase gene and a terminator; the carrier for expressing the heat-resistant scyllo-inositol dehydrogenase comprises a promoter, a heat-resistant scyllo-inositol dehydrogenase gene and a terminator.

Preferably, the promoter, the first promoter and/or the second promoter include, but are not limited to, T7 promoter, lac promoter, tac promoter, trc promoter, PR promoter, and the like. Most preferably, the promoter, the first promoter, the second promoter are independently from each other the T7 promoter.

Preferably, the terminator, first terminator and/or second terminator include, but are not limited to, the T7 terminator, the rrnB T1 terminator, the rrnB T2 terminator and the like. Most preferably, the terminator, the first terminator, the second terminator are independently from each other the T7 terminator.

Preferably, the vector is pET series, pGEX series, pACYCDuet-1, pRSFDuet-1, etc., most preferably pETDuet-1 or pET-29a (+).

Preferably, the host bacteria for preparing the engineering bacteria are escherichia coli, pichia pastoris, saccharomyces cerevisiae, bacillus subtilis and the like, and most preferably escherichia coli BL21(DE 3).

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 ℃; further preferably, the heat treatment temperature is 55-90 ℃; more preferably, the heat treatment temperature is 60-80 ℃; most preferably, the heat treatment temperature is 70 ℃.

Preferably, the heat treatment time is 1-40 min; further preferably, the heat treatment time is 5-30 min; more preferably, the heat treatment time is 10-25 min; most preferably, the heat treatment time is 20 min.

Preferably, the cell concentration at the time of the heat treatment is OD₆₀₀10-300; further preferably, the cell concentration is OD₆₀₀20-200; more preferably, the cell concentration is OD₆₀₀50-150; most preferably, the cell concentration is OD₆₀₀＝100。

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.

4. The method of any one of claims 1 to 3, wherein the substrate myo-inositol is present in a concentration of 1 to 400g/L in the reaction system for producing scyllo-inositol by catalyzing myo-inositol with permeable whole cells or a mixture of permeable whole cells, and the amount of permeable whole cells or a mixture of permeable whole cells added is OD₆₀₀1-100; more preferably, the concentration of myoinositol as substrate is 100-350g/L and the amount of permeable whole cells or mixture of permeable whole cells added is OD₆₀₀10-50; most preferably, the substrate myoinositol is present at a concentration of 250g/L and the permeable whole cells or mixture of permeable whole cells are added in an amount OD₆₀₀＝20-40。

Preferably, the ratio of permeable whole cells expressing a thermotolerant myo-inositol dehydrogenase to permeable whole cells expressing a thermotolerant scyllo-inositol dehydrogenase in the mixture of permeable whole cells is 0.1:1 to 10: 1; more preferably from 0.5:1 to 5: 1; most preferably 1: 1.

5. The method of any one of claims 1 to 3, wherein the substrate myo-inositol is present in a concentration of 1 to 400g/L in the reaction system for producing scyllo-inositol by catalyzing myo-inositol with a cell lysate or a mixture of cell lysates using whole cell OD₆₀₀The adding amount of the cracking solution is 5 to 95 percent of the total volume of the reaction solution when the reaction solution is 10 to 100 percent; more preferably, the concentration of myoinositol as substrate is 100-350g/L and the cell lysate or mixture of cell lysates is the whole cell OD₆₀₀The adding amount of the cracking solution is 50 to 90 percent of the total volume of the reaction solution when the reaction solution is 40 to 150 hours; most preferably, the concentration of myoinositol as substrate is 250g/L and the cell lysate or mixture of cell lysates is whole cell OD₆₀₀The amount of the lysate added was 90% of the total volume of the reaction solution, when the amount of the lysate was 100%.

Preferably, the ratio of whole cells used in whole cell lysates expressing a thermotolerant myo-inositol dehydrogenase to whole cells used in whole cell lysates expressing a thermotolerant scyllo-inositol dehydrogenase in the mixture of whole cell lysates is 0.1:1-10: 1; more preferably from 0.5:1 to 5: 1; most preferably 1: 1.

6. 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 0.5-72 hr; more preferably at pH 6.5-7.5, at 55-75 deg.C for 3-48 h; most preferably at pH7.0, 60-65 ℃ for 6-24 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.

7. A vector for co-expressing a thermotolerant myo-inositol dehydrogenase and a thermotolerant scyllo-inositol dehydrogenase.

Preferably, the thermotolerant myo-inositol dehydrogenase is an enzyme having a function of oxidizing myo-inositol to myo-inositol monoketone 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 dehydrogenase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant myo-inositol dehydrogenase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant myo-inositol dehydrogenase 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.

Preferably, the thermostable scyllo-inositol dehydrogenase is an enzyme having a function of reducing myoinositol monoketone to scyllo-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 thermostable scyllo-inositol dehydrogenase is derived from a thermophilic microorganism or the amino acid sequence of the thermostable scyllo-inositol dehydrogenase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermostable scyllo-inositol dehydrogenase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Geobacillus thermophiliovorans, Thermotoga maritima (Thermotoga maritima).

Preferably, the vector co-expressing the thermotolerant myoinositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase comprises a first promoter, a thermotolerant myoinositol dehydrogenase gene, a first terminator, a second promoter, a thermotolerant scyllo-inositol dehydrogenase gene and a second terminator, or comprises a first promoter, a thermotolerant myoinositol dehydrogenase gene, a first terminator, a second promoter, a thermotolerant myoinositol dehydrogenase gene and a second terminator, or comprises a promoter, a thermotolerant scyllo-inositol dehydrogenase gene, a thermotolerant myoinositol dehydrogenase gene and a terminator, or comprises a promoter, a thermotolerant myoinositol dehydrogenase gene, a thermotolerant scyllo-inositol dehydrogenase gene and a terminator.

Preferably, the first promoter and/or the second promoter include, but are not limited to, T7 promoter, lac promoter, tac promoter, trc promoter, PR promoter, and the like. Most preferably, the first promoter and the second promoter are independent of each other T7 promoter.

Preferably, the first terminator and/or the second terminator include, but are not limited to, a T7 terminator, a rrnB T1 terminator, a rrnB T2 terminator, and the like. Most preferably, the first terminator and the second terminator are independent of each other, and are T7 terminators.

Preferably, the vector is pET series, pGEX series, pACYCDuet-1, pRSFDuet-1, etc., most preferably pETDuet-1 or pET-29a (+).

8. A vector for expressing a thermotolerant myo-inositol dehydrogenase.

Preferably, the thermotolerant myo-inositol dehydrogenase is an enzyme having a function of oxidizing myo-inositol to myo-inositol monoketone 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 dehydrogenase is derived from a thermophilic microorganism or the amino acid sequence of the thermotolerant myo-inositol dehydrogenase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermotolerant myo-inositol dehydrogenase 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.

Preferably, the vector for expressing the thermotolerant myo-inositol dehydrogenase includes a promoter, a thermotolerant myo-inositol dehydrogenase gene, and a terminator.

Preferably, the promoter includes, but is not limited to, T7 promoter, lac promoter, tac promoter, trc promoter, PR promoter, and the like. Most preferably, the promoter is the T7 promoter.

Preferably, the terminator includes, but is not limited to, a T7 terminator, a rrnB T1 terminator, a rrnB T2 terminator, and the like. Most preferably, the terminator is the T7 terminator.

Preferably, the vector is pET series, pGEX series, pACYCDuet-1, pRSFDuet-1, etc., most preferably pETDuet-1 or pET-29a (+).

9. A vector for expressing a thermostable scyllo-inositol dehydrogenase.

Preferably, the thermostable scyllo-inositol dehydrogenase is an enzyme having a function of reducing myoinositol monoketone to scyllo-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 thermostable scyllo-inositol dehydrogenase is derived from a thermophilic microorganism or the amino acid sequence of the thermostable scyllo-inositol dehydrogenase is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a thermostable scyllo-inositol dehydrogenase derived from a thermophilic microorganism. More preferably, the thermophilic microorganism is selected from Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Geobacillus thermophiliovorans, Thermotoga maritima (Thermotoga maritima).

Preferably, the vector for expressing the heat-resistant scyllo-inositol dehydrogenase comprises a promoter, a heat-resistant scyllo-inositol dehydrogenase gene and a terminator.

Preferably, the promoter includes, but is not limited to, T7 promoter, lac promoter, tac promoter, trc promoter, PR promoter, and the like. Most preferably, the promoter is the T7 promoter.

Preferably, the terminator includes, but is not limited to, a T7 terminator, a rrnB T1 terminator, a rrnB T2 terminator, and the like. Most preferably, the terminator is the T7 terminator.

Preferably, the vector is pET series, pGEX series, pACYCDuet-1, pRSFDuet-1, etc., most preferably pETDuet-1 or pET-29a (+).

10. An engineering bacterium for co-expressing a thermotolerant myo-inositol dehydrogenase and a thermotolerant scyllo-inositol dehydrogenase, an engineering bacterium for expressing the thermotolerant myo-inositol dehydrogenase or an engineering bacterium for expressing the thermotolerant scyllo-inositol dehydrogenase.

Preferably, the engineered bacterium comprises the vector of claim 7, 8 or 9.

Preferably, the host bacteria for preparing the engineering bacteria are escherichia coli, pichia pastoris, saccharomyces cerevisiae, bacillus subtilis and the like, and most preferably escherichia coli BL21(DE 3).

Technical Field

The invention relates to the technical field of biological engineering, in particular to a preparation method of scyllo-inositol, and specifically relates to a method for converting scyllo-inositol into myo-inositol by catalyzing myo-inositol with whole cells of microorganisms and/or lysis solution thereof.

Background

Scyllo-inositol (SI) is a differential isomer of myo-inositol (MI) (FIG. 1), exists in plants, animals and some bacteria, but its content in nature is much lower than that of myo-inositol.Studies show that scyllo-inositol has amyloid β protein aggregation inhibition, can regulate central nerve signals dependent on myo-inositol, is a potential drug for treating Alzheimer's disease, and has been clinically tested by Transition Therapeutics Ireland Limited, and can improve cognitive level in people with Down syndrome.

In the traditional synthetic method, scyllo-inositol is mainly synthesized by taking myo-inositol as a raw material through complicated steps and adopting a chemical method, but the method needs protection and deprotection of hydroxyl, has complicated steps and is not beneficial to large-scale production. In recent years, with the development of biotechnology, the biological production of scyllo-inositol has become a new trend. It has been reported that a Cell factory of Bacillus subtilis (Bacillus subtilis) is used to produce scyllo-inositol (Yamaoka M, Osawa S, Morinaga T, Takenaka S, Yoshida K-i (2011) A Cell factory of Bacillus subtilis for the simple biochemical conversion of myo-inositol derivative microorganism Cell factory 10; Tanaka K, Tajima S, Takenaka S, Yoshida K-i (2013) enhanced Bacillus subtilis Cell factory for producing bacterial-inositol derivative, Bacillus subtilis Cell factory for producing Bacillus strain-inositol derivative, Bacillus strain culture Cell 331, Bacillus strain culture Cell factory for producing Bacillus strain-inositol derivative, Bacillus strain for producing Bacillus strain, Bacillus strain for producing Cell, Bacillus strain for producing strain, Bacillus strain for strain, Bacillus strain for producing strain, Bacillus strain, Cell strain, Bacillus strain K-strain for producing strain, Cell strain, Bacillus strain, Cell strain, Aspergillus strain, Cell strain, strain. This method requires a complicated metabolic flow control, and the yield of scyllo-inositol depends on the nutrient of the medium, which undoubtedly increases the complexity of the process.

Therefore, it is desired to develop a novel method for producing scyllo-inositol with a simple production process, a high yield and a low cost.

Disclosure of Invention

Aiming at the problems of the existing method for preparing scyllo-inositol, the invention provides a method for producing scyllo-inositol by catalyzing myo-inositol dehydrogenase (EC 1.1.1.18, IDH) and scyllo-inositol dehydrogenase (EC 1.1.1.370, SIDH) by using whole cells expressing the myo-inositol dehydrogenase (EC 1.1.1.18) and the scyllo-inositol dehydrogenase (EC 1.1.1.370, SIDH) and/or a lysate thereof. The method has the advantages of simple production process, low production cost, high yield and the like.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention, the present invention relates to a method for producing scyllo-inositol, comprising the steps of:

(1) constructing engineering bacteria for co-expressing thermotolerant myo-inositol dehydrogenase and thermotolerant scyllo-inositol dehydrogenase and/or engineering bacteria for respectively expressing the thermotolerant myo-inositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase;

(2) fermenting the engineering bacteria constructed in the step (1) to obtain whole cells;

(3) performing cell membrane permeability treatment and/or disruption on the whole cells obtained in the step (2) to obtain permeable whole cells and/or lysate;

(4) utilizing the whole cell obtained in step (3) to co-express a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a mixture of whole cells expressing a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a lysate of whole cells co-expressing a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a mixture of a lysate of whole cells expressing a thermomycent myo-inositol dehydrogenase and a lysate of whole cells expressing a thermovent scyllo-inositol dehydrogenase, and/or a mixed lysate of whole cells co-expressing a thermomycent myo-inositol dehydrogenase and a thermovent scyllo-inositol dehydrogenase, a lysate of whole cells expressing a thermomycent myo-inositol dehydrogenase and a lysate of whole cells expressing a thermovent scyllo-inositol dehydrogenase The compounds catalyze myo-inositol to produce scyllo-inositol.

According to the invention, the catalytic pathway comprises: from thermostable myo-inositol dehydrogenase in NAD⁺Oxidizing a substrate myo-inositol to an intermediate myo-inositol monoketone in the presence of a catalyst; the intermediate myoinositol monoketone is reduced to the product scyllo-inositol by a thermostable scyllo-inositol dehydrogenase in the presence of NADH, as shown in FIG. 2. In bookIn the catalytic pathway of the invention, the desired NAD⁺From the engineered bacterial cells, i.e., the method of the invention does not comprise additional NAD addition⁺The step (2).

Preferably, according to the present invention, in step (1), the engineered bacterium comprises a vector co-expressing a thermotolerant myo-inositol dehydrogenase and a thermotolerant scyllo-inositol dehydrogenase, or comprises both a vector expressing a thermotolerant myo-inositol dehydrogenase and a vector expressing a thermotolerant scyllo-inositol dehydrogenase, or comprises a vector expressing a thermotolerant myo-inositol dehydrogenase, or comprises a vector expressing a thermotolerant scyllo-inositol dehydrogenase. 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 constructing by recombinant DNA techniques to obtain the gene iolG encoding myo-inositol dehydrogenase and the gene iolX encoding scyllo-inositol dehydrogenase, constructing recombinant expression vectors, and transferring into host bacteria to obtain genetically engineered bacteria.

Preferably, according to the present invention, in step (1), "thermostable myo-inositol dehydrogenase" refers to an enzyme having a function of oxidizing myo-inositol to myo-inositol monoketone 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 myoinositol dehydrogenase is derived from a thermophilic microorganism, such as Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Thermotoga maritima (Thermotoga maritima), Pseudothermoga thermophilum, and the like; or the amino acid sequence of said thermotolerant myo-inositol dehydrogenase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity to a thermotolerant myo-inositol dehydrogenase derived from said thermophilic microorganism.

More preferably, the thermotolerant myo-inositol dehydrogenase is derived from Geobacillus kaustophilus; or the amino acid sequence of said thermotolerant myo-inositol dehydrogenase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity to a thermotolerant myo-inositol dehydrogenase derived from Geobacillus kaustophilus.

Preferably, according to the present invention, in step (1), "thermostable scyllo-inositol dehydrogenase" refers to an enzyme having a function of reducing myoinositol monoketone to scyllo-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 thermostable scyllo-inositol dehydrogenase is derived from a thermophilic microorganism, such as Geobacillus thermophilus (Geobacillus kaustophilus), Geobacillus stearothermophilus (Geobacillus stearothermophilus), Geobacillus thermoeovorans, Thermotoga maritima (Thermotogaritia) and the like; or the amino acid sequence of the thermostable scyllo-inositol dehydrogenase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the thermostable scyllo-inositol dehydrogenase derived from the thermophilic microorganism.

More preferably, the thermostable scyllo-inositol dehydrogenase is derived from Geobacillus kaustophilus; or the amino acid sequence of the thermostable scyllo-inositol dehydrogenase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with a thermostable scyllo-inositol dehydrogenase derived from Geobacillus kaustophilus.

Preferably, according to the present invention, the vector co-expressing the thermotolerant myoinositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase includes a first promoter, a thermotolerant myoinositol dehydrogenase gene, a first terminator, a second promoter, a thermotolerant scyllo-inositol dehydrogenase gene, and a second terminator, or includes a first promoter, a thermotolerant myoinositol dehydrogenase gene, a first terminator, a second promoter, a thermotolerant myoinositol dehydrogenase gene, and a second terminator, or includes a promoter, a thermotolerant scyllo-inositol dehydrogenase gene, a thermotolerant myoinositol dehydrogenase gene, a terminator; the vector for expressing the heat-resistant myoinositol dehydrogenase comprises a promoter, a heat-resistant myoinositol dehydrogenase gene and a terminator; the carrier for expressing the heat-resistant scyllo-inositol dehydrogenase comprises a promoter, a heat-resistant scyllo-inositol dehydrogenase gene and a terminator.

It will be appreciated by those skilled in the art that a variety of expression vectors known in the art may be used in the present invention, including, but not limited to, the pET series, pGEX series, pACYCDuet-1, pRSFDuet-1, and the like. Preferably, the vector is pETDuet-1, pET-29a (+).

It will be appreciated by those skilled in the art that a variety of host bacteria known in the art may be used in the present invention, including but not limited to E.coli, Pichia, Saccharomyces cerevisiae, Bacillus subtilis, and the like. Preferably, the host bacterium is escherichia coli BL21(DE 3).

One skilled in the art will appreciate that various promoters known in the art may be used as the promoter, first promoter, and/or second promoter of the present invention, including but not limited to the T7 promoter, lac promoter, tac promoter, trc promoter, PR promoter, and the like. Preferably, the promoter, the first promoter and the second promoter are independent of each other, and are T7 promoters.

One skilled in the art will appreciate that various terminators known in the art can be used as the terminator, first terminator, and/or second terminator of the present invention, including but not limited to the T7 terminator, the rrnB T1 terminator, the rrnB T2 terminator, and the like. Preferably, the terminator, the first terminator and the second terminator are independent of each other a T7 terminator.

According to the present invention, in step (2), 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 (3), 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. Preferably, the cell membrane permeability treatment is a heat treatment. The purpose of the permeability treatment of the cell membrane is to allow the entry of extracellular myoinositol into the cell through the cell membrane.

Preferably, the heat treatment temperature is 45-95 ℃; further preferably, the heat treatment temperature is 55-90 ℃; more preferably, the heat treatment temperature is 60-80 ℃; most preferably, the heat treatment temperature is 70 ℃.

Preferably, the heat treatment time is 1-40 min; further preferably, the heat treatment time is 5-30 min; more preferably, the heat treatment time is 10-25 min; most preferably, the heat treatment time is 20 min.

Preferably, the cell concentration at the time of heat treatment is OD₆₀₀10-300; further preferably, the cell concentration is OD₆₀₀20-200; more preferably, the cell concentration is OD₆₀₀50-150; most preferably, the cell concentration is OD₆₀₀＝100。

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.

According to the present invention, in step (3), the cell disruption is performed using a method known in the art. Cell disruption methods include, but are not limited to, sonication, high pressure homogenization, and the like.

Preferably, in the reaction system for preparing scyllo-inositol by catalyzing myoinositol with permeable whole cells or a mixture of permeable whole cells according to the present invention, in step (4), the concentration of myoinositol as a substrate is 1-400g/L, and the amount of myoinositol added to the permeable whole cells or the mixture of permeable whole cells is OD₆₀₀1-100; more preferably, the concentration of myoinositol as substrate is 100-350g/L and the amount of permeable whole cells or mixture of permeable whole cells added is OD₆₀₀10-50; most preferably, the substrate myoinositol is present at a concentration of 250g/L and the permeable whole cells or mixture of permeable whole cells are added in an amount OD₆₀₀＝20-40。

Preferably, according to the present invention, in step (4), the reaction conditions for catalyzing myoinositol with permeable whole cells or a mixture of permeable whole cells to produce scyllo-inositol are as follows: reacting at pH 6.0-8.0 and 50-80 deg.C for 0.5-72 hr; more preferably at pH 6.5-7.5, at 55-75 deg.C for 3-48 h; most preferably at a pH of 7.0 at 60-65 ℃ for 6-24 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, in the reaction system for preparing scyllo-inositol by catalyzing myoinositol with cell lysate or mixture of cell lysates in step (4), the concentration of myoinositol as substrate is 1-400g/L, and the cell lysate or mixture of cell lysates is whole-cell OD₆₀₀The adding amount of the cracking solution is 5 to 95 percent of the total volume of the reaction solution when the reaction solution is 10 to 100 percent; more preferably, the concentration of myoinositol as substrate is 100-300g/L and the cell lysate or mixture of cell lysates is the whole cell OD₆₀₀The adding amount of the cracking solution is 50 to 90 percent of the total volume of the reaction solution when the reaction solution is 40 to 150 hours; most preferably, the concentration of myoinositol as substrate is 250g/L and the cell lysate or mixture of cell lysates is whole cell OD₆₀₀The amount of the lysate added was 90% of the total volume of the reaction solution, when the amount of the lysate was 100%.

Preferably, according to the present invention, in step (4), the reaction conditions for catalyzing the production of scyllo-inositol from myoinositol using a cell lysate or a mixture of cell lysates are as follows: reacting at pH 6.0-8.0 and 50-80 deg.C for 0.5-72 hr; more preferably at pH 6.5-7.5, at 55-75 deg.C for 3-48 h; most preferably at a pH of 7.0 at 60-65 ℃ for 6-24 h.

According to the invention, the catalytic reaction by using the cell lysate or the mixture of the cell lysate can be carried out in a buffer-free system or a buffer system; preferably, the catalytic reaction is performed in a buffer system by using cell lysate or a mixture of cell lysates, and the buffer can be HEPES buffer, phosphate buffer, Tris buffer, acetate buffer and 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 (4), the ratio of permeable whole cells expressing a thermotolerant myo-inositol dehydrogenase to permeable whole cells expressing a thermotolerant scyllo-inositol dehydrogenase in the mixture of permeable whole cells is 0.1:1 to 10: 1; more preferably from 0.5:1 to 5: 1; most preferably 1: 1; the ratio of whole cells used by the whole cell lysate expressing the thermostable myo-inositol dehydrogenase to whole cells used by the whole cell lysate expressing the thermostable scyllo-inositol dehydrogenase in the mixture of the whole cell lysates is 0.1:1-10: 1; more preferably from 0.5:1 to 5: 1; most preferably 1: 1.

According to the invention, the scyllo-inositol can also be prepared by catalyzing myo-inositol with a mixture of permeable whole cells and cell lysate under the following reaction conditions: reacting at pH 6.0-8.0 and 50-80 deg.C for 0.5-72 hr; more preferably at pH 6.5-7.5, at 55-75 deg.C for 3-48 h; most preferably at a pH of 7.0 at 60-65 ℃ for 6-24 h.

According to another aspect of the present invention, the present invention relates to the above-mentioned vector for co-expressing a thermotolerant myo-inositol dehydrogenase and a thermotolerant scyllo-inositol dehydrogenase, the vector for expressing a thermotolerant myo-inositol dehydrogenase, and the vector for expressing a thermotolerant scyllo-inositol dehydrogenase.

According to another aspect of the invention, the invention relates to the engineering bacteria for co-expressing the thermotolerant myo-inositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase, the engineering bacteria for expressing the thermotolerant myo-inositol dehydrogenase and the engineering bacteria for expressing the thermotolerant scyllo-inositol dehydrogenase. According to the invention, the engineering bacteria for coexpressing the thermotolerant myo-inositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase comprise a carrier for coexpressing the thermotolerant myo-inositol dehydrogenase and the thermotolerant scyllo-inositol dehydrogenase, or comprise a carrier for expressing the thermotolerant myo-inositol dehydrogenase and a carrier for expressing the thermotolerant scyllo-inositol dehydrogenase; the engineering bacteria for expressing the thermotolerant myoinositol dehydrogenase comprise a vector for expressing the thermotolerant myoinositol dehydrogenase; the engineering bacteria for expressing the heat-resistant scyllo-inositol dehydrogenase comprise a carrier for expressing the heat-resistant scyllo-inositol dehydrogenase.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention utilizes the whole cells expressing the heat-resistant myo-inositol dehydrogenase and the heat-resistant scyllo-inositol dehydrogenase and/or the lysate thereof to catalyze myo-inositol to produce scyllo-inositol, and develops a novel method which is simple and easy to prepare scyllo-inositol in a large scale.

(2) The inventionNo need of additional NAD in the reaction system⁺Can realize NAD⁺The self-circulation of NADH is beneficial to reducing the production cost, and has important industrial application value.

(3) The preparation of scyllo-inositol in the method can be carried out at higher temperature, so that the solubility of substrate myo-inositol can be increased.

(4) The invention adopts the whole cells expressing the heat-resistant myo-inositol dehydrogenase and the heat-resistant scyllo-inositol dehydrogenase as the catalyst, and only needs heat treatment to change the permeability of cell membranes, thereby being beneficial to simplifying the production process and improving the safety of products.

(5) The conversion reaction of the scyllo-inositol in the method can be carried out in a buffer-free system or a buffer 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 scyllo-inositol product on the other hand.

Drawings

FIG. 1 shows the chemical structures of scyllo-inositol and myo-inositol.

FIG. 2 is a schematic diagram of the production of scyllo-inositol from myoinositol catalyzed by permeable whole cells and/or lysates according to the invention. MI: myo-inositol; and (3) SI: scyllo-inositol; IDH: myo-inositol dehydrogenase; and (3) SIDH: scyllo-inositol dehydrogenase.

FIGS. 3A-C are maps of recombinant expression vectors pETDuet-iolG, pET29a-iolX, and pETDuet-iolG-iolX, respectively.

FIG. 4 is an HPLC chromatogram of myoinositol and scyllo-inositol.

FIG. 5 shows the preparation of scyllo-inositol from whole cells heat-treated under different conditions.

FIG. 6 shows the preparation of scyllo-inositol at different reaction temperatures.

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.