A Bacillus amyloliquefaciens with high inositol production, its construction method and application

Abstract

The present invention discloses a strain of Bacillus amyloliquefaciens and a recombinant strain constructed using it as the starting strain. The name of the Bacillus amyloliquefaciens is: G2-9, the taxonomic name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC NO. 26351, and the preservation date is: December 27, 2022. On the basis of selecting the specific strain CGMCC NO. 26351 for constructing a high-yield inositol recombinant strain by genetic engineering means, the present invention further improves the inositol yield and stabilizes the fermentation process through fermentation process control technology. Using low-cost sucrose as the substrate, it has lower production costs and extremely high conversion rates. During the fermentation process, the utilization rate of the substrate sucrose is close to 100%, and the highest conversion rate can reach 143.33%. At the same time, the environmental pressure of waste discharge is smaller, making it more suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and metabolic process control, and in particular relates to a Bacillus amyloliquefaciens with high inositol yield, and a construction method and application thereof. Background Art

[0002] Inositol, also known as cyclohexanehexol, is widely distributed in animals and plants, and is a growth factor for animals and microorganisms. Free inositol mainly exists in muscles, heart, lungs and liver, and is a component of phosphatidylinositol, a kind of phospholipid, which has great practical value in the fields of medicine, food, feed, etc. In recent years, with the in-depth research on inositol and the continuous improvement of consumers' demands for healthy diet, the demand for inositol in domestic and foreign markets has been increasing year by year.

[0003] Traditionally, the production of inositol mainly relies on hydrolysis extraction method and chemical synthesis method. In recent years, with the increasing annual demand for inositol, the requirements of the ecological environment, and the demands for production costs, domestic and foreign scientific researchers tend to use the microbial method to solve the production contradiction of inositol.

[0004] With the continuous in-depth research on cell biology in recent decades, the metabolic pathways of inositol in Bacillus amyloliquefaciens, yeast, Escherichia coli, etc. have been basically clear. However, the inositol yield of naturally selected strains is usually low, and it is difficult and has a low success rate to simply construct high-inositol strains by genetic engineering. Therefore, considering both genetic engineering and fermentation metabolism control technology, Bacillus amyloliquefaciens with clear genomic information and mature gene operation tools is transformed, and fermentation metabolism process control means are also used to obtain high-inositol production strains and fermentation technologies.

[0005] Therefore, on the premise of clarifying the inositol metabolic pathway and pathway of the starting strain, recombinant strains are constructed by genetic engineering technology, and at the same time, fermentation metabolism control technology is combined to optimize the fermentation technology for producing inositol, so as to further increase the yield of inositol and solve the industrialization problems of low substrate utilization rate and high production cost for those skilled in the art.

[0006] Through retrieval, no patent publication documents related to the present invention patent application have been found. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Bacillus amyloliquefaciens with high inositol yield, and a construction method and application thereof.

[0008] The technical solution adopted by the present invention to solve its technical problems is:

[0009] A Bacillus amyloliquefaciens strain, named G2-9, with the classification name of Bacillus amyloliquefaciens, deposited under the accession number of CGMCC NO. 26351 on December 27, 2022, at the China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] A recombinant strain constructed using the above-mentioned Bacillus amyloliquefaciens as the starting strain.

[0011] The method for constructing the above-mentioned recombinant strain includes the following steps:

[0012] Using the wild-type Bacillus amyloliquefaciens CGMCC 26351 as the starting strain, gene editing was performed using a homologous recombination system to knockout the key enzyme inositol lyase EC 5.3.99.11 in the inositol degradation pathway, and the pathway affecting inositol synthesis was modified. Sequentially, inositol-3-phosphate synthase EC 5.5.1.4 was inserted, 6-phosphofructokinase EC 2.7.1.11 was replaced, phosphohexose isomerase EC 5.3.1.9 was knocked out, and glucosidase EC 2.7.1.2 was replaced to obtain the recombinant strain;

[0013] Among them, the gene sequence of inositol lyase EC 5.3.99.11 is SEQ ID NO.1, the gene sequences of inositol-3-phosphate synthase EC 5.5.1.4 are SEQ ID NO.2, SEQ ID NO.3 and / or SEQ ID NO.4, the gene sequence of 6-phosphofructokinase EC 2.7.1.11 is SEQ ID NO.5, the gene sequence of phosphohexose isomerase EC 5.3.1.9 is SEQ ID NO.9, and the gene sequence of glucosidase EC 2.7.1.2 is SEQ ID NO.10.

[0014] Furthermore, it specifically includes the following steps:

[0015] Using the homologous recombination system, the inositol-degrading enzyme EC5.3.99.11 of Bacillus amyloliquefaciens was knocked out. The coding genes of inositol-3-phosphate synthase EC5.5.1.4 from Streptomyces dextrose, Bifidobacterium adolescentis, and Rhodococcus were respectively integrated at the inositol-degrading enzyme gene locus; the 6-phosphofructokinase EC2.7.1.11 gene of Bacillus amyloliquefaciens was replaced with the 6-phosphofructokinase genes from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis respectively; the phosphohexose isomerase EC5.3.1.9 gene of Bacillus amyloliquefaciens was knocked out; the glucosidase EC2.7.1.2 gene of Bacillus amyloliquefaciens was replaced with the glucosidase genes from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis respectively.

[0016] Among them, the gene sequence of inositol-degrading enzyme EC5.3.99.11 is SEQ ID NO.1, the gene sequences of inositol-3-phosphate synthase EC5.5.1.4 are SEQ ID NO.2, SEQ ID NO.3, and / or SEQ ID NO.4, the gene sequences of the 6-phosphofructokinase genes from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis are SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 respectively, and the gene sequences of the glucosidase genes from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis are SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 respectively.

[0017] Use of the recombinant strain as described above in the fermentation production of inositol.

[0018] A method for producing inositol using the recombinant strain as described above, the method comprising using sucrose as a substrate and producing inositol by bioconversion with the recombinant strain.

[0019] Furthermore, the method further comprises: adding fermentation supplements during the fermentation of the recombinant strain, the fermentation supplements including fermentation supplement 1 and fermentation supplement 2, by volume:

[0020] Fermentation supplement 1 is: manganese sulfate 0.03 - 0.08 g / L, potassium nitrate 0.001 - 0.005 g / L;

[0021] Fermentation supplement 2 is: pyruvic acid 0.006 - 0.01 g / L, disodium 6-phosphofructose 0.01 - 0.02 g / L, acetic acid 0.005 - 0.01 g / L.

[0022] Furthermore, during the fermentation process for producing inositol by the recombinant strain, the fermentation supplement 1 is added to the fermentation medium at a fermentation time of 6 - 12 h, and the fermentation supplement 2 is added to the fermentation medium at a fermentation time of 24 - 30 h.

[0023] Furthermore, the fermentation supplement 1 is added to the fermentation medium at a fermentation time of 7.5 h, and the fermentation supplement 2 is added to the fermentation medium at a fermentation time of 26 h.

[0024] The advantages and positive effects achieved by the present invention are as follows:

[0025] 1. Based on the selection of a specific strain CGMCC NO.26351 and the use of genetic engineering means to construct a high - inositol - producing recombinant strain, the present invention further improves the inositol yield and stabilizes the fermentation process through fermentation process control technology. Using inexpensive sucrose as a substrate, it has lower production costs and extremely high conversion rates. During the fermentation process, the utilization rate of the substrate sucrose is close to 100%, and the highest conversion rate can reach 143.33%. At the same time, the environmental pressure of waste discharge is smaller, making it more suitable for industrial production. The reason for the extremely high conversion rate is that the recombinant strain preferentially decomposes sucrose into equimolar amounts of glucose and fructose, further converts all glucose and part of fructose into inositol, and part of fructose is used for the growth and metabolism of the cells.

[0026] 2. The present invention applies genetic engineering technology. Starting from the Bacillus amyloliquefaciens CGMCC 26351, a recombinant strain is constructed by introducing exogenous genes. When producing inositol using sucrose as a substrate, the addition of specific nutrient salts is controlled to achieve the purpose of fermentation metabolism control, which can significantly improve the substrate utilization rate and inositol yield. When the substrate is 40 g / L sucrose, the highest substrate utilization rate can reach 100%, and the highest inositol yield can reach 59.35 g / L. At the same time, when further adopting process feeding and related process control means, the inositol yield can be further improved. The present invention combines the construction of a high - producing recombinant strain with the fermentation - controlled nutrient salt technology. Through the construction of metabolic pathways and fermentation control means, the method is simple, easy to use, reduces production costs, and has good application prospects and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a verification diagram of PCR detection of recombinant Bacillus amyloliquefaciens 01 in the present invention; among them, lanes 1, 3, 5, and 7 are the PCR results of the starting strain, and lanes 2, 4, 6, and 8 are respectively the PCR results of recombinant Bacillus amyloliquefaciens 01Δ5.3.9.11::5.5.1.4, replacing 2.7.1.11, Δ5.3.1.9, replacing 2.7.1.2;

[0028] Figure 2Verification diagram of PCR detection of recombinant Bacillus amyloliquefaciens 02 in the present invention; among them, lanes 1, 3, 5, and 7 are the PCR results of the starting strain, and lanes 2, 4, 6, and 8 are respectively; the PCR results of recombinant Bacillus amyloliquefaciens 02Δ5.3.9.11::5.5.1.4, Δ5.3.1.9, replacing 2.7.1.2;

[0029] Figure 3 Verification diagram of PCR detection of recombinant Bacillus amyloliquefaciens 03 in the present invention; among them, lanes 1, 3, 5, and 7 are the PCR results of the starting strain, and lanes 2, 4, 6, and 8 are respectively; the PCR results of recombinant Bacillus amyloliquefaciens 03 inserting 5.5.1.4, replacing 2.7.1.11, Δ5.3.1.9, replacing 2.7.1.2. Detailed implementation mode

[0030] The embodiments of the present invention will be described in detail below. It should be noted that this embodiment is narrative and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0031] The raw materials used in the present invention are all conventional commercially available products without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0032] A strain of Bacillus amyloliquefaciens, the name of the Bacillus amyloliquefaciens is: G2-9, the classification name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC NO.26351, the preservation date is: December 27, 2022, the preservation unit: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. This Bacillus amyloliquefaciens was isolated from traditional Chinese vinegar vinegar grains and cultured on RCM medium. The colonies are round, light yellow, opaque, and have an irregular edge.

[0033] A recombinant strain constructed with the above-mentioned Bacillus amyloliquefaciens as the starting strain.

[0034] The construction method of the above-mentioned recombinant strain includes the following steps:

[0035] Using Bacillus amyloliquefaciens wild type CGMCC26351 as the starting strain, its gene was edited by the homologous recombination system, the key enzyme inositol-degrading enzyme EC5.3.99.11 in the inositol degradation pathway was knocked out, the pathway affecting inositol synthesis was transformed, and inositol-3-phosphate synthase EC5.5.1.4 was inserted in sequence, 6-phosphofructokinase EC2.7.1.11 was replaced, phosphohexose isomerase EC5.3.1.9 was knocked out, and glucosidase EC2.7.1.2 was replaced to obtain a recombinant strain;

[0036] Among them, the gene sequence of inositol-degrading enzyme EC5.3.99.11 is SEQ ID NO.1, the gene sequences of inositol-3-phosphate synthase EC5.5.1.4 are SEQ ID NO.2, SEQ ID NO.3 and / or SEQ ID NO.4, the gene sequence of 6-phosphofructokinase EC2.7.1.11 is SEQ ID NO.5, the gene sequence of phosphohexose isomerase EC5.3.1.9 is SEQ ID NO.9, and the gene sequence of glucosidase EC2.7.1.2 is SEQ ID NO.10.

[0037] Preferably, it specifically includes the following steps:

[0038] Using the homologous recombination system, knockout the inositol-degrading enzyme EC5.3.99.11 of Bacillus amyloliquefaciens. And integrate the coding genes of inositol-3-phosphate synthase EC5.5.1.4 derived from Streptomyces dextrose, Bifidobacterium adolescentis, and Rhodococcus erythropolis at the inositol-degrading enzyme gene locus respectively; and replace the 6-phosphofructokinase EC2.7.1.11 gene of Bacillus amyloliquefaciens with the 6-phosphofructokinase genes derived from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis respectively; and knockout the phosphohexose isomerase EC5.3.1.9 gene of Bacillus amyloliquefaciens; and replace the glucosidase EC2.7.1.2 gene of Bacillus amyloliquefaciens with the glucosidase genes derived from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis respectively;

[0039] Among them, the gene sequence of inositol-degrading enzyme EC5.3.99.11 is SEQ ID NO.1, the gene sequences of inositol-3-phosphate synthase EC5.5.1.4 are SEQ ID NO.2, SEQ ID NO.3 and / or SEQ ID NO.4, the gene sequences of the 6-phosphofructokinase genes derived from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis are SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 respectively, and the gene sequences of the glucosidase genes derived from Escherichia coli, Bacillus subtilis, and Bacillus licheniformis are SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 respectively.

[0040] Use of the recombinant strain as described above in the fermentation production of inositol.

[0041] A method for producing inositol using the recombinant strain as described above, the method comprising using sucrose as a substrate and producing inositol by bioconversion with the recombinant strain.

[0042] Preferably, the method further includes: adding a fermentation supplement during the fermentation of the recombinant strain, where the fermentation supplement includes fermentation supplement 1 and fermentation supplement 2, by volume:

[0043] Fermentation supplement 1 is: manganese sulfate 0.03 - 0.08 g / L, potassium nitrate 0.001 - 0.005 g / L;

[0044] Fermentation supplement 2 is: pyruvic acid 0.006 - 0.01 g / L, sodium fructose 1,6 - diphosphate 0.01 - 0.02 g / L, acetic acid 0.005 - 0.01 g / L.

[0045] Preferably, during the fermentation of inositol by the recombinant strain, fermentation supplement 1 is added to the fermentation medium at a fermentation time of 6 - 12 h, and fermentation supplement 2 is added to the fermentation medium at a fermentation time of 24 - 30 h.

[0046] Preferably, fermentation supplement 1 is added to the fermentation medium at a fermentation time of 7.5 h, and fermentation supplement 2 is added to the fermentation medium at a fermentation time of 26 h.

[0047] Specifically, the relevant preparation and detection examples are as follows:

[0048] Example 1:

[0049] Construct an inositol - highly productive strain 01. The gene - editing method used refers to the literature (Ruan L, Li L, Zou D. et al. Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S - adenosylmethionine by coupling of an engineered S - adenosylmethionine pathway and the tricarboxylic acid cycle. Biotechnol Biofuels 12, 211 (2019).), and the specific method is as follows:

[0050] 1. Construct a homologous recombination plasmid

[0051] Knock out inositol-degrading enzyme EC5.3.99.11 of Bacillus amyloliquefaciens, and integrate the coding gene of inositol-3-phosphate synthase (EC5.5.1.4) at the inositol-degrading enzyme gene locus. Select the genomic DNA of Bacillus amyloliquefaciens CGMCC NO.26351 as the template, and use upstream and downstream homologous arm primers to amplify the inositol-degrading enzyme EC5.3.99.11 homologous arm sequence fragments A and B respectively. At the same time, use the corresponding primers to amplify the inositol-3-phosphate synthase (EC5.5.1.4) fragment C from Bifidobacterium adolescentis. Use overlap PCR to integrate A, B and C to obtain an integration fragment for completing the insertion of inositol-degrading enzyme EC5.3.99.11 and inositol-3-phosphate synthase EC5.5.1.4. This fragment and the T2(2)-ori vector are simultaneously digested with restriction enzymes BamHI and XbaI, and after recovery, they are ligated with T4 DNA ligase to construct a recombinant plasmid T2Δ11::4, which is electrotransformed into Bacillus amyloliquefaciens competent cells.

[0052] 2. Screening of gene recombinant strains

[0053] Coat the electrotransformed competent cells on a solid LB plate containing kan. After PCR verification, inoculate the positive clones into an LB liquid medium containing kan, and culture them at 45 °C with shaking at 180 rpm for 8 h. After further kan resistance screening and PCR verification, pick the single crossover strains, transfer them into an LB liquid medium, culture them at 37 °C with shaking at 180 rpm for 8 h, and subculture them several times. Finally, spread the subcultured bacteria on an LB plate, and streak each single colony on an LB plate without Kan and an LB plate containing Kan to screen for Kan-sensitive colonies. Further PCR verification with primers checkF1 and checkR1 can find bands with different sizes from the starting strain, and they are consistent with the expected band size of 1264 bp, proving that the double crossover strain is successfully obtained.

[0054] 3. The subsequent gene replacement of 6-phosphofructokinase (EC2.7.1.11), gene knockout of phosphohexose isomerase (EC5.3.1.9), gene replacement of glucosidase (EC2.7.1.2), and gene knockout of ADP-glucose starch synthase (EC2.4.1.21) in Bacillus amyloliquefaciens are carried out by the same method.

[0055] Such as Figure 1As shown, through PCR verification, bands consistent with the theoretical results were obtained, proving the successful construction of recombinant Bacillus amyloliquefaciens 01. Finally, the myo-inositol-degrading enzyme EC5.3.99.11 of Bacillus amyloliquefaciens was knocked out, and the coding gene of inositol-3-phosphate synthase (EC5.5.1.4) derived from Bifidobacterium adolescentis was integrated at the myo-inositol-degrading enzyme gene locus; the 6-phosphofructokinase (EC2.7.1.11) gene of Bacillus amyloliquefaciens was replaced with the 6-phosphofructokinase gene derived from Bacillus subtilis; the phosphohexose isomerase (EC5.3.1.9) gene of Bacillus amyloliquefaciens was knocked out; and the glucosidase (EC2.7.1.2) gene of Bacillus amyloliquefaciens was replaced with the glucosidase gene derived from Bacillus licheniformis. The recombinant Bacillus amyloliquefaciens 01 is used for the efficient production of myo-inositol.

[0056] 4. The primers involved in the above strain construction process are shown in the following table

[0057] Table 1 PCR primers for the construction and verification of recombinant Bacillus amyloliquefaciens 01

[0058]

[0059]

[0060] Example 2:

[0061] 1. The following fermentation supplements 1 and 2 are selected for the fermentation production of myo-inositol, by volume:

[0062] Fermentation supplement 1: Manganese sulfate 0.05 g / L, potassium nitrate 0.003 g / L;

[0063] Fermentation supplement 2: Pyruvic acid 0.008 g / L, disodium 6-phosphofructose 0.015 g / L, acetic acid 0.008 g / L.

[0064] 2. The addition time of the fermentation supplements:

[0065] During the fermentation production of myo-inositol, fermentation supplement 1 is added to the fermentation medium at a fermentation time of 7.5 h, and fermentation supplement 2 is added to the fermentation medium at a fermentation time of 26 h.

[0066] Comparative example 1:

[0067] A strain 02 for the production of myo-inositol was constructed, and the gene editing method was the same as in Example 1.

[0068] As Figure 2As shown, through PCR verification, bands consistent with the theoretical results were obtained, proving the successful construction of recombinant Bacillus amyloliquefaciens 02. Finally, the inositol-degrading enzyme EC5.3.99.11 of Bacillus amyloliquefaciens was knocked out. The coding gene of inositol-3-phosphate synthase (EC5.5.1.4) derived from Bifidobacterium adolescentis was integrated at the inositol-degrading enzyme gene locus; the phosphohexose isomerase (EC5.3.1.9) gene of Bacillus amyloliquefaciens was knocked out; and the glucosidase (EC2.7.1.2) gene of Bacillus amyloliquefaciens was replaced with the glucosidase gene derived from Bacillus licheniformis. The recombinant Bacillus amyloliquefaciens 02 is used for inositol production.

[0069] The primers involved in the above strain construction process are shown in the following table

[0070] Table 2 PCR primers for the construction and verification of recombinant Bacillus amyloliquefaciens 02

[0071]

[0072]

[0073] Comparative Example 2:

[0074] Strain 03 for inositol production was constructed, and the gene editing method was the same as in Example 1.

[0075] As Figure 3 shown, through PCR verification, bands consistent with the theoretical results were obtained, proving the successful construction of recombinant Bacillus amyloliquefaciens 03. Finally, the coding gene of inositol-3-phosphate synthase (EC5.5.1.4) derived from Bifidobacterium adolescentis was integrated downstream of the inositol-degrading enzyme gene locus; the 6-phosphofructokinase (EC2.7.1.11) gene of Bacillus amyloliquefaciens was replaced with the 6-phosphofructokinase gene derived from Bacillus subtilis; the phosphohexose isomerase (EC5.3.1.9) gene of Bacillus amyloliquefaciens was knocked out; and the glucosidase (EC2.7.1.2) gene of Bacillus amyloliquefaciens was replaced with the glucosidase gene derived from Bacillus licheniformis. The recombinant Bacillus amyloliquefaciens 03 is used for inositol production.

[0076] The primers involved in the above strain construction process are shown in the following table

[0077] Table 3 PCR primers for the construction and verification of recombinant Bacillus amyloliquefaciens 03

[0078]

[0079]

[0080] Comparative Example 3: (Addition time comparison)

[0081] 1. For the fermentation production of inositol, the following fermentation supplements 1 and 2 are selected, by volume:

[0082] Fermentation supplement 1: Manganese sulfate 0.05 g / L, potassium nitrate 0.003 g / L;

[0083] Fermentation supplement 2: Pyruvic acid 0.008 g / L, sodium fructose-6-phosphate 0.015 g / L, acetic acid 0.008 g / L.

[0084] 2. Addition time of the fermentation supplements:

[0085] During the fermentation production of inositol, all the fermentation supplements are added at 0 h from the start of the fermentation time.

[0086] Comparative example 4: (Comparison of supplement formulations)

[0087] 1. For the fermentation production of inositol, the following fermentation supplements 1 and 2 are selected, by volume:

[0088] Fermentation supplement 1: Manganese sulfate 0.05 g / L;

[0089] Fermentation supplement 2: Pyruvic acid 0.008 g / L.

[0090] 2. Addition time of the fermentation supplements:

[0091] During the fermentation production of inositol, the fermentation supplement 1 is added to the fermentation medium at 7.5 h of the fermentation time, and the fermentation supplement 2 is added to the fermentation medium at 26 h of the fermentation time.

[0092] Comparative example 5: (Comparison of supplement formulations)

[0093] 1. For the fermentation production of inositol, the following fermentation supplements 1 and 2 are selected, by volume:

[0094] Fermentation supplement 1: Potassium nitrate 0.003 g / L;

[0095] Fermentation supplement 2: Sodium fructose-6-phosphate 0.015 g / L, acetic acid 0.008 g / L.

[0096] 2. Addition time of the fermentation supplements:

[0097] During the fermentation production of inositol, the fermentation supplement 1 is added to the fermentation medium at 7.5 h of the fermentation time, and the fermentation supplement 2 is added to the fermentation medium at 26 h of the fermentation time.

[0098] Experimental example:

[0099] Ferment inositol according to the strains and the addition methods of the fermentation supplements described in the above embodiments and comparative examples, wherein the fermentation medium comprises: 1% peptone, 0.5% yeast extract, 1% NaCl, 0.1% dipotassium hydrogen phosphate, 0.1% magnesium sulfate.

[0100] Activate the successfully constructed recombinant Bacillus amyloliquefaciens at 34-38 °C and inoculate it into LB medium, and culture it at 34-38 °C and 150-250 rpm for 15-20 h until the OD 660 is greater than 2.0, then it can be transferred to a fermenter for large-scale production of inositol. After fermentation termination, the fermentation broth is centrifuged at 10,000 rpm for 10 min, the supernatant is aspirated and filtered through a 0.22 μm filter membrane, and the inositol concentration is detected by RID-HPLC method. The liquid phase conditions are: Hi-PlexCa column (8 μm, 7.7×300 mm), column temperature 80 °C, mobile phase: H2O, detection temperature: 40 °C, detection time 35 min, flow rate 0.6 mL / min, injection volume 20 μL.

[0101] Use a 10 L fermenter to ferment and produce inositol. Inoculate the cultured seed liquid into the fermentation medium containing 40 g / L sucrose substrate at an inoculation amount of 5% for catalytic fermentation, and compare the effects of different recombinant strains and fermentation supplements on the inositol yield. The results are shown in Table 4. Different combinations of different ratios and addition methods of different strains and fermenting agents have different corresponding inositol yields, among which the inositol yields of the strains and fermenting agents described in Example 1 and Example 2 are the highest.

[0102] Supplementary note, sucrose is formed by the condensation and dehydration of one molecule of glucose and one molecule of fructose. It is speculated that part of the reason for the high inositol production in Example 1 is that it can almost completely utilize the glucose in sucrose and utilize part of the fructose and convert it into inositol. In addition, if the method of supplementing sucrose during fermentation is selected, the inositol yield can be further improved.

[0103] Table 4 Comparison of inositol yields

[0104]

[0105] The sequences used in the present invention can be as follows: 1.

[0107] EC5.3.99.11

[0108] Knockout sequence

[0109] DNA834

[0110] ATGAAACTTTGCTTTAATGAAGCGACTACGCTGGAGAATTCAAACCTGAAACAGGATTT

[0111] AGAACTGTGCGAAAAGCACGGCTATGATATATTGAAATCCGCACAATGGATAAACTGCC

[0112] GGAATACTTAAAGACCATTCACTGGCCGATCTGGCGGAATATTTTCGCACCCATCACATT

[0113] AAACCGCTTGCCCTGAACGCACTGGTTTTCTTCAACAACCGTGATGAGAAGGGCTATAG

[0114] GGAGATCATTTCGGAATTCAAAAGCATGATGGAAACCTGCAGGACGCTCGGCGTGAAAT

[0115] ATGTGGTGGCCGTGCCGCTTGTGACAGAGCGGAAGATCTTGAAAGAAGAGATCAAAAA

[0116] GAGCATAGAGGTGCTGACTGAGCTTTCGGACATCGCGGAGCCGTACGGCGTAAACATTG

[0117] CCCTTGAATTTGTCGGCCACCCGCAATGTACGGTCAATACATTTGAACAGGCGTATGACA

[0118] TCGTGAACGCTGTCGGCCGCGATAATGTCGGACTGGTGTTTGACAGCTTCCATTTCCACG

[0119] CGATGGGCTCCAATATTGAGAGCTTGAAGCAGGCGGACGGAAAGAAAATTTTCATCTAT

[0120] CATCGACGATACAGAAGACTTTCCGATCGGCTTTTTAACAGATGAAGACCGCGTCTGGC

[0121] CTGGACAGGGTGCGATTGACTTAGACGCCCATCTGTCAGCGTTAAAGAAATCGGATTCA

[0122] ATGATGTTGTCTCCGTAGAGCTGTTCAGACCTGAATATTATAAGCTGACTGCGGAGGAAA

[0123] CGATCAAAACGGCAAAGGAAACAACGGAAGCCGTCGTATCAAATACTTCATGAAGGAG

[0124] GCGTGA 2.

[0126] EC5.5.1.4

[0127] Insertion sequence Streptomyces drozdowiczii 1078

[0128] ATGGGTTCGGTTCGCGTGCCATCGTCGGCGTGGGCAACTGCGCCGCCTCGCTGGTCCAG

[0129] GGCGTCGAGTACTACAAGGACGCCGATCCGGCCGGCAAGGTGCCCGGCCTGATGCACG

[0130] TCCAGTTCGGCGACTACCACGTGCGGGACGTCGAGTTCGTCGCCGCCTTCGACGTCGAC

[0131] GCGAAGAAGGTCGGCCTCGACCTCTCGGACGCCATCGGTGCGAGCGAGAACAACACCA

[0132] TCAAGCTCTGCGACGTGCCGCACGCCGGTGTCACCGTCCAGCGCGGCCAACCCACGAC

[0133] GGGCTCGGCAAGTACTACCGCCAGACCATCGAGGAGTCGGCCGAGGCCCCGGTCGACG

[0134] TCGTCCAGGTCCTCAAGGACCGCAAGGTCGACGTCCTCGTCTGCTACCTCCCGGTGGGT

[0135] TCCGAGGTGGCCGCGAAGTTCTACGCCCAGTGCGCCATCGACGCCAAGGTCGCGTTCGT

[0136] CAACGCTCTCCCGGTCTTCATCGCCGGCACCAAGGAGTGGGCGGACAAGTTCACCGAG

[0137] GCCGGTGTCCCGATCGTCGGCGACGACATCAAGTCGCAGGTCGGCGCCACCATCACGC

[0138] ACCGCGTGATGGCGAAGCTCTTCGAGGACCGGGGCGTCATCCTGGACCGCACGATGCA

[0139] GCTGAACGTCGGCGGCAACATGGACTTCAAGAACATGCTCGAGCGTGAGCGCCTGGAG

[0140] TCCAAGAAGATCTCCAAGACGCAGGCCGTCACCTCGCAGATCCGTGACCGCGAGCTCG

[0141] GCGCGGACAACGTCCACATCGGCCCGTCGGACTACGTGGCCTGGCTGGACGACCGCAA

[0142] GTGGGCGTACGTGCGCCTCGAGGGCCGCGCCTTCGGCGACGTCCCGCTGAACCTGGAG

[0143] TACAAGCTCGAGGTCTGGGACTCCCCGAACTCCGCCGGTGTCATCATCGACGCCGTCCG

[0144] TGCCGCGAAGATCGCCAAGGACCGCGGCATCGGCGGCCCGATCCTCTCGGCCTCCTCGT

[0145] ACTTCATGAAGTCCCCGCCGGTCCAGTACTTCGACGACGAGGCCCGCGAGAACGTCGA

[0146] GAAGTTCATCCGCGGTGAGGTCTCCAAC 3.

[0148] EC5.5.1.4

[0149] Insertion sequence Bifidobacterium adolescentis 1139

[0150] ATGAGCATTCGAGTGGCTATCGCCGGCGTGGGCAATTGCGCCTCGTCGCTGGTGCAGGG

[0151] CGTTGAGTATTACAAGGACACCAAGGACGAGGATAAGATTCCCGGCCTGATGCACAACA

[0152] ATTTCGGTGGCTACCGCGTGCGTGACATCGAATTCGTCACCGCGTTCGACGTGGATGCG

[0153] CTGAAGGTGGGCAAGGATCTGTCCGAGGCGATTGGCGCTTCTCAGAACAACACCATCA

[0154] AATTCGCGGACGTGCCGAATCTGGGCGTGGAGGTGCTGCGTGGCCCGACTAACGACGG

[0155] CTTGGGCGAATACTACCGCCAGATGATCGAAGAGTCGGACGCCGAACCGGTGGACGTG

[0156] GCCCAGGTGCTGCGTGACAAGAAGGTCGACGTGCTGGTTTCCTACCTGCCGGTTGGTTC

[0157] CGAGAAGGCGGACAAGGCTTACGCTCAGGCCGCTATGGACGCTGGCTGCGCGTTCGTG

[0158] AACTGCCTGCCGGTGTTCATCGCCTCCGATCCGGAGTGGGCGCAGAAGTTCCGTGACGC

[0159] TGGTGTGCCGATTGTCGGCGACGATATCAAGTCGCAGGTCGGCGCCACCATCACGCACC

[0160] GCGTGCTGGCCCGTCTGTTCGAGGATCGCGGCGTGCGTCTGGATCGTACGTACCAGCTT

[0161] AACGTCGGCGGCAACATGGACTTCATGAACATGCTGCAGCGTTCCCGCCTGGAATCCAA

[0162] GAAGATCTCCAGACCCGCGCCGTCACTTCCGTGGTGCCGCACGAGATGGATCCGCACAA

[0163] CGTGCATATCGGCCCGTCCGATTACGTGGCTTGGCTTGACGACCGCAAGTTCGCCTTCGT

[0164] GCGTCTTGAGGGCACCACGTTCGGCGATGTGCCGCTGAACCTCGAATACAAGCTCGAGG

[0165] TGTGGGATTCCCCGAATTCCGCCGGTATTGTGATCGACGCCGTGCGTGCGGCCAAGATC

[0166] GCGCTCGATCGTCATCTGGCCGGTCCGGTTCTGGCTCCGAGCTCCTACTTCATGAAGTCT

[0167] CCGGCCGTGCAGCACGAGGATGGCGAGGCCCGCCGCTTGGTGGAGGAATTCATCAAGG

[0168] GTGAGGTCGAAGGCACCGAAGAGCAGCTTGACGCCGATGTGGCGGCCGCGAAGGCCG

[0169] CCGGCAAGGATGTGTGGCGCGCA 4.

[0171] EC5.5.1.4

[0172] Insertion sequence Rhodococcus 1081

[0173] ATGGCTGACACCAACCGCACGAGCGTGCGCGTCGCGATCGTGGGCGTGGGCAACTGTG

[0174] CCTCGTCCCTGGTCCAGGGCGTGCAGTACTACCAGGACGCCGACGAGAACGCGACGGT

[0175] CCCCGGCCTCATGCACGTCCGCTTCGGCCCTTACCACGTCCGCGACGTCGAGTTCGTCG

[0176] CCGCGTTCGACGTGGACGCCAAGAAGGTCGGCTTCGACCTGAACGAGGCGATCGTCTC

[0177] CAGCGAGAACAACACCATCAAGATCGCCGACGTGCCCCGACGGGCGTCATCGTCCAGC

[0178] GCGGCCCCACCAACGACGGTCTCGGCAAGTACTACCTGGAGACCATCACCGAGTCCGA

[0179] CGCCGAGCCGGTCGACATGGTCGCCGCGCTCAAGGACGCCGACGTCGACGTCCTGGTC

[0180] TGCTACCTCCCGGTGGGCTCGGAGGTCGCCGCCAAGCACTACGCGCAGTGCGCCATCGA

[0181] CGCGGGCGTGGCCTTCGTGAACGCCCTGCCGGTGTTCATCGCCTCCGACCCCGTGTGGG

[0182] CCAAGAAGTTCGAGGACGCGGGCGTGCCGATCGTCGGCGACGACATCAAGAGCCAGGT

[0183] GGGCGCGACCATCACCCACCGCGTGCTCGCCAAGCTGTTCGAGGACCGCGGCGTGCAG

[0184] CTCGACCGCACCATGCAGCTCAACGTCGGCGGCAACATGGACTTCAAGAACATGCTCG

[0185] AGCGCGAGCGCCTGGAGTCCAAGAAGATCTCCAAGACCCAGGCCGTCACGTCCAACCT

[0186] GCAGCGGGAAGTGGGCGCCAACAACGTCCACATCGGACCGTCGGGACTACGTCGCGTG

[0187] GCTCGACGACCGCAAGTGGGCCTACGTCCGCCTCGAGGGACGTGCGTTCGGTGACGTG

[0188] CCGCTGAGCCTGGAGTACAAGCTCGAGGTCTGGGACTCCCCGAACTCGGCCGGCATCAT

[0189] CATCGACGCCGTCCGGGCCGCGAAGATCGCCAAGGACCGCGGCATCGGTGGCCCCGTG

[0190] TTCGCCGCGGCCAGCTACCTGATGAAGTCGCCGCCGCGCCAGCTGGCCGACGACGTGG

[0191] CCCGCACCCAGCTCGAGGAGGTTCATCGCGGGC 5.

[0193] 敲除序列EC2.7.1.11

[0194] Bacillusamyloliquefaciens957

[0195] ATGAAGCGTATAGGAGTTTTAACGAGCGGCGGGGATTCCCCGGGTATGAACGCTGCGGT

[0196] GCGCGCGGTTGTGCGGAAAGCTATCTACCACGACGTCGAAGTGTACGGAATTTATAACG

[0197] GTTATTCAGGATTAATCAGCGGAAAAATAGAGAAACTGGAGCTTGGTTCGGTCGGTGAC

[0198] ATCATCCACCGCGGCGGAACAAAGCTGTATACGGCCAGATGTCCTGAATTCAAAACAGT

[0199] TGAAGGCCGTGAAAAAGGAATCGAAAATTTAAAGAAACTCGGCATTGAAGGTCTTGTC

[0200] GTCATCGGCGGAGACGGTTCATACATGGGTGCGAAAAAATTAACGGAACACGGGTTTCC

[0201] GTGTGTAGGTGTACCGGGTACGATAGATAATGACATCCCGGGAACTGATTTTACGATCGG

[0202] ATTTGATACGGCGCTAAACACCGTAATTGATGCGATTGATAAAATCCGTGACACCGCTAC

[0203] GTCTCATGAGCGTACATACGTTATTGAAGTGATGGGGCGCCATGCGGGCGACATTGCTTT

[0204] ATGGGCAGGTCTTGCAGGCGGAGCGGAATCAATTTTAATTCCTGAAGCGGACTACGATAT

[0205] GCAGGAAATTATCGGCCGTTTGAAGAGAGGGCACGACCGCGGCAAAAAACACAGTATC

[0206] ATCATCGTTGCAGAAGGGGTAGGCAGCGGAGTTGAATTCGGAAAACGCATCGAAGAAG

[0207] AAACGAACCTTGAAACACGGGTATCCGTTTTAGGGCACATCCAGCGCGGCGGATCTCCA

[0208] AGCGCTTCTGACCGGGTTCTTGCAAGCCGCCTCGGCGCTTACGCGGTTGAGCTTCTGCT

[0209] TAAAGGAAAAGGCGGACGCTGCGTAGGTATACAAAACAACAAGCTTGTAGACCATGATA

[0210] TTATAGAAATTTTAGAATCAAAACATACTGTTGAACCAAACATGTATCAGCTTTCAAAAG

[0211] AACTGTCTATT 6.

[0213] Replacement sequence EC2.7.1.11

[0214] Escherichiacoli960

[0215] ATGATTAAGAAAATCGGTGTGTTGACAAGCGGCGGTGATGCGCCAGGCATGAACGCCGC

[0216] AATTCGCGGGGTTGTTCGTTCTGCGCTGACAGAAGGTCTGGAAGTAATGGGTATTTATGA

[0217] CGGCTATCTGGGTCTGTATGAAGACCGTATGGTACAGCTAGACCGTTACAGCGTGTCTGA

[0218] CATGATCAACCGTGGCGGTACGTTCCTCGGTTCTGCGCGTTTCCCGGAATTCCGCGACG

[0219] AGAACATCCGCGCCGTGGCTATCGAAAACCTGAAAAAACGTGGTATCGACGCGCTGGT

[0220] GGTTATCGGCGGTGACGGTTCCTACATGGGTGCAATGCGTCTGACCGAAATGGGCTTCC

[0221] CGTGCATCGGTCTGCCTGGCACTATCGACAACGACATCAAAGGCACTGACTACACTATC

[0222] GGTTTCTTCACTGCGCTGAGCACCGTTGTAGAAGCGATCGACCGTCTGCGTGACACCTC

[0223] TTCTTCTCACCAGCGTATTTCCGTGGTGGAAGTGATGGGCCGTTATTGTGGCGATCTGAC

[0224] GTTGGCTGCGGCCATTGCCGGTGGCTGTGAATTCGTTGTGGTTCCGGAAGTTGAATTCA

[0225] GCCGTGAAGACCTGGTAAACGAAATCAAAGCGGGTATCGCGAAAGGTAAAAAACACGC

[0226] GATCGTGGCGATTACCGAACATATGTGTGATGTTGACGAACTGGCGCATTTCATCGAGAA

[0227] AGAAACCGGTCGTGAAACCCGCGCAACTGTGCTGGGCCACATCCAGCGCGGTGGTTCT

[0228] CCGGTGCCTTACGACCGTATTCTGGCTTCCCGTATGGGCGCTTACGCTATCGATCTGCTGC

[0229] TGGCAGGTTACGGCGGTCGTTGCGTAGGTATCCAGAACGAACAGCTGGTTCACCACGAC

[0230] ATCATCGACGCTATTGAAAACATGAAGCGTCCGTTCAAAGGCGACTGGCTGGACTGCGC

[0231] GAAAAAACTGTAT 7.

[0233] Replacement sequence EC2.7.1.11

[0234] Bacillus subtilis 957<(

[0235] ATGAAACGTATAGGGGTATTAACGAGCGGCGGGGATTCCCCGGGAATGAACGCAGCAGT

[0236] TCGCGCAGTAGTCAGAAAAGCGATTTATCATGACGTTGAAGTTTACGGCATTTACAACG

[0237] GATACGCAGGATTGATCAGCGGAAAGATTGAAAAGCTTGAACTCGGATCTGTAGGCGAT

[0238] ATTATACATCGCGGAGGAACAAAGCTTTATACGGCAAGATGTCCTGAATTCAAAACGGTT

[0239] GAAGGCCGTGAAAAAGGGATAGAAAACTTGAAGAAGCTCGGTATTGAAGGCCTTGTTG

[0240] TCATCGGCGGAGACGGTTCCTATATGGGTGCCAAAAAATTAACGGAACACGGGTTTCCA

[0241] It should be noted that there seems to be an incorrect tag format in line which is "<( ". It should probably be " ". This might cause issues in some systems or applications.TGTGTAGGTGTACCGGGTACAATTGATAACGACATTCCGGGCACTGATTTTACAATCGGC

[0242] TTCGATACAGCTTTAAATACAGTAATTGACGCAATTGATAAGATTCGCGACACAGCGACT

[0243] TCTCATGAACGTACATATGTGATCGAAGTAATGGGCCGCCATGCCGGCGATATCGCATTG

[0244] TGGGCCGGTCTTGCAGGGGGCGCGGAATCAATCTTGATCCCTGAGGCAGACTATGACAT

[0245] GCACGAAATCATTGCCCGCTTAAAACGCGGCCACGAACGCGGCAAGAAGCACAGTATTA

[0246] TTATTGTCGCCGAAGGTGTAGGCAGCGGTGTTGAATTCGGGAAACGCATTGAAGAAGAA

[0247] ACAAATCTTGAAACTAGGGTATCTGTATTGGGCCATATCCAGCGCGGAGGTTCTCCGAGC

[0248] GCTGCTGACCGCGTGCTGGCAAGCCGTCTTGGCGCATATGCAGTTGAACTGCTGCTTGA

[0249] AGGAAAAGGCGGACGCTGTGTAGGTATACAAAACAATAAGCTTGTAGACCATGATATTAT

[0250] AGAAATACTGGAAACAAAACACACAGTTGAGCAAAACATGTATCAGCTTTCAAAAGAA

[0251] CTGTCTATT 8.

[0253] Replacement sequence EC2.7.1.11

[0254] Bacillus licheniformis

[0255] DNA960

[0256] ATGAAACGTATCGGAGTATTGACAAGCGGCGGAGATTCCCCGGGAATGAACGCGGCGGT

[0257] CCGCGCGGTTGTCAGGAAAGCGATCTACCATGACGTAGAAGTATACGGAATTTACAACG

[0258] GGTATTCAGGTTTAATCAGCGGAAAAATAGAAAAGCTTGAAATCGGTTCTGTCGGAGAT

[0259] ATCATTCACCGCGGAGGAACGAAGCTATATACAGCAAGGTGTCCTGAGTTTAAAACGGT

[0260] AGAAGGACGGGAAAAAGGAATAGCGAATTTAAAAAAATATGGTATAGAAGGATTAGTCG

[0261] TCATTGGCGGCGACGGTTCTTATATGGGAGCCAAAAAGCTCACAGAACACGGTTTTCCT

[0262] TGTGTGGGAGTGCCGGGAACGATCGACAATGACATCCCAGGCACAGATCTGACGATCG

[0263] GATTCGATACGGCGCTAAATACCGTTATCGACGCCATCGATAAAATCAGGGATACAGCTA

[0264] CATCCCATGAACGTACATACGTGATTGAAGTAATGGGCCGCCATGCCGGGGACATTGCTT

[0265] TATGGTCAGGTTTGGCCGGAGGCGCGGAATCCATTTTGATTCCTGAAGCCGACTACGATA

[0266] TGGAAGAAATTATTGCAAGGCTGAAAAGAGGGCATGAGCGCGGCAAGAAACACAGCAT

[0267] CATCATTGTGGCGGAAGGCGTCGGCAGCGGCGTTGAATTCGGAAAAAGAATTGAAGAA

[0268] GCCACAAATCTCGAAACTAGAGTATCGGTGCTTGGACATATCCAGCGCGGCGGTTCCCC

[0269] GACTGCAGCAGACCGCGTATTGGCAAGCCGTCTAGGGGCATTTGCGGTTGAACTGCTTC

[0270] TTGAAGGAAAAGGCGGACGTTGTGTAGGAATTCAAAATAACCAGCTTGTGCATCATGAT

[0271] ATCATTGAAATACTCGAGCAAAAACATACAATTGATCAAAGCATGTACCGCTTGTCTCAA

[0272] GAACTGTCAATCTAG 9.

[0274] Knockout sequence EC5.3.1.9

[0275] Bacillussubtilis1353

[0276] ATGACGCATGTACGCTTTGACTACTCAAAAGCGTTGACTTTCTTCAACGAACATGAACTT

[0277] ACATACCTGCGGGACTTTGTAAAAACAGCACACCATAATATCCATGAGAAAACAGGCGC

[0278] GGGCAGCGATTTTCTAGGCTGGGTGGACCTCCCTGAACATTATGATAAAGAAGAATTCG

[0279] CGCGCATCAAAAAAAGCGCGGAAAAAATCAAATCTGACTCTGATGTCTTGCTTGTTGTC

[0280] GGCATCGGCGGTTCTTATCTTGGAGCGCGGGCAGCGATTGAAGCGCTGAATCACGCGTT

[0281] TTATAACACTTTGCCAAAAGCAAAACGCGGCAATCCGCAAGTCATTTTTATCGGGAACA

[0282] ACATCAGTTCATCTTATATGAGAGACGTCATGGATCTTCTTGAAGATGTTGACTTCTCTAT

[0283] TAATGTGATTTCTAAATCAGGTACGACAACTGAACCTGCAATCGCTTTCCGTATTTTCCGC

[0284] AAGCTTCTTGAAGAGAAATACGGTAAAGAAGAAGCGAAAGCGCGGATTTATGCAACAA

[0285] CTGATAAAGAGCGCGGCGCATTAAAAACGCTTTCTAACGAAGAAGGCTTTGAATCATTC

[0286] GTAATTCCTGACGATGTCGGCGGCCGTTATTCAGTTTTAACAGCTGTAGGTCTCTTGCCG

[0287] ATTGCTGTCAGCGGCGTCAACATTGACGACATGATGAAAGGCGCCCTGGATGCGAGCAA

[0288] AGATTTTGCAACATCTGAACTGGAAGATAACCCAGCATACCAATATGCGGTTGTTCGCAA

[0289] TGTCCTTTATAATAAGGGCAAAACAATTGAAATGCTCATCAACTACGAACCGGCGCTTCA

[0290] ATACTTTGCGGAATGGTGGAAGCAGCTGTTCGGAGAAAGCGAAGGGAAAGATGAGAAG

[0291] GGCATTTATCCTTCTTCAGCGAACTATTCAACAGACCTTCATTCTTTAGGCCAGTATGTAC

[0292] AAGAAGGCCGCAGAGATTTATTCGAAACGGTCCTGAACGTAGAGAAGCCTAAACATGA

[0293] ACTGACAATTGAGGAAGCGGATAACGATCTTGACGGCTTGAACTATTTAGCCGGTAAAA

[0294] CTGTTGATTTCGTTAACAAAAAAGCATTCCAAGGTACAATGCTTGCCCATACAGACGGA

[0295] AATGTTCCGAACTTAATCGTTAACATTCCTGAGCTGAATGCATATACTTTTGGATACCTTG

[0296] TATATTTCTTCGAAAAAGCCTGCGCGATGAGCGGTTACCTCCTTGGCGTCAATCCGTTTG

[0297] ACCAGCCTGGTGTAGAAGCGTATAAAGTCAATATGTTTGCGTTACTCGGCAAACCTGGCT

[0298] TTGAAGAGAAAAAAGCAGAGCTTGAAAAACGTCTGGAAGATTAA 10

[0300] Knockout sequence EC2.7.1.2

[0301] Bacillus amyloliquefaciens 963

[0302] ATGGAAGATACATGGTTTGCGGGGATTGATCTTGGCGGGACAACCATCAAGCTGGCCTT

[0303] CATCAACATGTACGGTGAAATTCAGCATAAATGGGAAGTTCCGACCGATAAATCAGGAA

[0304] ACACAATTACGGTCACGATCGCCAAAGCGCTTGACCAGAAGCTGGAAGAACTGAACAA

[0305] GCCGAAACGGATCGTAAAATGGATCGGAATGGGAGCGCCCGGACCCGTGGAAATGGCG

[0306] ACGGGAGTGGTCTATGAGACGACAAATATGGGGTGGAAAAACTATCCGCTGAAAGACCA

[0307] TCTCGAGGCGGAAACGGGTATTCCGGCCGTCATTGAAAACGATGCCAATATCGCGGCGC

[0308] TCGGAGAAATGTGGAAGGGTGCAGGTGACGGGGCCAAAGATGTCATTTTAGTGACGCT

[0309] CGGAACCGGAGTCGGCGGAGGCATCATCGTCAACGGAGAAATCGTTCACGGTAAAAAC

[0310] GGGGCCGGCGGAGAAATCGGTCATATTTGCAGTATTCCGGAAGGAGGAGCTCCGTGCAA

[0311] CTGCGGAAAATCCGGCTGCATCGAAACGATTGCGTCCGCAACCGGCATCGTCCGCATCG

[0312] CGAAAGAAAAGCTTGCGGCTGTTTCCGACTCTTCGCTCTTACAAGTGCGCGATCTGACG

[0313] GCACGTGACGTGTTCGAAGCGGCTAAACAGCAGGATAAGACAGCGCTTGAAGTCGTCG

[0314] ATTATGTCGCAAAACATTTAGGCCTCGTGCTCGGAAATCTGGCAAGCGCCATGAATCCGA

[0315] CGAAAATCGTGCTCGGCGGAGGCGTGTCCAAAGCGGGTGAAATCCTGCGGTCAAAAGT

[0316] GGAAGAGACTTTCAAAATCACCGCATTCCCGCGTTCTGCGGAAGCGGCGGATATTTCGA

[0317] TTGCGGCACTTGGAAATGACGCCGGAGTCATCGGCGGGGCGTGGATTGCCAAAAATGA

[0318] ATGGCTCAAACATCAAAACTGCTGA 11.

[0320] Replacement sequence EC2.7.1.2

[0321] Escherichia coli 966

[0322] ATGACAAAGTATGCATTAGTCGGTGATGTGGGCGGCACCAACGCACGTCTTGCTCTGTG

[0323] TGATATTGCCAGTGGTGAAATCTCGCAGGCTAAGACCTATTCAGGGCTTGATTACCCCAG

[0324] CCTCGAAGCGGTCATTCGCGTTTATCTTGAAGAACATAAGGTCGAGGTGAAAGACGGCT

[0325] GTATTGCCATCGCTTGCCCAATTACCGGTGACTGGGTGGCGATGACCAACCATACCTGGG

[0326] CGTTCTCAATTGCCGAAATGAAAAAGAATCTCGGTTTTAGCCATCTGGAAATTATTAACG

[0327] ATTTTACCGCTGTATCGATGGCGATCCCGATGCTGAAAAAAGAGCATCTGATTCAGTTTG

[0328] GTGGCGCAGAACCGGTCGAAGGTAAGCCTATTGCGGTTTACGGTGCCGGAACGGGGCT

[0329] TGGGGTTGCGCATCTGGTCCATGTCGATAAGCGTTGGGTAAGCTTGCCAGGCGAAGGCG

[0330] GTCACGTTGATTTTGCGCCGAATAGTGAAGAAGAGGCCATTATCCTCGAAATATTGCGTG <A

[0331] CGGAAATTGGTCATGTTTCGGCGGAGCGCGTGCTTTCTGGCCCTGGGCTGGTGAATTTGT

[0332] ATCGCGCAATTGTGAAAGCTGACAACCGCCTGCCAGAAAATCTCAAGCCAAAAGATATT

[0333] ACCGAACGCGCGCTGGCTGACAGCTGCACCGATTGCCGCCGCGCATTGTCGCTGTTTTG

[0334] CGTCATTATGGGCCGTTTTGGCGGCAATCTGGCGCTCAATCTCGGGACATTTGGCGGCGT

[0335] GTTTATTGCGGGCGGTATCGTGCCGCGCTTCCTTGAGTTCTTCAAAGCCTCCGGTTTCCG

[0336] TGCCGCATTTGAAGATAAAGGGCGCTTTAAAGAATATGTCCATGATATTCCGGTGTATCTC

[0337] ATCGTCCATGACAATCCGGGCCTTCTCGGTTCCGGTGCACATTTACGCCAGACCTTAGGT

[0338] CACATTCTGTAA 12.

[0340] Replacement sequence EC2.7.1.2

[0341] Bacillussubtilis

[0342] DNA966

[0343] ATGGACGAGATATGGTTTGCGGGCATTGACCTGGGAGGAACGACGATTAAACTCGCTTT

[0344] TATTAATCAATATGGCGAAATTCAGCATAAGTGGGAAGTTCCGACAGATAAAACCGGCG

[0345] ACACGATTACTGTCACAATTGCAAAAACAATCGACAGCAAGCTGGATGAGCTGCAAAA

[0346] ACCGAAGCACATCATCAAATACATCGGAATGGGTGCACCAGGCCCTGTAGATATGGCGG

[0347] CAGGAGTGGTTTATGAAACAGTAAATCTAGGGTGGAAAAATTATGCTTTGAAAAACCAT

[0348] CTGGAGACAGAAACCGGCATCCCAGCTGTTATAGAAAATGACGCGAATATTGCTGCGCT

[0349] CGGGGAAATGTGGAAGGGAGCGGGTGATGGCGCAAAAGACGTCATTCTCGTGACGCTT

[0350] GGCACAGGAGTTGGCGGCGGCATCATTGCAAATGGTGAAATTGTACATGGTATAAATGG

[0351] CGCCGGCGGAGAAATCGGCCATATTTGCAGCATCCCTGAAGGCGGAGCGCCCTGCAACT

[0352] GCGGCAAAACGGGCTGTATCGAAACAATTGCGTCAGCAACCGGAATTGTAAGAATTGCA

[0353] AAAGAAAAAATAGCAAATGCTAAAAAGACGACACGTTTAAAAGCAACCGAACAATTGT

[0354] CAGCGCGAGATGTGTTTGAAGCGGCGGGTGAAAATGATGAAATTGCCCTTGAGGTGGTT

[0355] GATTATGTAGCCAAGCATCTTGGTTTGGTGCTCGGAAATTTGGCAAGCTCGCTTAATCCA

[0356] TCCAAAATCGTTCTTGGCGGCGGCGTATCGAGAGCCGGAGAACTGCTGAGATCAAAAGT

[0357] CGAGAAAACATTCCGCAAATGCGCGTTTCCGCGGGCAGCCCAAGCTGCTGATATTTCAA

[0358] TCGCAGCACTTGGAAATGATGCCGGCGTTATCGGAGGCGCTTGGATCGCTAAAAATGAA

[0359] TGGCTGAAACATCAAAATTGTTAA 13.

[0361] Replacement sequence EC2.7.1.2

[0362] Bacilluslicheniformis

[0363] DNA975

[0364] ATGAATGACAGCTGGCTGGTCGGGGTCGACCTTGGAGGAACAACAGTAAAACTCGCTT

[0365] TTGTAAGCGCTTATGGCGAAATCCTGCACAAGTGGGAAATCCCGACCGATAAATCAGGG

[0366] AAAACGGTTACGGTAAGCATCGCGAAAGCCATCGACAGCAAATTGAACGAGCTCGGCA

[0367] AACCGAAGCACATCTTAAAATGGATCGGCATGGGCGCTCCCGGACCCGTCAATACGGAG

[0368] ACCGGCATCGTTTATAAAACGACGAATATGGGCTGGGAAAACTACCCGTTGAAAGACCA

[0369] TCTGGAAGCAGAGACAGGGATAGCTGCGGTCATTGAAAATGACGCGAACATCGCAGCT

[0370] CTCGGCGAAATGTGGAAGGGAGCGGGAGACGGCGCGAAGGATTTGATTCTTGTTACATT

[0371] GGGAACGGGCGTTGGAGGCGGCATTATTGTAAACGGGGAAATCGTCCGCGGGCAAAAC

[0372] GGAGCAGGCGGTGAAATCGGCCACATCTGTTCTGTTCCAGAAGGCGGAGCGCCGTGCA

[0373] ACTGCGGAAAAAGCGGCTGTATCGAAACGATCGCTTCGGCTACCGGCATCGTCAGGATT

[0374] GCCAAAGAAAAAATAGAATCTGACCATCGAGATACAAGCTTGCGTGAATGTCTGGACAT

[0375] AACGGCGCGCGACATCTTTGAAGCCGCCAGAAAAAACGATCCTGTTGCCGGCGAAGTC

[0376] GTCGATTATGTGGCGGGTCATTTAGGAATGGTCCTCGCTAACTTGGCGAGCTCGCTTAAT

[0377] CCTTCAAAGATCGTAATCGGCGGAGGCGTGTCAAAAGCGGGAGAAATCCTGCGCTCCA

[0378] AAGTTGAACAATCTTTCAAGCGCTTTGTATTCCCGCGGGCCGGCGAGGCGGCTGAGATC

[0379] GTAATCGCTTCACTTGGAAACGACGCAGGCGTGATCGGCGGTGCATGGATCGCAAAAAA

[0380] CCAATGGCTGAAAAGCCAGGCGCAGCCTGTTTTATAA

[0381] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), characterized in that: The name of the Bacillus amyloliquefaciens is: G2-9, the taxonomic name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC NO. 26351, the preservation date: December 27, 2022, the preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A recombinant strain constructed using the Bacillus amyloliquefaciens described in claim 1 as the starting strain, characterized in that: Its construction method includes the following steps: Using the wild-type Bacillus amyloliquefaciens CGMCC 26351 as the starting strain, its genes are edited by a homologous recombination system, knocking out the key enzyme inositol-degrading enzyme EC 5.3.99.11 in the inositol degradation pathway, modifying the pathway affecting inositol synthesis, and sequentially completing the insertion of inositol-3-phosphate synthase EC 5.5.1.4, replacing 6-phosphofructokinase EC 2.7.1.11, knocking out phosphohexose isomerase EC 5.3.1.9, and replacing glucosidase EC 2.7.1.2 to obtain a recombinant strain; Among them, the gene sequence of inositol-degrading enzyme EC 5.3.99.11 is SEQ ID NO.1, the gene sequence of inositol-3-phosphate synthase EC5.5.1.4 is SEQ ID NO. 3, the gene sequence of the replaced 6-phosphofructokinase EC 2.7.1.11 is SEQ ID NO.7, the gene sequence of phosphohexose isomerase EC 5.3.1.9 is SEQ ID NO.9, and the gene sequence of the replaced glucosidase EC 2.7.1.2 is SEQ ID NO.

13.

3. Use of the recombinant strain according to claim 2 in the fermentation production of inositol.

4. A method for producing inositol using the recombinant strain as described in claim 2, characterized in that: The method includes using sucrose as a substrate and using the recombinant strain to produce inositol through bioconversion.

5. The method for producing inositol according to claim 4, wherein: The method further includes: adding a fermentation supplement during the fermentation process of the recombinant strain, and the fermentation supplement includes fermentation supplement 1 and fermentation supplement 2, by volume: Fermentation supplement 1 is: manganese sulfate 0.03-0.08 g / L, potassium nitrate 0.001-0.005 g / L; Fermentation supplement 2 is: pyruvic acid 0.006-0.01 g / L, disodium 6-phosphofructose 0.01-0.02 g / L, acetic acid 0.005-0.01 g / L.

6. The method for producing inositol according to claim 4 or 5, characterized in that: During the fermentation process of the recombinant strain for producing inositol, fermentation supplement 1 is added to the fermentation medium at a fermentation time of 6-12 h, and fermentation supplement 2 is added to the fermentation medium at a fermentation time of 24-30 h.

7. The method for producing inositol according to claim 6, characterized in that: Fermentation supplement 1 is added to the fermentation medium at a fermentation time of 7.5 h, and fermentation supplement 2 is added to the fermentation medium at a fermentation time of 26 h.

Images