Method for constructing engineered rothia bacteria for producing inositol using glucose, glycerol and co2 as carbon sources and strains thereof

Abstract

The present application relates to the field of agricultural biotechnology, and in particular to a method for constructing an engineered strain of Rhodopseudomonas palustris for producing inositol using glucose, glycerol and CO2 as carbon sources, and the strain. The present application mutates the gene nagE encoding N-acetylglucosamine-specific phosphotransferase system in the genome of Rhodopseudomonas palustris (G265R), and knocks out the gene nagR encoding a transcriptional regulator of the GntR family, so that Rhodopseudomonas palustris can efficiently utilize glucose. On this basis, an engineered strain of Rhodopseudomonas palustris H16 is constructed for synthesizing inositol using renewable substrates (glucose and glycerol) and CO2 as carbon source. Based on this technology, not only the synthesis of bioactive substances can be achieved, but also a method for efficiently eliminating greenhouse gases in the atmosphere.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method and strain for constructing an engineered Rollstonella strain that produces inositol using glucose, glycerol, and CO2 as carbon sources. Background Technology

[0002] Climate change, caused by greenhouse gases (such as CO2) released from the extensive use of fossil fuels, is a major contributor to extreme weather events. Eliminating atmospheric CO2 through technological means while simultaneously utilizing it as a raw material for the production of fuels, sugars, and bioactive substances offers a win-win solution. Photocatalysis and electrocatalysis provide feasible pathways for converting CO2 into chemicals or dyes. However, such CO2 conversion requires substantial energy input. Carbon recovery technologies based on microbial cell factories hold even greater potential. Recent reports indicate that *R. ranktonii* H16, modified through metabolic engineering, can directly convert CO2 into high-value chemicals.

[0003] Inositol (cis-1,2,3,5-trans-4,6-cyclohexanol) is a cyclic polyol with five planar hydroxyl groups and one axial hydroxyl group. Currently, in vitro enzymatic synthesis of inositol has been established. However, in vitro enzymatic methods suffer from drawbacks such as short enzyme lifespan, difficulty in isolating inositol, and slow reaction rates. With the development of synthetic biology techniques, it is becoming increasingly possible to efficiently and inexpensively convert renewable raw materials into inositol using metabolic engineering. Reports of inositol production using metabolic engineering have been found using *Escherichia coli* and *Pichia pastoris* as host strains. Carbon sources include cellulose, starch, and glucose. However, there are currently no reports of inositol synthesis using inexpensive renewable carbon sources such as glycerol or CO2 (a greenhouse gas) as the sole carbon source.

[0004] *Cupriavidus necator* H16 is a facultative chemoautotrophic Gram-negative bacterial strain. Due to the absence of 6-phosphofructokinase and 6-phosphogluconate dehydrogenase, it lacks complete glycolysis and pentose phosphate pathways. Glucose-6-phosphate metabolism occurs via the Entner-Doudoroff (ED) pathway to pyruvate, which then enters the tricarboxylic acid cycle or supplies poly([R]-3-hydroxybutyrate) synthesis. As a potential candidate strain for "cellular agriculture," *Cupriavidus necator* H16 can utilize a wide range of organic matter as carbon sources (such as glycerol), and more importantly, it can directly use CO2 as a carbon source via the Calvin-Benson-Bassham (CBB) cycle. Due to the lack of glucose transporters, its wild-type strain cannot utilize glucose as a carbon source. *Cupriavidus necator* H16 possesses a natural PHB synthesis pathway; through metabolic pathway reconstruction, this portion of the carbon flow can be redirected to the synthesis of other high-value-added compounds. Currently, using metabolically engineered Rollstonella H16, the synthesis of isobutanol, methyl ketones, trehalose, and mannose using glycerol or CO2 as carbon sources has been successfully achieved. Summary of the Invention

[0005] To enable the production of inositol using renewable materials and the greenhouse gas CO2, the present invention aims to provide a method for constructing an engineered strain of Rollstonella that produces inositol using glucose, glycerol, and CO2 as carbon sources.

[0006] Another object of the present invention is to provide an engineered strain of Rollstonella that can be used to produce inositol from glucose, glycerol and CO2 as carbon sources.

[0007] The method for constructing an engineered Rollstonella strain that produces inositol according to the present invention includes the following steps:

[0008] (1) The gene nagE(H16_A0312) encoding the N-acetylglucosamine-specific phosphotransferase system in the genome of Rawlstonella is mutated so that the 265th amino acid of the protein it encodes is changed from G to R, and the transcriptional regulator of the GntR family, nagR(H16_A0310), is knocked out, so that Rawlstonella can efficiently utilize glucose. The gene encoding the N-acetylglucosamine-specific phosphotransferase system is nagE(H16_A0312), which encodes a protein with the amino acid sequence as shown in SEQ ID NO: 1 and its nucleotide sequence as shown in SEQ ID NO: 2. The gene encoding the transcriptional regulator of the GntR family is nagR(H16_A0310), whose nucleotide sequence is shown in SEQ ID NO: 3.

[0009] (2) Heterologous expression of the inositol-3-phosphate synthase (ScIPS) gene from Saccharomyces cerevisiae or the inositol monophosphatase (EcIMP) gene from Escherichia coli to construct mutant Rawlstonella chassis cells, wherein the inositol-3-phosphate synthase (ScIPS) gene from Saccharomyces cerevisiae is the inositol-3-phosphate synthase gene from Saccharomyces cerevisiae, and its nucleotide sequence is shown in SEQ ID NO: 4; the inositol monophosphatase (EcIMP) gene from Escherichia coli is the inositol monophosphatase gene from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO: 5.

[0010] The method for constructing an engineered Rollstonella strain that produces inositol according to the present invention further includes the following steps:

[0011] Knockout of key genes in the ED pathway and / or PHB synthesis pathway, among which,

[0012] The key genes of the ED pathway are the glucose-6-phosphate dehydrogenase encoding genes zwf1 (H16_A0316), zwf2 (H16_B1501), and zwf3 (H16_B2566). The nucleotide sequence of the glucose-6-phosphate dehydrogenase encoding gene zwf1 is shown in SEQ ID NO: 6, the nucleotide sequence of the zwf2 gene is shown in SEQ ID NO: 7, and the nucleotide sequence of the zwf3 gene is shown in SEQ ID NO: 8.

[0013] The key genes in the PHB synthesis pathway are the poly([R]-3-hydroxybutyrate) polymerase encoding gene phaC1 (H16_A1437), the acetyl-CoA acetyltransferase encoding gene phaA (H16_A1438), and the acetyl-CoA reductase encoding gene phaB1 (H16_A1439). The nucleotide sequence of the poly([R]-3-hydroxybutyrate) polymerase encoding gene phaC1 is shown in SEQ ID NO: 9, the nucleotide sequence of the acetyl-CoA acetyltransferase encoding gene phaA is shown in SEQ ID NO: 10, and the nucleotide sequence of the acetyl-CoA reductase encoding gene phaB1 is shown in SEQ ID NO: 11.

[0014] SEQ ID NO: 1 (nagE-encoded amino acid sequence)

[0015] SEQ ID NO: 1

[0016] 。

[0017] SEQ ID NO:2(nagE)

[0018] SEQ ID NO: 2:

[0019]

[0020] SEQ ID NO: 3 (nagR gene sequence)

[0021] Atggatcaacggctgcaggctctcaagccggacgaagcggaggcgacgccgatctacctgcaagtggcgcgcaggctggccgcggccatccaggccggccaatggcgggttggcgacgcgctgccgtccgagcgcacgctggtggattcactggagatttcacgcgtcacggcgcggcgcgcgctgcaggtgctggcagaggaaggcgcgatcacgcgcagccgcggcgcgggcacctttgtcgcgccgcgccctgagcagaaggcggcgcggctggacaacttcagcgagctggcgcgccggcgcggcatgacgccggccagcgaactggtggcgttcgaacgccgccgcgccacgccccaggaggctgcggcgctggcgctgcaggaaggggaagagattgtcagcctgacccgcctgcgcaaggccgacgggcaggtcttctggatggatgtcaccacgctggcactggccgtgctgcccgacgccagcgccatcggcgaatcgctgtacgcctacctggagcggatcggcaagccggtgctgcgcgtcaccgaaaggctgcgcgcgatcgtcgccggcgaagcactggccgcgcgcctgcagatcgcgcccggcgagccgctgctgcatatcctgcgcaccggctacacccatggcgaccagccggtcgaactgaccgacggctactgcctgaacgatttctacgagctgaagcagtag。

[0022] SEQ ID NO: 4 (ScIPS gene sequence)

[0023]

[0024] SEQ ID NO: 5 (EcIMP gene sequence),

[0025] Atgcacccgatgctgaacatcgccgtgcgcgccgcccgcaaggccggcaacctgatcgccaagaactacgagaccccggacgccgtggaggcctcgcagaagggctcgaacgacttcgtgaccaacgtggacaaggccgccgaggccgtgatcatcgacaccatccgcaagagctacccgcagcacaccatcatcaccgaggagtcgggcgagctggagggcaccgaccaggacgtgcagtgggtcattgacccgctggacggcaccaccaacttcatcaagcgcctgccgcacttcgccgtgagcatcgccgtgcgcatcaagggccgcaccgaggtggccgtggtgtacgacccgatgcgcaacgagctgttcaccgccacccgcggccagggcgcccagctgaacggctatcgcctgcgcggctcgaccgcccgcgacctggacggcacgatcctggccaccggcttcccgttcaaggccaagcagtacgccaccacctacatcaacatcgtgggcaagctgttcaacgagtgcgccgacttccgccgcaccggctcggccgccctggacctggcctatgtggccgccggccgcgtggacggcttcttcgaaatcggcctgcgcccgtgggacttcgccgccggcgagctgctggtgcgcgaagccggcggcatcgtgtcggacttcaccggcggccacaactacatgctgaccggcaacatcgtggccggcaacccgcgcgtggtgaaggccatgctggccaacatgcgcgacgagctgtcggacgccctgaagcgctaa。

[0026] SEQ ID NO: 6 (gene zwf1)

[0027]

[0028] SEQ ID NO: 7 (gene zwf2)

[0029]

[0030] SEQ ID NO: 8 (gene zwf3)

[0031]

[0032] SEQ ID NO: 9 (gene phaC1)

[0033]

[0034] SEQ ID NO: 10 (gene phaA)

[0035]

[0036] SEQ ID NO: 11(phaB1)

[0037] .

[0038] Another object of the present invention is to provide a method for producing inositol by fermentation, comprising the following steps: shaking flask fermentation of the above-mentioned engineered strain of Rollstonella that produces inositol to obtain inositol.

[0039] According to the method for producing inositol by fermentation according to the present invention, shake-flask fermentation is carried out using a culture medium with glucose, glycerol or CO2 as the sole carbon source.

[0040] According to the method for producing inositol by fermentation of the present invention, the fermentation medium is formulated as follows: 3.5 g / L Na2HPO4, 1.5 g / L KH2PO4, 1.0 g / L (NH4)2SO4, 80 mg / L MgSO4·7H2O, 1 mg / L CaSO4·2H2O, 0.56 mg / L NiSO4·7H2O, 0.4 mg / L ferric citrate, 200 mg / L NaHCO3, with 5 g / L glucose, glycerol, or a mixed gas of H2:O2:CO2≈8:1:1 added as carbon sources.

[0041] According to the method for producing inositol by fermentation according to the present invention, the final concentration of kanamycin antibiotic added to the fermentation medium is 200 mg / L.

[0042] According to the method for producing inositol by fermentation according to the present invention, the shake flask fermentation conditions are a temperature of 30°C and a rotation speed of 200 rpm.

[0043] Advantages of the technical solution in this application:

[0044] 1. The advantage of using the engineered strain of *Rolstonia* H16 for inositol synthesis lies in the fact that it cannot utilize inositol, thus eliminating the need for strain modification to achieve product accumulation. Furthermore, *Rolstonia* H16 possesses a broad substrate spectrum. It can utilize organic compounds such as glycerol as carbon sources for product synthesis and also exhibits highly efficient CO2 fixation capabilities, meaning it can directly utilize CO2 for inositol synthesis without the aid of electrochemical methods.

[0045] This application enables *R. rockburstii* to efficiently utilize glucose by mutating the gene *nagE* (G265R) encoding the N-acetylglucosamine-specific phosphotransferase system and knocking out *nagR*, which encodes a transcriptional regulator of the GntR family. Based on this, an engineered *R. rockburstii* strain H16 was constructed, capable of synthesizing inositol directly from renewable substrates (glucose and glycerol) and using CO2 as a carbon source. This technology not only enables the synthesis of bioactive substances but also provides an efficient method for eliminating greenhouse gases from the atmosphere.

[0046] 2. Inositol-3-phosphate synthases with high catalytic activity in *Rowstone* were screened. Inositol-3-phosphate synthases derived from *Trypanosoma brucei* or *Saccharomyces cerevisiae* were heterologously expressed in *Rowstone*, and inositol synthesis was carried out using endogenous inositol monophosphatase from *Rowstone* with glucose (5 g / L) as the sole carbon source. The results showed that *Trypanosoma brucei*-derived inositol-3-phosphate synthase (TbIPS) had no catalytic activity in *Rowstone*. The inositol yield of *Rowstone* strains heterologously expressed with *Saccharomyces cerevisiae*-derived inositol-3-phosphate synthase (ScIPS) was 122.5 mg / L.

[0047] 3. To increase inositol production in *Rowstone's* strain, *Escherichia coli*-derived inositol-3-phosphate synthase (ScIPS) gene was expressed heterologously in addition to *Saccharomyces cerevisiae* gene expression. Using glucose as the carbon source, compared to *Rowstone's* strain expressing only *Saccharomyces cerevisiae* gene expression, inositol production was increased by 2.2 times, reaching 394.2 mg / L.

[0048] 4. By knocking out key genes in the ED and PHB synthesis pathways individually or in combination, the diversion of glucose-6-phosphate, a precursor for glucose synthesis, was disrupted in each pathway, thus increasing the supply of product precursors in the chassis cells of mutant *R. roxtonii*. Inositol was produced by fermentation in a medium with glucose, glycerol, and CO2 as the sole carbon sources. The optimal strain achieved inositol yields of 520.2, 1076.3, and 1054.8 mg / L, respectively. Attached Figure Description

[0049] Figure 1 This shows the utilization of glucose and fructose and cell growth of wild-type and engineered Roldstone strains.

[0050] Figure 2 The cell dry weight of Rolstonia when screening for inositol-3-phosphate synthase from different sources using glucose as a carbon source is shown.

[0051] Figure 3 This shows the carbon source consumption of Rolstonia when screening for inositol-3-phosphate synthases from different sources using glucose as the carbon source.

[0052] Figure 4 This shows the results of inositol synthesis in Roldstone strain when glucose was used as a carbon source to screen inositol-3-phosphate synthases from different sources.

[0053] Figure 5 The results of cell dry weight determination of engineered Rawstone strains when synthesizing inositol using glucose as a carbon source are shown.

[0054] Figure 6 This shows the carbon source consumption results of engineered Rawstone strains when synthesizing inositol using glucose as a carbon source.

[0055] Figure 7 The results show that engineered strains of Rollstonella synthesize inositol using glucose as a carbon source.

[0056] Figure 8 The results of cell dry weight determination of engineered Rawlstonella strains synthesizing inositol using glycerol as a carbon source are shown.

[0057] Figure 9 This shows the carbon source consumption results of engineered Rawstone strains when synthesizing inositol using glycerol as a carbon source;

[0058] Figure 10 The results show that engineered strains of Rollstonella synthesize inositol using glycerol as a carbon source;

[0059] Figure 11 The results of cell dry weight determination of engineered Rawlstonella strains when synthesizing inositol using CO2 as a carbon source are shown.

[0060] Figure 12 This shows the results of engineered Rawstone strains synthesizing inositol using CO2 as a carbon source. Detailed Implementation

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0062] The modification process of Rollstonella in this application is shown in Table 1 below:

[0063] Table 1

[0064]

[0065]

[0066] Example 1: Construction of a Rawlstonella H16-RE strain with glucose utilization capability

[0067] Rawstone's H16 strain can utilize fructose, glycerol, and CO2 as carbon sources for growth. Glucose, as an inexpensive carbon source, has been used in metabolic engineering strains to produce various bioactive substances, such as γ-aminobutyric acid (GABA) and N-acetylglucosamine. However, due to the lack of transport proteins, Rawstone's H16 cannot utilize glucose. Therefore, this application constructs a glucose-utilizing Rawstone's H16-RE strain by mutating the gene nagE (G265R) encoding the N-acetylglucosamine-specific phosphotransferase system in the Rawstone's genome and knocking out the transcriptional regulator nagR of the GntR family. The strain was cultured in a medium with 10 g / L glucose or its natural carbon source, fructose, as the sole carbon source. The results of the strain's carbon source utilization and cell growth are as follows: Figure 1 As shown.

[0068] The specific method for amino acid mutation (G265R) of the gene nagE encoding the N-acetylglucosamine-specific phosphotransferase system in the Roldstone genome and knocking out the gene nagR encoding the transcriptional regulator of the GntR family is as follows:

[0069] 1. Construct the mutant plasmid pK18-nagE(G265R) with upstream and downstream homologous arms of the nagE gene sequence mutation point (G793C) and the mutant plasmid pK18-nagR with upstream and downstream homologous arms of the nagR gene sequence.

[0070] Primers were designed and synthesized based on the nagE (H16_A0312) and nagR (H16_A0310) gene sequences. Using the Rollstonella H16 genome as a template, the upstream and downstream homologous arms of the nagE and nagR genes were amplified using the designed primers, with fragment sizes of approximately 500 bp. The obtained fragments were ligated to the EcoRI / SmaI site of the pK18mobSacB plasmid using Gibson Assembly. The plasmid was electroporated into Escherichia coli S17-1 host, plated on LB agar plates (50 μg / mL kanamycin), and incubated overnight at 37°C. Positive clones were then screened, targeting a 1000 bp fragment. These clones were named pK18-nagE (G265R) and pK18-nagR.

[0071] 2. Constructing the pK18-nagE(G265R) plasmid and integrating it into the genome of Rawlstonella H16-E strain.

[0072] Escherichia coli S17-1 and Rawlstonella H16 strains carrying the pK18-nagE(G265R) plasmid were inoculated into LB liquid medium and cultured overnight, with appropriate antibiotics added (kanamycin 50 μg / mL or gentamicin 10 μg / mL). The cells were collected by centrifugation at 4600 rpm for 8 min and washed three times with LB medium. A mixture of E. coli S17-1 and Rawlstonella H16 strains carrying the pK18-nagE(G265R) plasmid was then spotted onto LB plates and incubated overnight at 30°C. The overnight mixed cells were washed off with LB medium and spread onto LB solid plates (kanamycin 200 μg / mL and gentamicin 10 μg / mL). After incubation at 30°C for 48 hours, positive clones were screened for the kanamycin resistance gene fragment, 500 bp in length. Positive clones were cultured overnight at 30°C in 1.5 mL EP tubes containing LB liquid medium.

[0073] 3. A Rowstonella H16-E strain with a successfully mutated nagE gene was constructed.

[0074] Dilute the bacterial culture to 10. -2 The culture was plated on LB agar plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to detect the target fragment size (1000 bp), and sequencing was performed to verify the result. Clones with successfully mutated target genes were screened. The successfully constructed strain was inoculated into LB liquid medium and named *Rowstoneella H16-E*.

[0075] 4. Constructing the pK18-nagR plasmid and integrating it into the genome of Rawlstonella H16-E strain

[0076] Escherichia coli S17-1 and Rawlstonella H16 strains carrying the pK18-nagR plasmid were inoculated into LB liquid medium and cultured overnight, with appropriate antibiotics added (kanamycin 50 μg / mL or gentamicin 10 μg / mL). The bacterial cells were collected by centrifugation at 4600 rpm for 8 min and washed three times with LB medium. A mixture of E. coli S17-1 and Rawlstonella H16 strains carrying the pK18-nagR plasmid was then spotted onto LB plates and incubated overnight at 30°C. The overnight mixed bacterial cells were washed off with LB medium and spread onto LB solid plates (kanamycin 200 μg / mL and gentamicin 10 μg / mL). After incubation at 30°C for 48 hours, positive clones were screened for the kanamycin resistance gene fragment (500 bp in length). The positive clones were then cultured overnight at 30°C in 1.5 mL EP tubes containing LB liquid medium.

[0077] 5. Construction of a Rawlstonella H16-RE strain with the nagR gene successfully knocked out.

[0078] Dilute the bacterial culture to 10. -2 The culture was plated on LB agar plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to detect the target fragment size (1000 bp), and clones with successfully knocked-out target genes were screened. The successfully constructed strain was inoculated into LB liquid medium and named *Rowstoneella H16-RE*.

[0079] The results showed that both wild-type and mutant strains exhibited a lag phase of up to 16 hours when cultured using glucose or fructose as the sole carbon source. After 32 hours of culture in a glucose-based medium, the wild-type strain showed no change in glucose levels or cell dry weight. However, in a fructose-based medium, the carbon source concentration decreased to 6.3 g / L, while the cell dry weight increased to 1.4 g / L. In the wild-type strain's fructose-based medium, fructose was depleted after 64 hours, with a maximum cell dry weight of 3.5 g / L, while glucose was never consumed, indicating that the wild-type strain lacked glucose utilization capabilities. For the mutant strain *Rowstoneella* H16-RE, after 32 hours of fermentation, the glucose concentration in the medium was 7.6 g / L, the fructose concentration was 5.8 g / L, and the cell dry weights were 0.8 g / L and 1.4 g / L, respectively. The glucose and fructose in the culture medium of the mutant strain were completely depleted after 64 hours of fermentation, and the cell dry weights reached 3.9 and 3.8 g / L, respectively. This indicates that the mutant strain has the ability to utilize glucose as a carbon source, and its rate of glucose utilization is comparable to that of fructose, the optimal natural carbon source for Rawlstonella.

[0080] Example 2: Screening of inositol-3-phosphate synthase suitable for inositol synthesis by Rollstonella

[0081] The enzymatic synthesis of inositol requires a two-step catalytic reaction. First, glucose-6-phosphate is converted to inositol-3-phosphate by inositol-3-phosphate synthase, and then this product is converted to the final product inositol by inositol monophosphatase. Inositol-3-phosphate synthase is the rate-limiting step in this reaction. The suhB(H16_A1214) gene encoding inositol monophosphatase has been identified in the *Rowstoneella* genome (GenBank: AM260479.1), but the inositol-3-phosphate synthase gene is absent. Therefore, inositol-3-phosphate synthases from different sources were screened.

[0082] 1. Construct expression vectors TbIPS-pBBR1 and ScIPS-pBBR1 containing the TbIPS or ScIPS gene.

[0083] Primers were designed and synthesized based on the sequence of the PphaC1 promoter from *Ralstonia solanacearum*. Using the *Ralstonia solanacearum* genome as a template, the PphaC1 promoter fragment was amplified, with a fragment size of 342 bp. The inositol-3-phosphate synthase genes from *Trypanosoma brucei* and *Saccharomyces cerevisiae* were synthesized after codon optimization. Primers were designed and synthesized based on the gene sequences. Using the synthesized gene fragments as templates, the inositol-3-phosphate synthase gene fragments from *Trypanosoma brucei* and *Saccharomyces cerevisiae* were amplified using the designed primers, with fragment sizes of 1587 bp and 1602 bp, respectively. The obtained promoter fragments and gene fragments were ligated to the EcoRI / SmaI site of the pBBR1-MCS2 vector plasmid using Gibson Assembly to obtain the ScIPS-pBBR1 and TbIPS-pBBR1 expression vectors.

[0084] 2. Construction of Rawlstonella H16-RE-TbIPS and Rawlstonella H16-RE-ScIPS strains

[0085] Rawstone's H16-RE strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 200 mL of LB agar and incubated at 30°C and 200 rpm until OD500 was reached. 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. Add 400 ng of TbIPS-pBBR1 or ScIPS-pBBR1 plasmid to 100 μL of competent cells and transfer to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at 2.5 kV. Add 200 μL of LB broth to resuspend the cells and incubate at 30°C and 200 rpm for 2 h on a shaker. Spread the mixture onto LB agar plates containing 200 μg / mL kanamycin and 10 μg / mL gentamicin. Positive clones were identified by PCR. Finally, *Rowstoneella* H16-RE-TbIPS and *Rowstoneella* H16-RE-ScIPS strains were obtained.

[0086] 3. Screening of inositol-3-phosphate synthases suitable for synthesizing inositol in Ralstonia

[0087] Each mutant strain was fermented in a saline medium with glucose at a concentration of 5 g / L as the sole carbon source. A negative control was obtained by transforming pBBR1-MCS2 into *Rowstoneella H16-RE* to obtain *Rowstoneella H16-RE*-pBBR1. The growth of each strain in the fermentation medium is shown below. Figure 2 , Figure 3 As shown.

[0088] After one day of fermentation, the glucose in the medium for *Ralstonia solanacearum* H16-RE-pBBR1 and *Ralstonia solanacearum* H16-RE-TbIPS was completely depleted, with final cell dry weights of 1.5 and 1.4 g / L, respectively. At this point, the glucose concentration in the medium for *Ralstonia solanacearum* H16-RE-ScIPS was 2.1 g / L. After two days of fermentation, the cell dry weight of *Ralstonia solanacearum* H16-RE-ScIPS reached 1.3 g / L, at which point the glucose in the medium was completely depleted.

[0089] Although previous studies have shown that inositol-3-phosphate synthase derived from Trypanosoma brevicornu is more efficient than that derived from Saccharomyces cerevisiae and has been applied to the synthesis of inositol in Escherichia coli, in this application, the product inositol can only be detected in the supernatant of the culture medium of Ralstonia solanacearum H16-RE-ScIPS strain. Figure 4 This indicates that inositol-3-phosphate synthase (TbIPS) derived from Trypanosoma brevicornu is inactive or its gene cannot be expressed in *Rowstoneella*. As a two-step enzymatic reaction, product synthesis is slightly delayed relative to carbon source consumption. After 5 days of fermentation, the inositol concentration in the *Rowstoneella* H16-RE-ScIPS fermentation broth was 122.5 mg / L.

[0090] Example 3: Construction of the inositol synthesis pathway in Rollstonella

[0091] Based on the previously constructed *Rowstoneella* H16-RE-ScIPS strain capable of synthesizing inositol from glucose as a carbon source, this application further investigates the effect of inositol monophosphatase on inositol production. The inositol monophosphatase (EcIMP) gene from *Escherichia coli*, possessing high catalytic activity, was ligated into the pre-constructed *ScIPS-pBBR1* gene and regulated by the PphaC1 promoter, which regulates *ScIPS* expression, resulting in the expression vector *ScIPS-EcIMP-pBBR1*. This expression vector was then introduced into *Rowstoneella* H16-RE-ScIPS to construct *Rowstoneella* H16-RE-ScIPS-EcIMP* for inositol production.

[0092] 1. Construct the expression vector ScIPS-EcIMP-pBBR1 containing the ScIPS and EcIMP genes.

[0093] Primers were designed and synthesized based on the sequence of the PphaC1 promoter from *Ralstonia solanacearum*. Using the *Ralstonia solanacearum* genome as a template, the PphaC1 promoter fragment was amplified, with a size of approximately 342 bp. The inositol-3-phosphate synthase gene from *Saccharomyces cerevisiae* and the inositol monophosphatase gene from *Escherichia coli* were synthesized after codon optimization. Primers were designed and synthesized based on the gene sequences. Using the synthesized gene fragments as templates, the 1602 bp inositol-3-phosphate synthase gene fragment from *Saccharomyces cerevisiae* and the 804 bp inositol monophosphatase gene fragment from *E. coli* were amplified using the designed primers. The obtained promoter fragment and gene fragments were ligated to the EcoRI / SmaI site of the pBBR1-MCS2 vector plasmid using GibsonAssembly to obtain the ScIPS-EcIMP-pBBR1 expression vector.

[0094] 2. Construction of Rollstonella H16-RE-ScIPS-EcIMP strain

[0095] Rawstone's H16-RE strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 200 mL of LB agar and incubated at 30°C and 200 rpm until OD500 was reached. 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. Add 400 ng of ScIPS-EcIMP-pBBR1 plasmid to 100 μL of competent cells and transfer to a 2 mm Bio-Rad electroporation cuvette for electroporation at 2.5 kV. Add 200 μL of LB broth to resuspend the cells and incubate at 30°C and 200 rpm for 2 h on a shaker. Spread the mixture onto LB agar plates containing 200 μg / mL kanamycin and 10 μg / mL gentamicin. Positive clones were identified by PCR. The final strain of *Rowstoneella* H16-RE-ScIPS-EcIMP was obtained.

[0096] The strain was fermented in a saline medium with 5 g / L glucose as the sole carbon source, and the results were as follows: Figure 2 , Figure 3 and Figure 4 As shown. After one day of fermentation, its maximum cell dry weight reached 1.4 g / L, which is comparable to and superior to the Rawstone's H16-RE-ScIPS strain. After overexpression of Escherichia coli-derived inositol monophosphatase, the inositol yield increased from 122.5 mg / L to 394.2 mg / L.

[0097] Example 4: Construction of an engineered Rawlstonella strain that produces inositol

[0098] The engineered *Rowstone* strain producing inositol is a mutant strain expressing genes for enzymes required in the inositol synthesis pathway, and having key genes knocked out in the ED pathway and PHB synthesis pathway. The enzyme genes in the inositol synthesis pathway are inositol-3-phosphate synthase from *Saccharomyces cerevisiae* and inositol monophosphatase from *Escherichia coli*. The key genes in the ED pathway are the glucose-6-phosphate dehydrogenase encoding genes zwf1 (H16_A0316), zwf2 (H16_B1501), and zwf3 (H16_B2566). The key genes in the PHB synthesis pathway are the poly([R]-3-hydroxybutyrate) polymerase encoding gene phaC1 (H16_A1437), the acetyl-CoA acetyltransferase encoding gene phaA (H16_A1438), and the acetyl-CoA reductase encoding gene phaB1 (H16_A1439).

[0099] I. Construction of Rollstonella H16-RE△zwf-ScIPS-EcIMP strain

[0100] The zwf gene knockout vectors pK18-zwf1, pK18-zwf2, and pK18-zwf3 were sequentially introduced into the *Ralstonia solanacearum* H16-RE host using a binding transfer method. A first round of screening was performed using kanamycin to obtain host strains where the knockout vectors were successfully integrated into the strain genome. A second round of screening was then performed using sucrose to obtain *Ralstonia solanacearum* H16-RE△zwf strains with the zwf gene successfully knocked out. The specific methods are as follows:

[0101] 1. Construct knockout plasmids pK18-zwf1, pK18-zwf2, and pK18-zwf3 containing upstream and downstream homologous arms of the zwf1, zwf2, and zwf3 gene sequences.

[0102] Primers were designed and synthesized based on the gene sequences of zwf1 (H16_A0316), zwf2 (H16_B1501), and zwf3 (H16_B2566). Using the *Rowstoneella* H16 genome as a template, the upstream and downstream homologous arms of the *glk* gene were amplified using the designed primers, with fragments approximately 500 bp in size. The obtained fragments were ligated to the EcoRI / SmaI site of the pK18mobSacB plasmid using Gibson Assembly. The plasmid was electroporated into *E. coli* S17-1 host, plated on LB agar plates (50 μg / mL kanamycin), and incubated overnight at 37°C. Positive clones were then screened, targeting a 1000 bp fragment. These clones were named pK18-zwf1, pK18-zwf2, and pK18-zwf3.

[0103] 2. Constructing the pK18-zwf1 plasmid and integrating it into the genome of the Rawlstonella H16-RE strain.

[0104] Escherichia coli S17-1 and Rawlstonella H16-RE strains carrying the pK18-zwf1 plasmid were inoculated into LB liquid medium and cultured overnight, with appropriate antibiotics added (kanamycin 50 μg / mL or gentamicin 10 μg / mL). The bacterial cells were collected by centrifugation at 4600 rpm for 8 min and washed three times with LB medium. A mixture of E. coli S17-1 and Rawlstonella H16-RE strains carrying the pK18-zwf1 plasmid was then spotted onto LB plates and incubated overnight at 30°C. The overnight mixed bacterial cells were washed off with LB medium and spread onto LB solid plates (kanamycin 200 μg / mL and gentamicin 10 μg / mL). After incubation at 30°C for 48 hours, positive clones were screened for the kanamycin resistance gene fragment, 500 bp in length. Positive clones were cultured overnight at 30°C in 1.5 mL EP tubes containing LB liquid medium.

[0105] 3. Construction of a Rawlstonella H16-RE strain with the zwf1 gene successfully knocked out.

[0106] Dilute the bacterial culture to 10. -2 The culture was plated on LB agar plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to screen clones with successfully knocked-out target gene fragments (1000 bp). The successfully constructed strain was inoculated into LB liquid medium and named *Rowstoneella H16-RE△zwf1*.

[0107] 4. Construction of a Rawlstonella H16-RE strain with successful knockout of zwf2 and zwf3 genes.

[0108] Following the steps described above, using *Rolstonia H16-RE△zwf1* and *Rolstonia H16-RE△zwf1△zwf2* as hosts, zwf2 and zwf3 were knocked out, respectively. The final *Rolstonia H16-RE△zwf* strain was obtained.

[0109] 5. Construction of a Rawstone strain H16-RE△zwf-ScIPS-EcIMP that synthesizes inositol

[0110] Rawstone's H16-RE△zwf strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 200 mL of LB agar and incubated at 30°C and 200 rpm until OD200 was reached. 600≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. Add 400 ng of ScIPS-EcIMP-pBBR1 plasmid to 100 μL of competent cells and transfer to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at 2.5 kV. Add 200 μL of LB broth to resuspend the cells and incubate at 30°C and 200 rpm for 2 h on a shaker. Spread the mixture onto LB agar plates containing 200 μg / mL kanamycin and 10 μg / mL gentamicin. Positive clones were identified by PCR. The final strain of *Rowstoneella* H16-RE△zwf-ScIPS-EcIMP was obtained.

[0111] II. Construction of Rawlstonella H16-RE△PHB-ScIPS-EcIMP strain

[0112] The target knockout vector pK18-PHB, containing the phaC1AB1 operon (and its phaC1, phaA, and phaB1 genes), was introduced into the *Ralstonia solanacearum* H16-RE host using a binding transfer method. A first round of screening was performed using kanamycin to obtain host strains where the knockout vector had successfully integrated into the genome. A second round of screening was then performed using sucrose to obtain the *Ralstonia solanacearum* H16-RE△PHB strain, which successfully knocked out the phaC1AB1 operon.

[0113] The specific method is as follows:

[0114] 1. Construct the knockout plasmid pK18-phaC1AB1 with upstream and downstream homologous arms of the phaC1AB1 operon sequence.

[0115] Primers were designed and synthesized based on the sequences of the phaC1AB1 operon (phaC1(H16_A1437), phaA(H16_A1438), and phaB1(H16_A1439)). Using the Rollstonella H16 genome as a template, the upstream and downstream homologous arms of the operon gene were amplified using the designed primers, with fragments approximately 500 bp in size. The obtained fragments were ligated to the EcoRI / SmaI site of the pK18mobSacB plasmid using Gibson Assembly. The plasmid was electroporated into Escherichia coli S17-1 host, plated on LB agar plates (50 μg / mL kanamycin), and incubated overnight at 37°C. Positive clones were then selected, targeting a 1000 bp fragment. This clone was named pK18-phaC1AB1.

[0116] 2. Constructing the pK18-phaC1AB1 plasmid and integrating it into the genome of the Rawlstonella H16-RE strain.

[0117] Escherichia coli S17-1 and Rawlstonella H16-RE strains carrying the pK18-phaC1AB1 plasmid were inoculated into LB liquid medium and cultured overnight, with appropriate antibiotics added (kanamycin 50 μg / mL or gentamicin 10 μg / mL). The cells were collected by centrifugation at 4600 rpm for 8 min and washed three times with LB medium. A mixture of E. coli S17-1 and Rawlstonella H16-RE strains carrying the pK18-phaC1AB1 plasmid was then spotted onto LB plates and incubated overnight at 30°C. The overnight mixed cells were washed off with LB medium and spread onto LB solid plates (kanamycin 200 μg / mL and gentamicin 10 μg / mL). After incubation at 30°C for 48 hours, positive clones were screened for the kanamycin resistance gene fragment, 500 bp in length. Positive clones were cultured overnight at 30°C in 1.5 mL EP tubes containing LB liquid medium.

[0118] 3. Constructing a Rawlstonella H16-RE strain with the phaC1, phaA, and phaB1 genes knocked out.

[0119] Dilute the bacterial culture to 10. -2 The culture was spread onto LB agar plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to screen clones with successfully knocked-out target genes (target fragment size 1000 bp). The successfully constructed strain was inoculated into LB liquid medium and named *Rowstoneella H16-RE-△PHB*.

[0120] 4. Construction of a Rawstone strain H16-RE△PHB-ScIPS-EcIMP for synthesizing inositol

[0121] Rawstone's H16-RE△PHB strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 200 mL of LB agar and incubated at 30°C and 200 rpm until OD500 was reached. 600≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. Add 400 ng of ScIPS-EcIMP-pBBR1 plasmid to 100 μL of competent cells and transfer to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at 2.5 kV. Add 200 μL of LB broth to resuspend the cells and incubate at 30°C and 200 rpm for 2 h on a shaker. Spread the mixture onto LB agar plates containing 200 μg / mL kanamycin and 10 μg / mL gentamicin. Positive clones were identified by PCR. The final strain of *Rowstoneella* H16-RE△PHB-ScIPS-EcIMP was obtained.

[0122] III. Construction of Rawlstonella H16-RE△zwf△PHB-ScIPS-EcIMP strain

[0123] The target knockout vector pK18-PHB, containing the phaC1AB1 operon (containing the phaC1, phaA, and phaB1 genes), was introduced into the *Ralstonia solanacearum* H16-RE△zwf host using a binding transfer method. A first round of screening was performed using kanamycin to obtain host strains where the knockout vector was successfully integrated into the strain's genome. A second round of screening was then performed using sucrose to obtain the *Ralstonia solanacearum* H16-RE△zwf△PHB strain with the phaC1AB1 operon successfully knocked out. The specific methods are as follows:

[0124] 1. Constructing the pK18-phaC1AB1 plasmid and integrating it into the genome of the Rawlstonella H16-RE△zwf strain.

[0125] Escherichia coli S17-1 and Rawlstonella H16-RE△zwf strains carrying the pK18-phaC1AB1 plasmid were inoculated into LB liquid medium and cultured overnight, with appropriate antibiotics added (kanamycin 50 μg / mL or gentamicin 10 μg / mL). The cells were collected by centrifugation at 4600 rpm for 8 min and washed three times with LB medium. A mixture of E. coli S17-1 and Rawlstonella H16-RE△zwf strains carrying the pK18-phaC1AB1 plasmid was then spotted onto LB plates and incubated overnight at 30°C. The overnight mixed cells were washed off with LB medium and spread onto LB solid plates (kanamycin 200 μg / mL and gentamicin 10 μg / mL). After incubation at 30°C for 48 hours, positive clones were screened for the kanamycin resistance gene fragment, 500 bp in length. Positive clones were cultured overnight at 30°C in EP tubes containing 1.5 mL of LB liquid medium.

[0126] 2. A Rawlstonella H16-RE△zwf strain with successful knockout of the phaC1, phaA, and phaB1 genes was constructed.

[0127] Dilute the bacterial culture to 10. -2 The culture was plated on LB agar plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to screen clones with successfully knocked-out target genes (target fragment size 1000 bp). The successfully constructed strain was inoculated into LB liquid medium and named *Rowstoneella H16-RE△zwf△PHB*.

[0128] 3. Construction of a Rawstoneella H16-RE△zwf△PHB-ScIPS-EcIMP strain for synthesizing inositol

[0129] Rawstone's H16-RE△zwf△PHB strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 200 mL of LB agar and incubated at 30°C and 200 rpm until OD500 was reached. 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. Add 400 ng of ScIPS-EcIMP-pBBR1 plasmid to 100 μL of competent cells and transfer to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at 2.5 kV. Add 200 μL of LB broth to resuspend the cells and incubate at 30°C and 200 rpm for 2 h on a shaker. Spread the mixture onto LB agar plates containing 200 μg / mL kanamycin and 10 μg / mL gentamicin. Positive clones were identified by PCR. The final strain of *Rowstoneella* H16-RE△zwf△PHB-ScIPS-EcIMP was obtained.

[0130] Example 5: Production of inositol by fermentation using an engineered strain of Rollstonella using glucose as a carbon source.

[0131] The engineered strains of Rawlstonella H16-RE-pBBR1 and the inositol-producing Rawlstonella H16-RE-ScIPS-EcIMP, Rawlstonella H16-RE△zwf-ScIPS-EcIMP, Rawlstonella H16-RE△PHB-ScIPS-EcIMP, and Rawlstonella H16-RE△zwf△PHB-ScIPS-EcIMP were inoculated into 50 mL of LB liquid medium supplemented with 200 mg / L kanamycin and rejuvenated at 30°C and 200 rpm for 16 hours.

[0132] Preparation of fermentation medium with glucose as the sole carbon source: Accurately weigh 3.5g Na2HPO4, 1.5g KH2PO4, 1.0g (NH4)2SO4, 80mg MgSO47H2O, 1mg CaSO4·2H2O, 0.56mg NiSO4·7H2O, 0.4mg ferric citrate, 200mg NaHCO3, and 5g glucose. Make up to 1L with water as solvent. Autoclave at 115℃ for 30 minutes to obtain fermentation medium with glucose as the sole carbon source.

[0133] Engineered strains of *Ralstonia solanacearum* H16-RE-pBBR1, H16-RE-ScIPS, H16-RE-ScIPS-EcIMP, H16-RE△zwf-ScIPS-EcIMP, H16-RE△PHB-ScIPS-EcIMP, and H16-RE△zwf△PHB-ScIPS-EcIMP were transferred at a 1% inoculum to 200 mL of fermentation medium supplemented with 200 mg / L kanamycin and cultured overnight in shake flasks at 30°C and 200 rpm. OD... 600 Once the OD value reaches 1.0 or higher, collect the bacterial cells by centrifugation at 4600 rpm for 8 min. After washing three times with PBS, resuspend the bacterial cells in fermentation medium and adjust the OD value. 600 ≈1.0, and shake-flask fermentation was carried out at 30℃ and 200 rpm. Samples were taken periodically to determine the concentrations of glucose and inositol in the culture medium.

[0134] Bacterial liquid OD 600 Measurement: The absorbance of the bacterial culture at a wavelength of 600 nm was measured using a visible light spectrophotometer. Cell dry weight was calculated according to the formula reported in the literature: Cell dry weight (g / L) / OD 600 The value was calculated to be approximately 0.363 g / L.

[0135] Glucose and inositol concentrations were determined using high-performance liquid chromatography (HPLC). A Shimadzu parallax refractive index detector was used, and an Agilent Hi-Plex Ca column (300 mm × 7.7 mm) was employed at a column temperature of 80 °C. Ultrapure water was used as the mobile phase at a flow rate of 0.5 mL / min, with an injection volume of 10 μL.

[0136] like Figure 5After one day of fermentation, the cell dry weights of *Ralstonia solanacearum* H16-RE-pBBR1, *Ralstonia solanacearum* H16-RE-ScIPS-EcIMP, and *Ralstonia solanacearum* H16-RE△PHB-ScIPS-EcIMP reached their maximum values, at 1.50, 1.45, and 0.86 g / L, respectively. However, the dry weights of *Ralstonia solanacearum* H16-RE△zwf-ScIPS-EcIMP and *Ralstonia solanacearum* H16-RE△zwf△PHB-ScIPS-EcIMP* strains did not reach their maximum values ​​until five days of fermentation, at 0.77 and 1.23 g / L, respectively. Truncation of the ED pathway resulted in a relatively long lag phase for the strains. Truncation of either the ED pathway or the PHB synthesis pathway alone led to relatively low biomass, while the destination of the carbon flux remains unknown. The maximum cell dry weight of *Rolstonia solanacearum* H16-RE△zwf△PHB-ScIPS-EcIMP, which simultaneously interrupts both the ED and PHB synthesis pathways, is higher than that of strains with only the ED pathway interrupted (*Rolstonia solanacearum* H16-RE△zwf-ScIPS-EcIMP). This may be because the carbon flux supplied to the strain's growth is increased after the PHB pathway is interrupted.

[0137] Based on glucose utilization rates, the strains can be divided into three categories: *Rolstonia* H16-RE-pBBR1, *Rolstonia* H16-RE-ScIPS-EcIMP, and *Rolstonia* H16-RE△PHB-ScIPS-EcIMP, all of which deplete glucose in their fermentation broth after one day of fermentation. At this point, the glucose levels in the fermentation broths of *Rolstonia* H16-RE△zwf-ScIPS-EcIMP and *Rolstonia* H16-RE△zwf△PHB-ScIPS-EcIMP were 4.5 g / L and 4.7 g / L, respectively, and the glucose was not depleted until five days into fermentation. This is consistent with the data from cell dry weight measurements.

[0138] Inositol detection results showed that, except for Rawlstonella H16-RE-pBBR1, inositol was detected in the supernatant of the fermentation broth of all other strains. Figure 7As shown, when glucose is used as the sole carbon source, glucose consumption mainly occurs via the ED pathway. Truncation of the ED pathway increases the supply of inositol precursors (glucose-6-phosphate), thus significantly increasing inositol production. After 7 days of fermentation, the inositol yield of *Rolstonia hygroscopici* H16-RE△zwf-ScIPS-EcIMP reached 520.2 mg / L, 1.4 times that of the *Rolstonia hygroscopici* H16-RE-ScIPS-EcIMP strain. However, truncation of the PHB synthesis pathway did not increase inositol production in *Rolstonia hygroscopici*, a result consistent with studies on methyl ketone synthesis. This may be because the carbon flux is lost as metabolites such as pyruvate after PHB knockout. The inositol yields of *Ralstonia solanacearum* strains H16-RE△PHB-ScIPS-EcIMP and H16-RE△zwf△PHB-ScIPS-EcIMP were 213.5 and 248.6 mg / L, respectively, significantly lower than those of strain H16-RE-ScIPS-EcIMP. In conclusion, truncating the ED pathway is beneficial for *Ralstonia solanacearum* to synthesize inositol using glucose as a carbon source, while truncating the PHB synthesis pathway leads to complex metabolic changes in the strain, thus negatively impacting inositol synthesis.

[0139] Example 6: Production of inositol by fermentation using engineered strains of Rollstonella using glycerol as a carbon source.

[0140] The engineered strains of Rawlstonella H16-RE-pBBR1 and the inositol-producing Rawlstonella H16-RE-ScIPS-EcIMP, Rawlstonella H16-RE△zwf-ScIPS-EcIMP, Rawlstonella H16-RE△PHB-ScIPS-EcIMP, and Rawlstonella H16-RE△zwf△PHB-ScIPS-EcIMP were inoculated into 50 mL of LB liquid medium supplemented with 200 mg / L kanamycin and rejuvenated at 30°C and 200 rpm for 16 hours.

[0141] Preparation of fermentation medium with glycerol as the sole carbon source: Accurately weigh 3.5g Na2HPO4, 1.5g KH2PO4, 1.0g (NH4)2SO4, 80mg MgSO4·7H2O, 1mg CaSO4·2H2O, 0.56mg NiSO4·7H2O, 0.4mg ferric citrate, 200mg NaHCO3, and 5g glycerol. Make up to 1L with water as solvent. Autoclave at 115℃ for 30 minutes to obtain fermentation medium with glycerol as the sole carbon source.

[0142] Rawstone's H16-RE-pBBR1 and engineered strains of Rawstone's H16-RE-ScIPS-EcIMP, H16-RE△zwf-ScIPS-EcIMP, H16-RE△PHB-ScIPS-EcIMP, and H16-RE△zwf△PHB-ScIPS-EcIMP producing inositol were transferred at a 1% inoculum to 200 mL of fermentation medium supplemented with 200 mg / L kanamycin and cultured overnight in shake flasks at 30°C and 200 rpm. OD was then calculated. 600 Once the OD value reaches 1.0 or higher, collect the bacterial cells by centrifugation at 4600 rpm for 8 min. After washing three times with PBS, resuspend the bacterial cells in fermentation medium and adjust the OD value. 600 ≈1.0, and shake-flask fermentation was carried out at 30℃ and 200 rpm. Samples were taken periodically to determine the concentrations of inositol and glycerol in the culture medium.

[0143] Bacterial liquid OD 600 Measurement: The absorbance of the bacterial culture at a wavelength of 600 nm was measured using a visible light spectrophotometer. Cell dry weight was calculated according to the formula reported in the literature: Cell dry weight (g / L) / OD 600 The value was calculated to be approximately 0.363 g / L.

[0144] Glycerol and inositol concentrations were determined using high-performance liquid chromatography (HPLC). A Shimadzu parallax refractive index detector was used, and an Agilent Hi-Plex Ca column (300 mm × 7.7 mm) was employed at a column temperature of 80 °C. Ultrapure water was used as the mobile phase at a flow rate of 0.5 mL / min, with an injection volume of 10 μL.

[0145] like Figure 8As shown, after 4 days of fermentation, the maximum cell dry weights of *Ralstonia solanacearum* H16-RE-pBBR1 and *Ralstonia solanacearum* H16-RE-ScIPS-EcIMP strains were 1.3 and 1.1 g / L, respectively. The cell dry weights of *Ralstonia solanacearum* H16-RE△zwf-ScIPS-EcIMP and *Ralstonia solanacearum* H16-RE△PHB-ScIPS-EcIMP strains reached their highest values ​​after 5 days of fermentation, at 1.0 and 0.8 g / L, respectively. The *Ralstonia solanacearum* H16-RE△zwf△PHB-ScIPS-EcIMP strain, which had its ED and PHB synthesis pathways interrupted, continued to grow until 7 days later, with a maximum cell dry weight of 0.8 g / L. Glycerol in the medium of *Rolstonia solanacearum* H16-RE-pBBR1 was depleted after four days of fermentation, while the glycerol concentrations in the fermentation broths of *Rolstonia solanacearum* H16-RE-ScIPS-EcIMP, *Rolstonia solanacearum* H16-RE△zwf-ScIPS-EcIMP, *Rolstonia solanacearum* H16-RE△PHB-ScIPS-EcIMP, and *Rolstonia solanacearum* H16-RE△zwf△PHB-ScIPS-EcIMP were 0.4, 0.9, 1.8, and 3.8 g / L, respectively.

[0146] Except for the Rawlstonella H16-RE-pBBR1 strain, inositol was detected in the fermentation broth of all other strains. Previous reports have shown that Rawlstonella metabolizes glycerol via gluconeogenesis and the endogenous glycogenotoxic (ED) pathway. Truncation of the ED pathway results in relatively low biomass, but the increased supply of product precursors leads to higher inositol production. Compared to the 539.8 mg / L inositol production of the Rawlstonella H16-RE-ScIPS-EcIMP strain, the inositol production of the Rawlstonella H16-RE△zwf-ScIPS-EcIMP strain (1076.3 mg / L) increased by 99.4% (e.g., ...). Figure 10 (As shown). However, the inositol production of *Rowstoneella* strain H16-RE△PHB-ScIPS-EcIMP decreased to 386.8 mg / L. This may be attributed to the reduced biomass resulting from the knockout of the PHB synthesis pathway. Furthermore, according to literature reports, the knockout of the PHB synthesis pathway leads to the loss of carbon flux in the form of pyruvate, which may also be one of the reasons for the decreased inositol production. In addition, although the double truncation of the ED pathway and the PHB synthesis pathway severely affected bacterial growth and resulted in slow carbon source consumption (e.g., ... Figure 10 (as shown), but this also blocks the loss of carbon flux, thereby increasing the inositol production to a final yield of 753.8 mg / L.

[0147] Example 7: Production of inositol by fermentation using engineered strains of Rollstonella with CO2 as a carbon source

[0148] The engineered strains of Rawlstonella H16-RE-pBBR1 and the inositol-producing Rawlstonella H16-RE-ScIPS-EcIMP, Rawlstonella H16-RE△zwf-ScIPS-EcIMP, Rawlstonella H16-RE△PHB-ScIPS-EcIMP, and Rawlstonella H16-RE△zwf△PHB-ScIPS-EcIMP were inoculated into 50 mL of LB liquid medium supplemented with 200 mg / L kanamycin and rejuvenated at 30°C and 200 rpm for 16 hours.

[0149] Preparation of fermentation medium with CO2 as the sole carbon source: Accurately weigh 3.5g Na2HPO4, 1.5g KH2PO4, 1.0g (NH4)2SO4, 80mg MgSO4·7H2O, 1mg CaSO4·2H2O, 0.56mg NiSO4·7H2O, 0.4mg ferric citrate and 200mg NaHCO3, and dilute to 1L with water as solvent. Autoclave at 115℃ for 30 minutes.

[0150] Rawstone's H16-RE-pBBR1 and engineered strains of Rawstone's H16-RE-ScIPS-EcIMP, H16-RE△zwf-ScIPS-EcIMP, H16-RE△PHB-ScIPS-EcIMP, and H16-RE△zwf△PHB-ScIPS-EcIMP producing inositol were transferred to 200 mL of fermentation medium at an inoculum rate of 1% and cultured overnight in shake flasks at 30°C and 200 rpm. OD was then calculated. 600 Once the OD value reaches 1.0 or higher, collect the bacterial cells by centrifugation at 4600 rpm for 8 min. After washing three times with PBS, resuspend the bacterial cells in fermentation medium and adjust the OD value. 600 The inositol concentration was approximately 1.0, and anaerobic shake-flask fermentation was carried out at 30℃ and 200 rpm. A mixed gas (H2:O2:CO2≈8:1:1) was introduced daily to supplement the carbon source, and the inositol concentration in the culture medium was measured periodically.

[0151] Bacterial liquid OD 600 Measurement: The absorbance of the bacterial culture at a wavelength of 600 nm was measured using a visible light spectrophotometer. Cell dry weight was calculated according to the formula reported in the literature: Cell dry weight (g / L) / OD 600 The value was calculated to be approximately 0.363 g / L.

[0152] Inositol concentration determination: Glycerol and inositol in the sample were detected using high-performance liquid chromatography (HPLC). A Shimadzu parallax refractive index detector was used, and an Agilent Hi-Plex Ca column (300 mm × 7.7 mm) was employed at a column temperature of 80 °C. Ultrapure water was used as the mobile phase at a flow rate of 0.5 mL / min, with an injection volume of 10 μL.

[0153] During the first 8 days of gas fermentation, all strains exhibited rapid growth rates, with *Rowstoneella* H16-RE-pBBR1 showing the fastest growth. Its cell dry weight reached 0.8 g / L, while the cell dry weights of other strains ranged from 0.5 to 0.6 g / L. Figure 11 As shown. Subsequently, the growth rate of each strain slowed down. After 64 days of fermentation, the cell dry weights of *Ralstonia solanacearum* H16-RE-pBBR1, *Ralstonia solanacearum* H16-RE-ScIPS-EcIMP, *Ralstonia solanacearum* H16-RE△zwf-ScIPS-EcIMP, and *Ralstonia solanacearum* H16-RE△PHB-ScIPS-EcIMP were 1.1, 1.0, 0.9, 0.8, and 0.8 g / L, respectively.

[0154] like Figure 12As shown, except for the Rawlstonella H16-RE-pBBR1 strain, inositol was detected in the fermentation broth of all other strains. Truncation of the ED and PHB synthesis pathways helps to increase the supply of glucose-6-phosphate, a precursor for inositol synthesis, thereby increasing the product yield. After 24 days of gaseous fermentation, compared to the Rawlstonella H16-RE-ScIPS-EcIMP strain, the inositol yields in the fermentation broths of the Rawlstonella H16-RE△zwf-ScIPS-EcIMP, Rawlstonella H16-RE△PHB-ScIPS-EcIMP, and Rawlstonella H16-RE△zwf△PHB-ScIPS-EcIMP strains were 498.6, 579.5, and 574.4 mg / L, respectively, representing increases of 13.3%, 31.7%, and 30.6%. However, after 40 days of fermentation, due to the increased cell count of *Rolstonia solanacearum* H16-RE-ScIPS-EcIMP, its inositol production reached a peak of 720.2 mg / L. The negative effects of the ED and PHB synthesis pathways on inositol production began to emerge. Inositol synthesis in *Rolstonia solanacearum* H16-RE△zwf△PHB-ScIPS-EcIMP reached a plateau. After 64 days of fermentation, the final inositol production of *Rolstonia solanacearum* H16-RE-ScIPS-EcIMP, *Rolstonia solanacearum* H16-RE△zwf-ScIPS-EcIMP, *Rolstonia solanacearum* H16-RE△PHB-ScIPS-EcIMP, and *Rolstonia solanacearum* H16-RE△zwf△PHB-ScIPS-EcIMP* were 1054.8, 874.0, 978.0, and 711.2 mg / L, respectively.

[0155] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for constructing an engineered Rhizobium robusf strain producing inositol using glucose, glycerol and CO2 as carbon sources, characterized in that, The method comprises the following steps: (1) Mutating the gene encoding the N-acetylglucosamine-specific phosphotransferase system in the wild-type Lysinibacillus sphaericus H16 genome nagE so that the 265th amino acid of the encoded protein is mutated from G to R and knocking out the gene encoding a GntR family transcriptional regulator nagR to enable the Lysinibacillus sphaericus to efficiently utilize glucose, wherein the gene encoding the N-acetylglucosamine-specific phosphotransferase system is nagE, a nucleotide sequence as shown in SEQ ID NO: 3; and nagR a protein encoding an amino acid sequence as shown in SEQ ID NO:

1. (2) Heterologously expressing a myo-inositol-3-phosphate synthase derived from Saccharomyces cerevisiae Sc an IPS gene and a myo-inositol monophosphatase derived from Escherichia coli Ec an IMP gene, constructing a mutant Rolstonia plagiocladiis chassis cell, wherein the myo-inositol-3-phosphate synthase derived from Saccharomyces cerevisiae Sc The nucleotide sequence of the IPS gene is shown as SEQ ID NO: 4, and the myo-inositol monophosphatase derived from Escherichia coli Ec The nucleotide sequence of the IMP gene is shown as SEQ ID NO:

5.

2. The method for constructing the engineered Rhizobium robusfum producing inositol with glucose, glycerol and CO2 as carbon sources according to claim 1, characterized in that, The method further comprises the following steps: knocking out key genes in the ED pathway and / or the PHB synthesis pathway, wherein, The ED pathway key gene is a coding gene of glucose-6-phosphate dehydrogenase zwf1, zwf2 and zwf3 The nucleotide sequence of the coding gene of glucose-6-phosphate dehydrogenase zwf1 is shown as SEQ ID NO: 6, the nucleotide sequence of the gene zwf2 is shown as SEQ ID NO: 7, and the nucleotide sequence of the gene zwf3 is shown as SEQ ID NO: 8; The PHB synthesis pathway key genes are poly([R]-3-hydroxybutyric acid) polymerase encoding gene phaC1 , acetyl-CoA acetyltransferase encoding gene phaA (H16_A1438), and acetoacetyl-CoA reductase encoding gene phaB1 The nucleotide sequence of the poly([R]-3-hydroxybutyric acid) polymerase encoding gene phaC1 is shown as SEQ ID NO: 9, the nucleotide sequence of the acetyl-CoA acetyltransferase encoding gene phaA is shown as SEQ ID NO: 10, and the nucleotide sequence of the acetoacetyl-CoA reductase encoding gene phaB1 is shown as SEQ ID NO:

11.

3. An engineered strain of Rhizobium robusiim producing myo-inositol with glucose, glycerol and CO2 as carbon sources, characterized in that, The engineered strain is obtained by a method comprising the following steps: (1) Mutating the gene encoding the N-acetylglucosamine-specific phosphotransferase system in the genome of wild-type Raoultella ornithinolytica nagE to make the 265th amino acid of the encoded protein mutate from G to R and knock out the gene encoding a GntR family transcriptional regulator nagR so that the Raoultella ornithinolytica can efficiently utilize glucose, wherein the gene encoding the N-acetylglucosamine-specific phosphotransferase system is nagE, a nucleotide sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 1, and the gene encoding the GntR family transcriptional regulator nagR has a nucleotide sequence as shown in SEQ ID NO: 3; (2) Heterologously expressing a myo-inositol-3-phosphate synthase derived from Saccharomyces cerevisiae Sc an IPS gene and a myo-inositol monophosphatase derived from Escherichia coli Ec an IMP gene, constructing a mutant Rolstonia plattcaria chassis cell, wherein the myo-inositol-3-phosphate synthase derived from Saccharomyces cerevisiae Sc The nucleotide sequence of the IPS gene is shown as SEQ ID NO: 4, and the myo-inositol monophosphatase derived from Escherichia coli Ec The nucleotide sequence of the IMP gene is shown as SEQ ID NO:

5.

4. The engineered strain of Rhizobium rothii producing inositol from glucose, glycerol and C02 according to claim 3, characterized in that, The method further comprises the following steps: knocking out key genes in the ED pathway and / or the PHB synthesis pathway, wherein, The ED pathway key gene is a coding gene of glucose-6-phosphate dehydrogenase zwf1, zwf2 and zwf3 The nucleotide sequence of the coding gene of glucose-6-phosphate dehydrogenase zwf1 is shown as SEQ ID NO: 6, the zwf2 nucleotide sequence of the gene is shown as SEQ ID NO: 7, the zwf3 nucleotide sequence of the gene is shown as SEQ ID NO: 8; The PHB synthesis pathway key genes are poly([R]-3-hydroxybutyric acid) polymerase encoding gene phaC1 , acetyl-CoA acetyltransferase encoding gene phaA (H16_A1438), and acetoacetyl-CoA reductase encoding gene phaB1 The nucleotide sequence of the poly([R]-3-hydroxybutyric acid) polymerase encoding gene phaC1 is shown as SEQ ID NO: 9, the nucleotide sequence of the acetyl-CoA acetyltransferase encoding gene phaA is shown as SEQ ID NO: 10, and the nucleotide sequence of the acetoacetyl-CoA reductase encoding gene phaB1 is shown as SEQ ID NO:

11.

5. A method for the fermentative production of inositol, characterized in that, The method comprises the step of shake flask fermentation of the engineered strain of Raoultella sp. producing inositol with glucose, glycerol and CO2 as carbon sources according to claim 3 or 4.

6. The method for the fermentative production of inositol according to claim 5, characterized in that, Fermentation is carried out using a medium with different carbon sources, wherein the formula of the carbon source medium is: 3.5 g / L Na2HPO4, 1.5 g / L KH2PO4, 1.0 g / L (NH4)2SO4, 80 mg / L MgSO4·7H2O, 1 mg / L CaSO4·2H2O, 0.56 mg / L NiSO4·7H2O, 0.4 mg / L ferric citrate, 200 mg / L NaHCO3, 5 g / L of glucose or glycerol, or mixed gas of H2:O2:CO2=8:1:1 as carbon source is added respectively.

7. The method of fermentative production of inositol according to claim 6, characterized in that, The carbon source medium contains kanamycin antibiotic with a final concentration of 200 mg / L.

8. The method of fermentative production of inositol according to claim 6, characterized in that, The fermentation conditions are temperature of 30℃ and rotation speed of 200 rpm.

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