Expression regulatory element for realizing construction of engineering strain for producing inositol, engineering strain and production method of inositol

Abstract

The invention belongs to the technical field of microbial engineering, and discloses an expression regulatory element for realizing construction of an engineering strain for producing inositol, the engineering strain and a production method of inositol. The expression regulatory element in the invention comprises an element for blocking the expression of endogenous pgi and zwf in a strain and an element for expressing Ino1, IMP1 and PPGK with a specific structure. The expression regulation element can construct a metabolic network for efficiently driving glucose to enter cells, converting glucose into G6P and carrying out inositol synthesis by using G6P in common engineering strains such as escherichia coli, and the metabolic network has good suitability with a background metabolic network of the common engineering strains; the method can be used for constructing an inositol-producing engineering strain with excellent inositol synthesis capability, and has a very excellent application prospect in realizing large-scale, high-efficiency and high-yield fermentation production of inositol.

Description

Technical Field

[0001] This invention belongs to the technical field of microbial engineering, and particularly relates to an expression regulatory element for constructing an inositol-producing engineered strain, an engineered strain, and a method for producing inositol. Background Technology

[0002] Inositol, also known as cyclohexanehexol, is a water-soluble B vitamin and an essential substance for the growth of humans, animals, and microorganisms. It is widely used in the pharmaceutical, food, and animal feed industries. Currently, global demand is approximately 5,000 tons per year. Existing methods for inositol production include hydrolysis, chemical synthesis, and enzymatic hydrolysis. However, these methods either suffer from drawbacks such as high energy consumption, long production cycles, and environmental unfriendliness, or are limited by technology and cannot yet be industrialized. Microbial fermentation is considered the most promising emerging method for inositol production with significant research potential and industrial production value, offering advantages such as simple operation, high raw material utilization, and low environmental pollution.

[0003] Therefore, obtaining an engineered strain with efficient inositol synthesis capabilities is of great significance for realizing large-scale fermentation production of inositol. Summary of the Invention

[0004] The primary objective of this invention is to construct an engineered strain capable of efficiently synthesizing inositol, and to provide an expression regulatory element for constructing an engineered strain that produces inositol.

[0005] A second objective of the present invention is to provide the application of the above-mentioned expression regulatory element in improving the ability of engineered strains to synthesize inositol.

[0006] A third objective of this invention is to provide an engineered bacterial strain.

[0007] A fourth objective of this invention is to provide a method for constructing the aforementioned engineered strain.

[0008] The fifth objective of this invention is to provide the application of the above-mentioned expression regulatory elements or engineered strains in the field of inositol preparation.

[0009] The sixth objective of this invention is to provide a method for producing inositol.

[0010] Specifically, the expression regulatory elements provided by the present invention for constructing an inositol-producing engineered strain include: (1) an element for blocking the expression of endogenous phosphoglucose isomerase in the inositol-producing engineered strain; (2) an element for blocking the expression of endogenous glucose-6-phosphate 1-dehydrogenase in the inositol-producing engineered strain; (3) an element for expressing or overexpressing inositol-3-phosphate synthase; (4) an element for expressing or overexpressing inositol monophosphatase; and (5) an element for expressing polyphosphorylase, wherein the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO:15 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:15.

[0011] Further, the element for expressing or overexpressing inositol-3-phosphate synthase includes at least one of the following technical features: (1) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:13; (2) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:22 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:22; (3) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:23 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:23; (4) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:24 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:24; (5) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:25 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:23. (6) The amino acid fragment shown in NO:25 has at least 90% sequence identity with a variant fragment; (7) The inositol-3-phosphate synthase comprises an amino acid fragment with the sequence shown in SEQ ID NO:26 or a variant fragment with at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:26; (8) The inositol-3-phosphate synthase comprises an amino acid fragment with the sequence shown in SEQ ID NO:28 or a variant fragment with at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:28; (9) The inositol-3-phosphate synthase comprises an amino acid fragment with the sequence shown in SEQ ID NO:29 or a variant fragment with at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:29; (10) The inositol-3-phosphate synthase comprises an amino acid fragment with the sequence shown in SEQ ID NO:30 or a variant fragment with at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:26. The amino acid fragment shown in NO:30 is a variant fragment with at least 90% sequence identity.

[0012] Further, the element for expressing or overexpressing inositol monophosphatase includes at least one of the following technical features: (1) the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:14 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:14; (2) the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:31 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:31; (3) the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:32 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:32; (4) the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:33 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:33; (5) the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:34 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:33. (6) The inositol monophosphatase comprises an amino acid fragment with the sequence shown in SEQ ID NO:35 or a variant fragment with the sequence shown in SEQ ID NO:35 or a variant fragment with the sequence shown in SEQ ID NO:35 or a variant fragment with the sequence shown in SEQ ID NO:35 or a variant fragment with the sequence shown in SEQ ID NO:35 or a variant fragment with the sequence shown in SEQ ID NO:36 or a variant fragment with the sequence shown in SEQ ID NO:36 or a variant fragment with the sequence shown in SEQ ID NO:36 or a variant fragment with the sequence shown in SEQ ID NO:37 or a variant fragment with the sequence shown in SEQ ID NO:37 or a variant fragment with the sequence shown in SEQ ID NO:37 or a variant fragment with the sequence shown in SEQ ID NO:37 or a variant fragment with the sequence shown in SEQ ID NO:38 or a variant fragment with the sequence shown in SEQ ID NO:38 or a variant fragment with the sequence shown in SEQ ID NO:38 or a variant fragment with the sequence shown in SEQ ID NO:39 ... The amino acid fragment shown in NO:39 has a variant fragment with at least 90% sequence identity; (11) the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:40 or a variant fragment with at least 90% sequence identity to the amino acid fragment shown in SEQ ID NO:40.

[0013] Further, the element for expressing polyphosphorylase includes at least one of the following technical features: (1) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:15; (2) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:41; (3) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:42; (4) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:43; (5) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:44; (6) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:45; (7) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:46; (8) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:47; (9) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:48; (10) the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:45. The amino acid fragment shown in NO:49; (11) The polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:50.

[0014] Further, the expression regulatory element includes at least one of the following technical features: (1) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:15; (2) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:43; (3) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:44; (4) the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:15. The amino acid fragment shown in NO:14, the polyphosphorylase includes the amino acid fragment with the sequence shown in SEQ ID NO:45; (5) the inositol-3-phosphate synthase includes the amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes the amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphorylase includes the amino acid fragment with the sequence shown in SEQ ID NO:48.

[0015] This invention provides the application of the above-mentioned expression regulatory elements in improving the ability of engineered strains to synthesize inositol.

[0016] The engineered strains provided by this invention include the above-mentioned expression regulatory elements.

[0017] The method for constructing the engineered strain provided by the present invention includes: introducing the above-mentioned expression regulatory element into Escherichia coli to obtain the engineered strain.

[0018] This invention provides the application of the above-mentioned expression regulatory elements or engineered strains in the field of inositol preparation.

[0019] The method for producing inositol provided by the present invention includes: fermenting the above-mentioned inositol-producing engineered strain to obtain the inositol.

[0020] Beneficial effects: This invention provides an expression regulatory element for constructing inositol-producing engineered strains. Specifically, the expression regulatory element includes an element for blocking the expression of phosphoglucose isomerase (PGI) in the engineered inositol-producing strain, an element for blocking the expression of glucose-6-phosphate 1-dehydrogenase (ZWF) in the engineered inositol-producing strain, an element for expressing or overexpressing inositol-3-phosphate synthase (Ino1), an element for expressing or overexpressing inositol monophosphatase (IMP1), and an element for expressing or overexpressing polyphosphorylase (PPGK). This expression regulatory element can be used to construct common engineered strains such as *Escherichia coli* and *Saccharomyces cerevisiae*. Metabolic modifications were performed on the strain by blocking the expression of pgi and zwf and expressing / overexpressing specific polyphosphorylases to knock out or block intracellular glycolysis and pentose phosphate pathways, as well as to enhance the synthesis and accumulation of polyphosphates. Simultaneously, the overexpression of Ino1 and IMP1 was synergistically employed to construct an intracellular metabolic network capable of efficiently driving glucose into the cell and converting it into G6P, and utilizing G6P for efficient inositol synthesis. This endows the resulting engineered strain with excellent inositol synthesis capabilities. These expression regulatory elements show great promise for the construction of highly efficient, high-yield inositol-producing engineered strains.

[0021] In some specific embodiments, when the expression regulatory element is applied to the construction of engineered Escherichia coli strains producing inositol, the metabolic network constructed by the expression regulatory element, which efficiently drives glucose into the cell and converts it into G6P, and utilizes G6P for inositol synthesis, has good compatibility with the endogenous glycerol metabolic pathway of Escherichia coli cells. The resulting engineered Escherichia coli strain can well enter the dual carbon source utilization metabolic mode of inositol production-cell growth under mixed carbon source culture conditions of glucose + glycerol, and has excellent ability to efficiently utilize glucose to synthesize inositol. It has very promising application prospects in realizing large-scale, high-efficiency and high-yield fermentation production of inositol.

[0022] In some specific embodiments, when the expression regulatory element is applied to the construction of an inositol-producing Escherichia coli engineered strain, Ino1 preferably includes an amino acid fragment with the sequence shown in SEQ ID NO:13, and IMP1 preferably includes an amino acid fragment with the sequence shown in SEQ ID NO:14. In this case, the metabolic network constructed by Ino1, IMP1, and PPGK that drives glucose into the cell and converts it into G6P and uses G6P for inositol synthesis makes the resulting Escherichia coli engineered strain more suitable for the inositol production-cell growth dual module constructed with glucose and glycerol as mixed carbon sources, thereby achieving more efficient biosynthesis of inositol.

[0023] In some specific embodiments, when the expression regulatory element is applied to the construction of an inositol-producing *E. coli* engineered strain, Ino1 preferably includes an amino acid fragment with the sequence shown in SEQ ID NO:13, IMP1 preferably includes an amino acid fragment with the sequence shown in SEQ ID NO:14, and PPGK preferably includes one or more of the amino acid fragments with the sequences shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:48. At this time, the catalytic activities of Ino1, IMP1, and PPGK reach a relatively ideal balance. The metabolic network constructed by them, which drives glucose into the cell and converts it into G6P, and utilizes G6P for inositol synthesis, has excellent compatibility with the background metabolic network in *E. coli* cells, giving the resulting *E. coli* engineered strain a more efficient ability to synthesize inositol from glucose. Detailed Implementation

[0024] Based on addressing the problems of low inositol yield, long fermentation cycle, and low raw material utilization in existing inositol-producing engineered strains, the inventors of this invention aim to obtain an inositol-producing engineered strain capable of efficiently utilizing glucose for inositol synthesis. Through extensive and in-depth research and numerous experiments, and a comprehensive understanding of the background metabolic networks of common engineered strains such as *E. coli* and *Saccharomyces cerevisiae*, a novel expression regulatory element with good applicability to these common engineered strains was creatively obtained. This expression regulatory element enables metabolic modification of the engineered strain, effectively constructing a metabolic network within the cell that converts cells to G6P and utilizes G6P for inositol synthesis, thereby endowing the resulting engineered strain with excellent and efficient inositol production capabilities. Based on this, the technical solution of this invention is obtained.

[0025] In a first aspect, the present invention provides an expression regulatory element for constructing an inositol-producing engineered strain. The expression regulatory element specifically includes: (1) an element for blocking the expression of endogenous phosphoglucose isomerase in the inositol-producing engineered strain; (2) an element for blocking the expression of endogenous glucose-6-phosphate 1-dehydrogenase in the inositol-producing engineered strain; (3) an element for expressing or overexpressing inositol-3-phosphate synthase; (4) an element for expressing or overexpressing inositol monophosphatase; and (5) an element for expressing polyphosphorylase.

[0026] In this invention, the element used to block the expression of endogenous phosphoglucose isomerase in the engineered inositol-producing strain refers to a functional molecule, nucleic acid sequence, or genetic tool that targets a certain step (such as transcription, mRNA processing, translation, etc.) of the expression of the endogenous pgi gene in the engineered inositol-producing strain to reduce or terminate the expression of the pgi gene. The core function of this element is to precisely shut down the expression of the endogenous pgi gene in the engineered inositol strain.

[0027] In this invention, the element used to block the expression of endogenous phosphoglucose isomerase in the engineered inositol-producing strain is determined based on the sequence information of the chassis cells of the engineered inositol-producing strain and the pgi gene contained therein, and is limited to the ability to block the expression of endogenous pgi gene in the engineered inositol-producing strain. This invention does not impose any particular limitation on it.

[0028] In some specific embodiments, specific examples of the element for blocking the expression of endogenous phosphoglucose isomerase in engineered inositol-producing strains include, but are not limited to: a repressive promoter for replacing the natural promoter in the pgi gene, a strong terminator insertion sequence for inserting upstream of the pgi gene or inside the open reading frame, an expression cassette containing sgRNA used to knock out the pgi gene, an expression cassette containing asRNA complementary to the mRNA transcribed from the pgi gene, and an expression cassette containing siRNA or shRNA targeting the pgi gene, or one or more of these.

[0029] In this invention, the element used to block the expression of endogenous glucose-6-phosphate 1-dehydrogenase in the inositol-producing engineered strain refers to a functional molecule, nucleic acid sequence, or genetic tool that targets a certain step (such as transcription, mRNA processing, translation, etc.) of the expression of the endogenous zwf gene in the inositol-producing engineered strain to reduce or terminate the expression of the zwf gene. The core function of this element is to precisely shut down the expression of the endogenous zwf gene in the inositol-producing engineered strain.

[0030] In this invention, the element used to block the expression of endogenous glucose-6-phosphate 1-dehydrogenase in the engineered inositol-producing strain is determined based on the chassis cells of the engineered inositol-producing strain and the sequence information of the zwf gene contained therein, and is limited to the ability to block the expression of endogenous zwf gene in the engineered inositol-producing strain. This invention does not impose any particular limitation on it.

[0031] In some specific embodiments, specific examples of the element for blocking the expression of endogenous glucose-6-phosphate 1-dehydrogenase in engineered strains producing inositol include, but are not limited to: a repressive promoter for replacing the natural promoter in the zwf gene, a strong terminator insertion sequence for inserting upstream of the zwf gene or inside an open reading frame, an expression cassette containing sgRNA used to knock out the zwf gene, an expression cassette containing asRNA targeting the zwf gene, and an expression cassette containing siRNA / shRNA targeting the zwf gene, or one or more of these.

[0032] In this invention, the element for expressing or overexpressing inositol-3-phosphate synthase refers to an expression cassette capable of enhancing the expression of the endogenous Ino1 gene in engineered inositol-producing strains to achieve overexpression of endogenous inositol-3-phosphate synthase and / or achieving the expression of heterologous inositol-3-phosphate synthase in engineered inositol-producing strains.

[0033] In this invention, when the element for expressing or overexpressing inositol-3-phosphate synthase is used to achieve overexpression of endogenous inositol-3-phosphate synthase, specific examples of the element include, but are not limited to, one or more of the following: an expression cassette containing an endogenous Ino1 gene; an expression cassette containing an optimized Ino1 gene sequence with a strong promoter replacing the natural promoter; an expression cassette containing an optimized Ino1 gene sequence with an inserted enhancer; an expression cassette containing sgRNA used to knock out the Ino1 gene negative regulator gene; an expression cassette containing asRNA targeting the Ino1 gene negative regulator gene; and an expression cassette containing siRNA / shRNA targeting the Ino1 gene negative regulator gene. The above-mentioned expression cassette is a conventionally used technique in genetic engineering. Those skilled in the art can adaptively select and design the structure and functional fragments of the expression cassette according to actual needs. This invention does not impose any particular limitations, and specific examples include, but are not limited to, one or more of linear DNA, plasmids, mRNA, and circRNA.

[0034] In this invention, when the element for expressing or overexpressing inositol-3-phosphate synthase is used to achieve the expression of heterologous inositol-3-phosphate synthase, the element is specifically an expression cassette containing a nucleic acid fragment encoding heterologous inositol-3-phosphate synthase, and the nucleic acid fragment encoding heterologous inositol-3-phosphate synthase is designed based on the amino acid sequence of heterologous inositol-3-phosphate synthase encoded by the heterologous Ino1 gene and the codon preference of inositol-producing engineered strains. Specific examples of the heterologous Ino1 gene include, but are not limited to, bacteria derived from the genus *Acidobacterium* (…). Acidilobus sp. The INO1-As gene, derived from bacteria of the genus Thermophyton ( Thermophilum sp. The INO1-TS gene, derived from Methanocaldococcus villosus The INO1-Mv gene, derived from bacteria of the genus *Pyrococcus* ( Pyrococcus sp. The INO1-Ps gene, derived from bacteria of the genus Thermostylus ( Thermophilum sp. The INO1-Tfs gene, derived from *Homo haemolyticus* ( Hospital firefly The INO1-Ih gene, derived from bacteria of the genus Thermococcus ( Thermococcus sp. The INO1-Tms gene, derived from Thermoproteota archaeon The INO1-Tpa gene, derived from Thermoproteota archaeon The INO-Ta gene and derived from Candidate Aenigmatarchaeota archaeon One or more of the INO-Caa genes.

[0035] More specifically, the source mentioned Acidilobus sp. The nucleotide sequence of the INO1-As gene is shown in the table below. The inositol-3-phosphate synthase INO1-As encoded by the INO1-As gene comprises an amino acid fragment as shown in SEQ ID NO:13 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:13. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:13, and the substitution, deletion, or addition does not affect the conformational structure of inositol-3-phosphate synthase INO1-As or has minimal impact on the enzyme activity of inositol-3-phosphate synthase INO1-As. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:13 can be 90%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0036]

[0037] More specifically, the source mentioned Thermophilum sp.The nucleotide sequence of the INO1-TS gene is shown in the table below. The inositol-3-phosphate synthase INO1-TS encoded by the INO1-TS gene comprises an amino acid fragment as shown in SEQ ID NO:22 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:22. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:22, and the substitution, deletion, or addition does not affect the conformational structure of the inositol-3-phosphate synthase INO1-TS or has minimal impact on the enzyme activity of the inositol-3-phosphate synthase INO1-TS. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:22 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0038]

[0039] More specifically, the source mentioned Methanocaldococcus villosus The nucleotide sequence of the INO1-Mv gene is shown in the table below. The inositol-3-phosphate synthase INO1-Mv encoded by the INO1-Mv gene comprises an amino acid fragment as shown in SEQ ID NO:23 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:23. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:23, and the substitution, deletion, or addition does not affect the conformational structure of inositol-3-phosphate synthase INO1-Mv or has minimal impact on the enzyme activity of inositol-3-phosphate synthase INO1-Mv. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:23 can be 90%, 91%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0040]

[0041] More specifically, the source mentioned Pyrococcus sp.The nucleotide sequence of the INO1-Ps gene is shown in the table below. The inositol-3-phosphate synthase INO1-Ps encoded by the INO1-Ps gene comprises an amino acid fragment as shown in SEQ ID NO:24 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:24. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:24, and the substitution, deletion, or addition does not affect the conformational structure of the inositol-3-phosphate synthase INO1-Ps or has minimal impact on the enzyme activity of the inositol-3-phosphate synthase INO1-Ps. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:24 can be 90%, 91%, 95%, 97%, 99%, 99.9999%, or any value in between.

[0042]

[0043] More specifically, the source mentioned Thermophilum sp. The nucleotide sequence of the INO1-Tfs gene is shown in the table below. The inositol-3-phosphate synthase INO1-Tfs encoded by the INO1-Tfs gene comprises an amino acid fragment as shown in SEQ ID NO:25 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:25. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:25, and the substitution, deletion, or addition does not affect the conformational structure of the inositol-3-phosphate synthase INO1-Tfs or has minimal impact on the enzyme activity. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:25 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0044]

[0045] More specifically, the source mentioned Hospital fireflyThe nucleotide sequence of the INO1-Ih gene is shown in the table below. The inositol-3-phosphate synthase INO1-Ih encoded by the INO1-Ih gene comprises an amino acid fragment as shown in SEQ ID NO:26 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:26. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:26, and the substitution, deletion, or addition does not affect the conformational structure of inositol-3-phosphate synthase INO1-Ih or has minimal impact on the enzyme activity. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:26 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0046]

[0047] More specifically, the source mentioned Thermococcus sp. The nucleotide sequence of the INO1-Tms gene is shown in the table below. The inositol-3-phosphate synthase INO1-Tms encoded by the INO1-Tms gene comprises an amino acid fragment as shown in SEQ ID NO:27 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:27. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:27, and the substitution, deletion, or addition does not affect the conformational structure of the inositol-3-phosphate synthase INO1-Tms or has minimal impact on the enzyme activity. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:27 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0048]

[0049] More specifically, the source mentioned Thermoproteota archaeonThe nucleotide sequence of the INO1-Tpa gene is shown in the table below. The inositol-3-phosphate synthase INO1-Tpa encoded by the INO1-Tpa gene comprises an amino acid fragment as shown in SEQ ID NO:28 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:28. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:28, and the substitution, deletion, or addition does not affect the conformational structure of inositol-3-phosphate synthase INO1-Tpa or has minimal impact on the enzyme activity of inositol-3-phosphate synthase INO1-Tpa. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:28 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0050]

[0051] More specifically, the source mentioned Thermoproteota archaeon The nucleotide sequence of the INO1-Ta gene is shown in the table below. The inositol-3-phosphate synthase INO1-Ta encoded by the INO1-Ta gene comprises an amino acid fragment as shown in SEQ ID NO:29 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:29. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:29, and the substitution, deletion, or addition does not affect the conformational structure of inositol-3-phosphate synthase INO1-Ta or has minimal impact on its enzyme activity. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:29 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0052]

[0053] More specifically, the source mentioned Candidate Aenigmatarchaeota archaeonThe nucleotide sequence of the INO1-Caa gene is shown in the table below. The inositol-3-phosphate synthase INO1-Caa encoded by the INO1-Caa gene comprises an amino acid fragment as shown in SEQ ID NO:30 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:30. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:30, and the substitution, deletion, or addition does not affect the conformational structure of inositol-3-phosphate synthase INO1-Caa or has minimal impact on the enzyme activity of inositol-3-phosphate synthase INO1-Caa. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:30 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0054]

[0055] In this invention, the element for expressing or overexpressing inositol monophosphatase refers to an expression cassette capable of enhancing the expression of the endogenous IMP1 gene in engineered inositol-producing strains to achieve overexpression of endogenous inositol monophosphatase and / or achieving the expression of heterologous inositol monophosphatase in engineered inositol-producing strains.

[0056] In this invention, when the element for expressing or overexpressing inositol monophosphatase is used to achieve overexpression of endogenous inositol monophosphatase, specific examples of the element include, but are not limited to, one or more of the following: an expression cassette containing an endogenous IMP1 gene; an expression cassette containing an optimized IMP1 gene sequence with a strong promoter replacing the natural promoter; an expression cassette containing an optimized IMP1 gene sequence with an inserted enhancer; an expression cassette containing sgRNA used to knock out the negative regulator gene of the IMP1 gene; an expression cassette containing asRNA targeting the negative regulator gene of the IMP1 gene; and an expression cassette containing siRNA / shRNA targeting the negative regulator gene of the IMP1 gene. The above-mentioned expression cassettes are a conventionally used technique in genetic engineering. Those skilled in the art can adaptively select and design the structure and functional fragments of the expression cassette according to actual needs. This invention does not impose any particular limitations, and specific examples include, but are not limited to, one or more of linear DNA, plasmids, mRNA, and circRNA.

[0057] In this invention, when the element for expressing or overexpressing inositol monophosphatase is used to achieve the expression of heterologous inositol monophosphatase, the element is specifically an expression cassette containing a nucleic acid fragment encoding heterologous inositol monophosphatase, and the nucleic acid fragment encoding heterologous inositol monophosphatase is designed based on the amino acid sequence of heterologous inositol monophosphatase encoded by the heterologous IMP1 gene and the codon preference of the inositol-producing engineered strain. Specific examples of the heterologous IMP1 gene include, but are not limited to, those derived from *Thermophyton floccosum* (…). Thermotoga maritime The IMP1-Tm gene, derived from *Thermophyton floccosum* ( Thermosipho japonicus The IMP1-Tsj gene, derived from extreme thermophiles ( Thermosipho melanesiensis The IMP1-Tsm gene, derived from bacteria of the genus Curvularia ( Rose-flexed sp. The IMP1-Rs gene, derived from fibrophagocytophyte bacteria ( Deltaproteobacteria bacterium The IMP1-Db gene, derived from Candidate Kryptonium sp. The IMP1-Cks gene, derived from alkalophilic halophilic bacilli ( Alkalihalobacillus sp. IMP1-Aks, derived from Candidatus Peregrinibacteria bacteria The IMP1-Cpb gene, derived from Kosmotogales bacterium The IMP1-Kb gene, derived from bacteria of the genus *Vibrio thermodesulfurans* ( Thermodesulfovibrio sp. The IMP1-Tds gene and the gene derived from nitrifying spirilla ( Nitrospirota bacterium One or more of the IMP1-Nb genes.

[0058] More specifically, the source mentioned Thermotoga maritime The nucleotide sequence of the IMP1-Tm gene is shown in the table below. The inositol monophosphatase IMP1-Tm encoded by this gene comprises an amino acid fragment as shown in SEQ ID NO:14 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:14. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:14, and the substitution, deletion, or addition does not affect the conformational structure of inositol monophosphatase IMP1-Tm or has minimal impact on its enzyme activity. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:14 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0059]

[0060] More specifically, the source Thermosipho japonicus The nucleotide sequence of the IMP1-Tsj gene is shown in the table below. The inositol monophosphatase IMP1-Tsj encoded by the IMP1-Tsj gene comprises an amino acid fragment as shown in SEQ ID NO:31 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:31. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:31, and the substitution, deletion, or addition does not affect the conformational structure of inositol monophosphatase IMP1-Tsj or has minimal impact on the enzyme activity of inositol monophosphatase IMP1-Tsj. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:31 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0061]

[0062] More specifically, the source mentioned Thermosipho melanesiensis The nucleotide sequence of the IMP1-Tsm gene is shown in the table below. The inositol monophosphatase IMP1-Tsm encoded by the IMP1-Tsm gene comprises an amino acid fragment as shown in SEQ ID NO:32 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:32. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:32, and the substitution, deletion, or addition does not affect the conformational structure of inositol monophosphatase IMP1-Tsm or has minimal impact on the enzyme activity of inositol monophosphatase IMP1-Tsm. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:32 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0063]

[0064] More specifically, the source mentioned Roseiflexus sp.The nucleotide sequence of the IMP1-Rs gene is shown in the table below. The inositol monophosphatase IMP1-Rs encoded by this gene comprises an amino acid fragment as shown in SEQ ID NO:33 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:33. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:33, and the substitution, deletion, or addition does not affect the conformational structure of the inositol monophosphatase IMP1-Rs or has minimal impact on its enzyme activity. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:33 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0065]

[0066] More specifically, the source mentioned Deltaproteobacteria bacterium The nucleotide sequence of the IMP1-Db gene is shown in the table below. The inositol monophosphatase IMP1-Db encoded by the IMP1-Db gene comprises an amino acid fragment as shown in SEQ ID NO:34 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:34. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:34, and the substitution, deletion, or addition does not affect the conformational structure of inositol monophosphatase IMP1-Db or has minimal impact on the enzyme activity of inositol monophosphatase IMP1-Db. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:34 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0067]

[0068] More specifically, the source mentioned Candidate Kryptonium sp.The nucleotide sequence of the IMP1-Cks gene is shown in the table below. The inositol monophosphatase IMP1-Cks encoded by the IMP1-Cks gene comprises an amino acid fragment as shown in SEQ ID NO:35 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:35. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:35, and the substitution, deletion, or addition does not affect the conformational structure of the inositol monophosphatase IMP1-Cks or has minimal impact on the enzyme activity of the inositol monophosphatase IMP1-Cks. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:35 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0069]

[0070] More specifically, the source mentioned Alkalihalobacillus sp. The nucleotide sequence of the IMP1-Aks gene is shown in the table below. The inositol monophosphatase IMP1-Aks encoded by the IMP1-Aks gene comprises an amino acid fragment as shown in SEQ ID NO:36 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:36. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:36, and the substitution, deletion, or addition does not affect the conformational structure of the inositol monophosphatase IMP1-Aks or has minimal impact on the enzyme activity of the inositol monophosphatase IMP1-Aks. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:36 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0071]

[0072] More specifically, the source mentioned Candidatus Peregrinibacteria bacteriumThe nucleotide sequence of the IMP1-Cpb gene is shown in the table below. The inositol monophosphatase IMP1-Cpb encoded by the IMP1-Cpb gene comprises an amino acid fragment as shown in SEQ ID NO:37 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:37. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:37, and the substitution, deletion, or addition does not affect the conformational structure of inositol monophosphatase IMP1-Cpb or has minimal impact on the enzyme activity of inositol monophosphatase IMP1-Cpb. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:37 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0073]

[0074] More specifically, the source mentioned Kosmotogales bacterium The nucleotide sequence of the IMP1-Kb gene is shown in the table below. The inositol monophosphatase IMP1-Kb encoded by this gene comprises an amino acid fragment as shown in SEQ ID NO:38 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:38. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:38, and the substitution, deletion, or addition does not affect the conformational structure of inositol monophosphatase IMP1-Kb or has minimal impact on its enzyme activity. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:38 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0075]

[0076] More specifically, the source mentioned Thermodesulfovibrio sp.The nucleotide sequence of the IMP1-Tds gene is shown in the table below. The inositol monophosphatase IMP1-Tds encoded by the IMP1-Tds gene comprises an amino acid fragment as shown in SEQ ID NO:39 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:39. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:39, and the substitution, deletion, or addition does not affect the conformational structure of the inositol monophosphatase IMP1-Tds or has minimal impact on the enzyme activity of the inositol monophosphatase IMP1-Tds. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:39 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0077]

[0078] More specifically, the source mentioned Nitrospirota bacterium The nucleotide sequence of the IMP1-Nb gene is shown in the table below. The inositol monophosphatase IMP1-Nb encoded by the IMP1-Nb gene comprises an amino acid fragment as shown in SEQ ID NO:40 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:40. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:40, and the substitution, deletion, or addition does not affect the conformational structure of inositol monophosphatase IMP1-Nb or has minimal impact on the enzyme activity of inositol monophosphatase IMP1-Nb. The homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:40 can be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value in between.

[0079]

[0080] In this invention, the element used to express polyphosphorylase refers to an engineered strain capable of producing inositol derived from thermophilic actinomycetes (…). Thermobifida fuscaAn expression cassette for expressing a polyphosphorylase; specifically, the expression cassette includes a nucleic acid fragment encoding the polyphosphorylase, and the nucleic acid fragment encoding the polyphosphorylase is designed based on the amino acid sequence of the polyphosphorylase and the codon preference of the inositol-producing engineered strain. The above-mentioned expression cassette is a commonly used technique in genetic engineering. Those skilled in the art can adaptively select and design the structure and functional fragments of the expression cassette according to actual needs. This invention does not impose any particular limitations, and specific examples include, but are not limited to, one or more of linear DNA, plasmids, mRNA, and circRNA.

[0081] In this invention, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:15 or a variant fragment having at least 90% sequence identity with the amino acid fragment shown in SEQ ID NO:15. The variant fragment is obtained by substituting, deleting, or adding one or more amino acids to the amino acid fragment shown in SEQ ID NO:15, and the homology between the variant fragment and the amino acid fragment shown in SEQ ID NO:15 can specifically be 90%, 92.5%, 93%, 97%, 99%, 99.9999%, or any value intermediate thereto.

[0082] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:41. More specifically, the polyphosphorylase is obtained by mutating an amino acid fragment as shown in SEQ ID NO:15 via a V52K mutation, wherein the V52K mutation is a substitution mutation from valine to lysine at amino acid residue 52 of the amino acid fragment shown in SEQ ID NO:15.

[0083] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:42. More specifically, the polyphosphorylase is obtained by mutating an amino acid fragment as shown in SEQ ID NO:15 through an R196A mutation, wherein the R196A mutation is a substitution mutation of arginine to alanine at amino acid residue 196 of the amino acid fragment shown in SEQ ID NO:15.

[0084] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:43. More specifically, the polyphosphorylase is obtained by mutating the amino acid fragment shown in SEQ ID NO:15 through a V150A mutation, wherein the V150A mutation is a substitution mutation from valine to alanine at amino acid residue 150 of the amino acid fragment shown in SEQ ID NO:15.

[0085] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:44. More specifically, the polyphosphorylase is obtained by mutating the amino acid fragment shown in SEQ ID NO:15 through an E122S mutation, wherein the E122S mutation is a substitution mutation from glutamic acid to serine at amino acid residue 122 of the amino acid fragment shown in SEQ ID NO:15.

[0086] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:45. More specifically, the polyphosphorylase is obtained from an amino acid fragment shown in SEQ ID NO:15 through V52K and E122S mutations.

[0087] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:46. More specifically, the polyphosphorylase is obtained from an amino acid fragment shown in SEQ ID NO:15 through V52K and V150A mutations.

[0088] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:47. More specifically, the polyphosphorylase is obtained from an amino acid fragment shown in SEQ ID NO:15 through V52K and R196A mutations.

[0089] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:48. More specifically, the polyphosphorylase is obtained from an amino acid fragment shown in SEQ ID NO:15 through E122S mutation and V150A mutation.

[0090] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:49. More specifically, the polyphosphorylase is obtained from an amino acid fragment shown in SEQ ID NO:15 through E122S mutation and R196A mutation.

[0091] In some specific embodiments, the polyphosphorylase specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:50. More specifically, the polyphosphorylase is obtained from an amino acid fragment shown in SEQ ID NO:15 through V150A and R196A mutations.

[0092] In this invention, the expression regulatory element preferably includes the following technical features: the inositol-3-phosphate synthase comprises an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase comprises an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphorylase comprises an amino acid fragment with the sequence shown in SEQ ID NO:15; that is, the inositol-producing engineered strain constructed using this expression regulatory element can simultaneously express the aforementioned inositol-3-phosphate synthase, inositol monophosphatase, and polyphosphorylase with specific structures. At this time, the metabolic network that the expression regulatory element can construct, driving glucose into the cell and converting it into G6P, and utilizing G6P for inositol synthesis, has good compatibility with the background metabolic network of *E. coli*, and has excellent potential to improve the ability of *E. coli* to synthesize inositol from glucose. It has excellent application prospects in the construction of high-performance inositol-producing engineered *E. coli* strains.

[0093] In this invention, the expression regulatory element preferably includes the following technical features: the inositol-3-phosphate synthase comprises an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase comprises an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphorylase comprises one or more segments of amino acid fragments with the sequences shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:48; that is, the inositol-producing engineered strain constructed using this expression regulatory element can simultaneously express the above-mentioned inositol-3-phosphate synthase, inositol monophosphatase, and polyphosphorylase with the special structures described above. At this point, in the metabolic network that the expression regulatory element can construct to drive glucose into the cell and convert it into G6P, and to utilize G6P for inositol synthesis, the catalytic activities of inositol-3-phosphate synthase, inositol monophosphatase, and polyphosphorylase are in a relatively ideal balance. This is conducive to Escherichia coli entering a dual carbon source utilization mode of "using glycerol for growth" and "using glucose for inositol synthesis". It has excellent application prospects in the construction of high-performance inositol-producing engineered Escherichia coli.

[0094] In some specific embodiments, the expression regulatory element specifically includes the following technical features: the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:43.

[0095] In some specific embodiments, the expression regulatory element specifically includes the following technical features: the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:44.

[0096] In some specific embodiments, the expression regulatory element specifically includes the following technical features: the inositol-3-phosphate synthase includes an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase includes an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphorylase includes an amino acid fragment with the sequence shown in SEQ ID NO:45.

[0097] In some specific embodiments, the inositol-3-phosphate synthase comprises an amino acid fragment with the sequence shown in SEQ ID NO:13, the inositol monophosphatase comprises an amino acid fragment with the sequence shown in SEQ ID NO:14, and the polyphosphorylase comprises an amino acid fragment with the sequence shown in SEQ ID NO:48.

[0098] In this invention, the expression regulatory element is well-suited for constructing inositol-producing engineered strains based on chassis cells conventionally used in existing genetic engineering techniques. Specific examples of chassis cells include, but are not limited to, one or more of Escherichia coli, Saccharomyces cerevisiae, lactic acid bacteria, Corynebacterium glutamicum, Pichia pastoris, and Bacillus subtilis.

[0099] Secondly, this invention provides the application of the above-mentioned expression regulatory element in improving inositol production in *Escherichia coli*. In this application, by introducing the expression regulatory element into *E. coli*, a metabolic network is constructed within its cells to drive glucose entry into the cell and its conversion to G6P, utilize G6P for inositol synthesis, and utilize glycerol for growth. This effectively modifies the metabolic pathways within *E. coli* cells, thereby endowing the resulting recombinant strain with excellent inositol production capacity.

[0100] Thirdly, the present invention provides an engineered strain of *Escherichia coli*. Specifically, the engineered *E. coli* strain includes the aforementioned expression regulatory elements, wherein the endogenous pgi gene and zwf gene in the engineered *E. coli* strain are knocked out, and its cells simultaneously express or overexpress the aforementioned inositol-3-phosphate synthase, inositol monophosphatase, and polyphosphorylase.

[0101] Fourthly, the present invention provides a method for constructing the above-mentioned engineered Escherichia coli strain. The construction method specifically includes: introducing the above-mentioned expression regulatory element into Escherichia coli to obtain the engineered Escherichia coli strain.

[0102] In this invention, the method of introducing the above-mentioned expression regulatory element into Escherichia coli is a conventional technique used in existing genetic engineering technology. Those skilled in the art can make adaptive selections and designs according to actual needs, and this invention does not impose any particular limitations on it.

[0103] In this invention, specific examples of the *Escherichia coli* include, but are not limited to, one or more of the following: *Escherichia coli* DH5α, *Escherichia coli* BL21(DE3), *Escherichia coli* TOP10, *Escherichia coli* MG1655, and *Escherichia coli* W3110.

[0104] Fifthly, the present invention provides the application of the above-mentioned expression regulatory elements or engineered Escherichia coli strains in the field of inositol preparation.

[0105] Sixthly, the present invention provides a method for producing inositol. The method comprises: fermenting and culturing the aforementioned engineered strain of *Escherichia coli* to obtain the inositol.

[0106] In this invention, the fermentation culture refers to the biological reaction process in which engineered strains of *Escherichia coli* grow, reproduce, and synthesize inositol in a specific fermentation medium. Specific examples of the fermentation culture methods include, but are not limited to, shake-flask fermentation and / or fed-batch fermentation.

[0107] In this invention, when the fermentation culture is specifically a shake flask fermentation culture, the fermentation medium used in the shake flask fermentation culture preferably includes: 1% (v / v)~2% (v / v) glycerol, 2% (w / v)~4% (w / v) glucose, 2 g / L~5 g / L yeast extract, 0.5 mmol / L~1.5 mmol / L IPTG, and basal medium. The basal medium can be any medium conventionally used in existing E. coli culture techniques. Those skilled in the art can make adaptive selections and designs according to actual needs. This invention does not impose any particular limitation, and specific examples include, but are not limited to, one or more of LB medium, M9 medium, and EMB medium.

[0108] In some specific embodiments, the conditions for the shake flask fermentation culture include a temperature preferably of 20℃~40℃, a shaking frequency preferably of 180rpm~220rpm, and a culture time preferably of 24h~48h.

[0109] In this invention, when the fermentation culture is specifically a fed-batch fermentation culture, the fermentation medium used in the fed-batch fermentation culture preferably includes: 2% (v / v)~3% (v / v) glycerol, 4% (w / v)~6% (w / v) glucose, 6 g / L~8 g / L yeast extract, 0.5 mmol / L~1.5 mmol / L IPTG, and basal medium; the fed-batch solution preferably includes: 200 g / L~600 g / L glucose, 100 g / L~300 g / L glycerol, and basal medium. The fermentation medium and the basal medium used in the fed-batch solution can be the same or different, and can be any medium conventionally used in existing E. coli culture techniques. Those skilled in the art can make adaptive selections and designs according to actual needs. This invention does not impose any particular limitation, and specific examples include, but are not limited to, one or more of LB medium, M9 medium, and EMB medium.

[0110] In some specific embodiments, the conditions for the continuous fed fermentation culture include a temperature preferably of 20℃~40℃, an aeration ratio preferably of 0.5vvm~2vvm, a solution stirring speed preferably of 300rpm~700rpm, a dissolved oxygen threshold preferably of 5%~15%, and a feed flow rate of 1~2g / L / h.

[0111] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0112] Example 1 This embodiment illustrates the construction of the deletion strain BL21(DE3)Δpgi-Δzwf, which is obtained by knocking out the pgi and zwf genes of Escherichia coli BL21(DE3) using CRISPR / Cas9 technology, starting from E. coli BL21(DE3). The specific steps include: 1. Construction of homologous fragments for gene knockout (1) Using the genomic DNA of Escherichia coli BL21(DE3) as a template, PCR amplification was performed using the primers shown in Table 1 to obtain the upstream and downstream homologous arms of the pgi gene and zwf gene.

[0113] Table 1.

[0114] The PCR amplification reaction system was prepared according to Takara PrimeSTAR®HS DNA Polymerase (Takara, catalog number R010A, the same below); the PCR amplification program included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min.

[0115] (2) Using the upstream and downstream homologous arms of the pgi gene and zwf gene as templates, PCR amplification was performed using the primers shown in Table 2 to obtain homologous fragments for knocking out the pgi gene and zwf gene.

[0116] Table 2.

[0117] The PCR amplification reaction system was prepared according to Takara PrimeSTAR® HS DNA Polymerase; the PCR amplification program included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min.

[0118] 2. Construction of gene knockout plasmids Using plasmid pTargetF as a template, PCR amplification was performed using the primers shown in Table 3 to obtain gene knockout plasmids containing the sgRNA used to knock out each gene.

[0119] Table 3.

[0120] The PCR amplification reaction system was prepared according to Takara PrimeSTAR® HS DNA Polymerase; the PCR amplification program included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 15 s, 75℃ extension for 1 min, 30 cycles; and 72℃ extension for 10 min.

[0121] 3. Construction of the deletion strain BL21(DE3)Δpgi-Δzwf (1) Escherichia coli BL21(DE3) containing pCas9 plasmid was made into electrocompetent cells. The plasmid pTargetF-pig and the homologous fragment for knocking out the pig gene were transferred into the electrocompetent cells by electroporation. The cells were then inoculated onto LB solid plates containing 50 μg / mL kanamycin sulfate and 50 μg / mL spectinomycin and cultured at 37°C until colonies grew. Single colonies were picked and sent for sequencing. The results showed that the PGI gene was successfully knocked out and the deletion strain BL21(DE3)Δpgi was obtained.

[0122] (2) Based on the deletion strain BL21(DE3)Δpgi, the zwf gene was knocked out using the method provided in (1) to obtain the deletion strain BL21(DE3)Δpgi-Δzwf.

[0123] Example 2 This example illustrates the construction of the engineered Escherichia coli strain KDY-JC-01. This engineered Escherichia coli strain KDY-JC-01 was constructed using the deletion strain BL21(DE3)Δpgi-Δzwf provided in Example 1 as the starting strain. It was constructed by introducing the recombinant plasmid PPGK-Ino1-IMP1-pETduet-1, which includes nucleic acid fragments encoding inositol-3-phosphate synthase (Ino1-As, SEQ ID NO:13), inositol monophosphatase (IMP1-Tm, SEQ ID NO:14), and polyphosphorylase (PPGK, SEQ ID NO:15). The amino acid sequences of each enzyme molecule and their sources are shown in Table 4.

[0124] Table 4.

[0125] The construction of the engineered Escherichia coli strain KDY-JC-01 specifically includes the following steps: 1. Construction of recombinant plasmid Ino1-IMP1-pETduet-1 (1) Based on the amino acid sequences of inositol-3-phosphate synthase and inositol monophosphatase, the codons of Escherichia coli were optimized and sent to Sangon Biotech (Shanghai) Co., Ltd. for whole-gene synthesis to obtain the inositol-3-phosphate synthase gene and the inositol monophosphatase gene. The above genes were carried on the pET28a vector to obtain the Ino1-As-pET28a plasmid and the IMP1-Tm-pET28a plasmid.

[0126] (2) Using Ino1-As-pET28a plasmid and IMP1-Tm-pET28a plasmid as templates, PCR amplification was performed using the primers shown in Table 5 to obtain PCR amplification products.

[0127] Table 5.

[0128] The PCR amplification reaction system was prepared according to Takara PrimeSTAR® HS DNA Polymerase; the PCR amplification program included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min.

[0129] (3) The expression vector pETduet-1 was digested with endonucleases Nde I and Xho I (purchased from New England Biolabs, the same below) according to the instructions to obtain the digested product.

[0130] (4) Homologous recombination seamless cloning kit (purchased from Beijing TransGen Biotech, the same below) was used to take PCR amplification products and enzyme digestion products for homologous recombination according to the instructions. Trans5α competent cells were then transformed and seeded into LB liquid medium. The cells were cultured overnight at 37℃ and 160 rpm with shaking. The plasmid was extracted using a plasmid extraction kit (purchased from Axygen, the same below) according to the instructions to obtain the recombinant plasmid Ino1-IMP1-pETduet-1.

[0131] 2. Construction of recombinant plasmid PPGK-Ino1-IMP1-pETduet-1 (1) Based on the amino acid sequence of polyphosphorylase, the codon of Escherichia coli was optimized and sent to Sangon Biotech (Shanghai) Co., Ltd. for whole-gene synthesis to obtain the polyphosphorylase gene, which was carried on the pET28a vector to obtain the PPGK-pET28a plasmid.

[0132] (2) Using the PPGK-pET28a plasmid as a template, PCR amplification was performed using the primers shown in Table 6 to obtain the PCR amplification product.

[0133] Table 6.

[0134] The PCR amplification reaction system was prepared according to Takara PrimeSTAR® HS DNA Polymerase; the PCR amplification program included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min.

[0135] (3) The recombinant plasmid Ino1-IMP1-pETduet-1 was digested with endonucleases Nde I and Xho I according to the instructions to obtain the digested product.

[0136] (4) After homologous recombination and seamless cloning kit, PCR amplification products and enzyme digestion products were taken and homologously recombinated according to the instructions. Trans5α competent cells were then transformed and seeded into LB liquid medium. The cells were cultured overnight at 37°C and 160 rpm with shaking. The plasmid was extracted using a plasmid extraction kit according to the instructions to obtain the recombinant plasmid PPGK-Ino1-IMP1-pETduet-1.

[0137] 3. Construction of engineered Escherichia coli strain KDY-JC-01 Electrocompetent cells were prepared from the deletion strain BL21(DE3)Δpgi-Δzwf. The recombinant plasmid PPGK-Ino1-IMP1-pETduet-1 was transformed into the electrocompetent cells via electroporation. The cells were then inoculated onto LB agar plates containing 100 μg / mL ampicillin and cultured at 37°C for 12 h. Single colonies were picked and sequenced. The results showed that the recombinant plasmid PPGK-Ino1-IMP1-pETduet-1 was successfully introduced, and the engineered Escherichia coli strain KDY-JC-01 was obtained. The cells were stored in glycerol tubes at -80°C.

[0138] Example 3 This embodiment illustrates the construction of engineered Escherichia coli strains KDY-JC-02~KDY-JC-10. These engineered Escherichia coli strains KDY-JC-02~KDY-JC-10 are essentially the same as the engineered Escherichia coli strain KDY-JC-01 provided in Example 2. The difference lies in the expression of inositol-3-phosphate synthase by the engineered Escherichia coli strains KDY-JC-02~KDY-JC-10, as shown in Table 7. Under the same conditions, engineered Escherichia coli strains KDY-JC-02~KDY-JC-10 were constructed.

[0139] Table 7.

[0140] Example 4 This embodiment illustrates the construction of engineered Escherichia coli strains KDY-JC-11 to KDY-JC-20. These engineered Escherichia coli strains KDY-JC-11 to KDY-JC-20 are essentially the same as the engineered Escherichia coli strain KDY-JC-01 provided in Example 2. The difference lies in the expression of inositol monophosphatase by the engineered Escherichia coli strains KDY-JC-11 to KDY-JC-20, as shown in Table 8. Under the same conditions, engineered Escherichia coli strains KDY-JC-11 to KDY-JC-20 were constructed.

[0141] Table 8.

[0142] Example 5 This embodiment illustrates the construction of the PPGK mutant, which is based on the amino acid sequence of the polyphosphorylase provided in Example 2, and is constructed by mutating one or more sites. The specific mutations and amino acid sequences of each PPGK mutant compared to the original polyphosphorylase are shown in Table 9. The construction and enzyme activity verification of the PPGK mutant are as follows: (1) Using the PPGK-pET28a plasmid provided in Example 2 as a template, PCR amplification was performed by designing corresponding mutation primers according to the mutations shown in Table 9, and PCR amplification products containing the coding information of the corresponding mutation sites and homologous arms were obtained.

[0143] The PCR amplification reaction system was prepared according to Takara PrimeSTAR® HS DNA Polymerase; the PCR amplification program included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min.

[0144] (2) The expression vector pETduet-1 was digested with endonucleases Nde I and Xho I according to the instructions to obtain the digested product.

[0145] (3) Using the homologous recombination seamless cloning kit and following the instructions, the PCR amplification products and enzyme digestion products were homologously recombinated and transformed into competent Escherichia coli BL21(DE3) to construct the PPGK mutant expression strain.

[0146] (4) The PPGK mutant expression strain was inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C and 160 rpm with shaking. Then, 1% of the culture solution was inoculated into fresh LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 160 rpm with shaking until OD. 600 The concentration was 0.8, and IPTG was added to a final concentration of 0.1 mM. The mixture was then incubated at 25 °C for 16 h to obtain the fermentation broth.

[0147] (5) Centrifuge the fermentation culture at 4℃ and 10000rpm for 10min and collect the cell slurry; resuspend the cell slurry in 15mL of sodium phosphate buffer (50mM, pH=7.5), sonicate the cells in an ice bath for 10min in the form of working 4s and intermittent 4s, centrifuge at 4℃ and 10000rpm for 10min, collect the supernatant, and purify the supernatant using a Ni-NTA affinity chromatography column (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain the PPGK mutant pure enzyme solution.

[0148] (6) The enzyme activity of each PPGK mutant was determined by using the PPGK enzyme activity assay kit (purchased from Sobrinos) according to the instructions. The relative activity (unit: %) was calculated with the polyphosphorylase provided in Example 2 as a control. The results are shown in Table 9.

[0149] Table 9.

[0150] As shown in Table 9, referring to the amino acid sequence of the polyphosphorylase shown in SEQ ID NO:15, the enzyme activity of PPGK mutants with V52K mutation or R196A mutation is reduced, while the enzyme activity of PPGK mutants with V150A mutation and / or E122S mutation is significantly increased.

[0151] Example 6 This embodiment illustrates the construction of engineered Escherichia coli strains KDY-JC-01-01 to KDY-JC-01-10. These engineered Escherichia coli strains KDY-JC-01-01 to KDY-JC-01-10 are essentially the same as the engineered Escherichia coli strain KDY-JC-01 provided in Example 2. The difference lies in the polyphosphorylase expressed by the engineered Escherichia coli strains KDY-JC-01-01 to KDY-JC-01-10, as shown in Table 10. Under the same conditions, each PPGK mutant engineered Escherichia coli strain was constructed.

[0152] Table 10.

[0153] Example 7 This embodiment illustrates the ability of the engineered Escherichia coli strain provided in the above embodiments to synthesize inositol from glucose. The specific tests include: 1. Shake flask fermentation (1) Various engineered Escherichia coli strains were streaked onto LB solid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 12 h. Six single colonies were then picked and inoculated into 100 mL of M9 liquid medium containing 100 μg / mL ampicillin, 2% (w / v) glucose, 2 g / L yeast extract and 1% (v / v) glycerol. The cultures were then cultured at 37℃ and 220 rpm for 48 h. When their OD values ​​reached 100 μg / mL, the culture was collected. 600 When the value was 0.6, IPTG was added at a final concentration of 1 mmol / L to obtain the inositol fermentation broth of various Escherichia coli engineered strains.

[0154] (2) Each inositol fermentation broth was centrifuged at 12000 rpm for 15 min. The supernatant was collected and filtered through a 0.22 μm filter membrane. The obtained filtrate was analyzed by HPLC using a differential detector of high performance liquid chromatography (HPLC) to obtain the inositol yield in the sample (unit: g / L). The results are shown in Table 11.

[0155] The HPLC analysis conditions included: a Waters Sugar-Pak I 300mm × 6.5mm column, a column temperature of 70℃, an injection flow rate of 0.4mL / min, and mobile phases A and B being aqueous solutions containing 50mg / L of calcium disodium ethylenediaminetetraacetate.

[0156] Table 11.

[0157] As shown in Table 11, compared with the deletion strain BL21(DE3)Δpgi-Δzwf, gene modification to overexpress exogenous inositol-3-phosphate synthase, inositol monophosphatase and polyphosphorylase in the deletion strain can modify its intracellular metabolic pathway, thus endowing the resulting Escherichia coli engineered strain with a better inositol synthesis ability. Under the same shake-flask fermentation conditions, the inositol yield was improved.

[0158] Among them, when the engineered strain of Escherichia coli expresses a substance derived from... Acidilobus sp. Ino1-As, derived from Thermotoga maritime IMP1-Tm and derived from Thermobifida fusca When PPGK (also known as KDY-JC-01) is used, the inositol synthesis pathway and glucose-driving pathway in the cells of the engineered E. coli strain KDY-JC-01 are better suited to the inositol production-cell growth dual module constructed with glucose and glycerol as mixed carbon sources, thereby enabling more efficient biosynthesis of inositol.

[0159] Furthermore, based on the engineered Escherichia coli strain KDY-JC-01, point mutations were performed on the polyphosphorylase expressed therein. By adjusting the activity of the polyphosphorylase to coordinate the balance of catalytic activities among inositol-3-phosphate synthase, inositol monophosphatase, and polyphosphorylase, the engineered Escherichia coli strains KDY-JC-01-03, KDY-JC-01-04, KDY-JC-01-05, and KDY-JC-01-08 were constructed, which have a more efficient ability to synthesize inositol.

[0160] 2. Continuous Feeding Fermentation (1) Escherichia coli engineered strains KDY-JC-01, KDY-JC-01-03, KDY-JC-01-04, KDY-JC-01-05 and KDY-JC-01-08 were streaked onto LB solid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 12 h. Six single colonies were then picked and inoculated into 100 mL of M9 liquid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm until the solution reached OD. 600 A value of 2 yields seed solutions of various engineered Escherichia coli strains.

[0161] (2) At an inoculation rate of 1% (v / v), each of the various seed cultures was inoculated into a 5L fermenter containing 2L of fermentation medium. The culture was carried out for 12h at a temperature of 30℃, a pH of 7.0, an aeration ratio of 1vvm, an initial rotation speed of 400rpm (range of 300rpm~700rpm), a dissolved oxygen threshold of 10%, and a glucose concentration not higher than 5g / L. Then, after cooling to 30℃, IPTG was added at a final concentration of 1mmol / L for induced enzyme production fermentation culture for 96h. During the induced enzyme production fermentation culture, feed solution was added in batches at a flow rate of 1.25g / L / h to obtain the inositol fermentation broth of various Escherichia coli engineered strains.

[0162] The fermentation medium consisted of 3% (v / v) glycerol, 6% (w / v) glucose, 8 g / L yeast extract, and M9 medium. The feed solution consisted of 400 g / L glucose, 200 g / L glycerol, and M9 medium, with a pH of 7.0.

[0163] (3) The inositol fermentation broth was centrifuged at 12000 rpm for 15 min. The supernatant was collected and filtered through a 0.22 μm filter membrane. The obtained filtrate was analyzed by HPLC using a differential detector of high performance liquid chromatography (HPLC) to obtain the inositol yield in the sample (unit: g / L). The results are shown in Table 12.

[0164] The HPLC analysis conditions included: a Waters Sugar-Pak I 300mm × 6.5mm column, a column temperature of 70℃, an injection flow rate of 0.4mL / min, and mobile phases A and B being aqueous solutions containing 50mg / L of calcium disodium ethylenediaminetetraacetate.

[0165] Table 12.

[0166] As shown in Table 12, compared with the engineered E. coli strain KDY-JC-01, the inositol production of engineered E. coli strains KDY-JC-01-03, KDY-JC-01-04, KDY-JC-01-05, and KDY-JC-01-08 increased by 123.2% to 173.4%. This indicates that the inositol synthesis pathway and glucose-driving pathway introduced in the above-mentioned engineered E. coli strains can still effectively drive glucose into the cell and convert it into inositol under scale-up culture conditions. They have an excellent ability to synthesize inositol using glucose as a carbon source and have very promising application prospects in achieving large-scale, high-yield fermentation production of inositol.

[0167] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. An expression regulatory element for implementing construction of an inositol-producing engineered strain, characterized by, The expression regulatory elements include: (1) an element for blocking expression of endogenous phosphoglucose isomerase in the myo-inositol-producing engineered strain; (2) an element for blocking expression of endogenous glucose-6-phosphate 1-dehydrogenase in the myo-inositol-producing engineered strain; (3) an element for expressing or over-expressing myo-inositol-3-phosphate synthase; (4) an element for expressing or over-expressing myo-inositol monophosphatase; (5) an element for expressing polyphosphorylase, and the polyphosphorylase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 15 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO:

15.

2. The expression regulatory element for implementing the construction of an inositol-producing engineered strain according to claim 1, characterized in that, The element for expressing or over-expressing myo-inositol-3-phosphate synthase includes at least one of the following technical features: (1) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 13 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 13; (2) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 22 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 22; (3) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 23 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 23; (4) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 24 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 24; (5) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 25 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 25; (6) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 26 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 26; (7) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 27 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 27; (8) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 28 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 28; (9) the myo-inositol-3-phosphate synthase includes an amino acid fragment having a sequence as set forth in SEQ ID NO: 29 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 29; (10) the myo-inositol-3-phosphate synthase comprises an amino acid fragment as set forth in SEQ ID NO: 30 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO:

30.

3. The expression regulatory element for implementing the construction of an inositol-producing engineered strain according to claim 1, characterized in that, The element for expressing or over-expressing the myo-inositol monophosphatase comprises at least one of the following technical features: (1) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 14 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 14; (2) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 31 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 31; (3) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 32 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 32; (4) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 33 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 33; (5) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 34 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 34; (6) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 35 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 35; (7) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 36 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 36; (8) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 37 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 37; (9) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 38 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 38; (10) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 39 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO: 39; (11) the myo-inositol monophosphatase comprises an amino acid fragment as set forth in SEQ ID NO: 40 or a variant fragment having at least 90% sequence identity to the amino acid fragment as set forth in SEQ ID NO:

40.

4. The expression regulatory element for implementing the construction of a myo-inositol production engineered strain according to claim 1, characterized in that, The element for expressing the polyphosphorylase comprises at least one of the following technical features: (1) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 15; (2) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 41; (3) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 42; (4) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 43; (5) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 44; (6) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 45; (7) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 46; (8) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 47; (9) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 48; (10) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 49; (11) the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO:

50.

5. The expression regulatory element for implementing the construction of an inositol-producing engineered strain according to claim 1, characterized in that, The expression regulatory element comprises at least one of the following technical features: (1) the myo-inositol-3-phosphate synthase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 13, the myo-inositol monophosphatase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 14, and the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 15; (2) the myo-inositol-3-phosphate synthase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 13, the myo-inositol monophosphatase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 14, and the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 43; (3) the myo-inositol-3-phosphate synthase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 13, the myo-inositol monophosphatase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 14, and the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 44; (4) the myo-inositol-3-phosphate synthase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 13, the myo-inositol monophosphatase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 14, and the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 45; (5) the myo-inositol-3-phosphate synthase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 13, the myo-inositol monophosphatase comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 14, and the polyphosphorylase comprises an amino acid fragment with a sequence as shown in SEQ ID NO:

48.

6. Use of the expression regulatory element according to any one of claims 1-5 in improving the ability of an engineered strain to synthesize myo-inositol.

7. An engineered bacterial strain, characterized in that, The engineered strain comprises the expression regulatory element according to any one of claims 1-5.

8. The method of constructing an engineered bacterial strain of claim 7, characterized in that, The construction method comprises: introducing the expression regulatory element according to any one of claims 1-5 into E. coli to obtain the engineered strain.

9. Use of the expression regulatory element according to any one of claims 1-5 or the engineered strain according to claim 7 in the field of myo-inositol production.

10. A method for producing myo-inositol, characterized by, The production method comprises: fermenting and culturing the engineered strain according to claim 7 to obtain the myo-inositol.

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