Method for synthesizing inositol by using maltose and application

Abstract

The invention aims to provide a method for synthesizing inositol by catalyzing maltose and metahexaphosphate through a multi-enzyme system. The method realizes high-conversion-rate synthesis of inositol from low-value raw materials, maltose is taken as an initial raw material, and a multi-enzyme cascade catalytic reaction system is designed, so that the inositol is efficiently synthesized.

Description

TECHNICAL FIELD

[0001] The application provides a kind of synthetic inositol multi-enzyme cascade catalytic reaction system, the multi-enzyme cascade catalytic reaction system of described by maltose phosphorylase, polyphosphate glucokinase, beta-glucose phosphorclavase, glucose-6-phosphate-1-epimerase, inositol-3-phosphate synthase, inositol monophosphatase Composition.It is maltose phosphorylase (Maltose phosphorylase, MP) with maltose as substrate, catalyzing maltose hydrolysis into glucose and beta-glucose-1-phosphate;Polyphosphate glucokinase (Polyphosphate glucokinase, PPGK) with glucose as substrate, catalyzing glucose to generate alpha-glucose-6-phosphate;Beta-phosphoglucomutase (Beta-phosphoglucomutase, BPGM) with beta-glucose-1-phosphate as substrate, catalyzing beta-glucose-1-phosphate to generate beta-glucose-6-phosphate;Glucose-6-phosphate 1-epimerase (glucose-6-phosphate 1-epimerase, G6PE) with beta-glucose-6-phosphate as substrate, catalyzing beta-glucose-6-phosphate isomerization into alpha-glucose-6-phosphate, inositol-3-phosphate synthase (inositol-3-phosphate synthase, IPS) with alpha-glucose-6-phosphate as substrate, catalyzing alpha-glucose-6-phosphate to synthesize inositol-3-phosphate;Inositol-1-monophosphatase (inositol-1-monophosphatase, IMP) with inositol-3-phosphate as substrate, catalyzing inositol-3-phosphate to synthesize inositol. BACKGROUND

[0002] Myo-inositol, also known as cyclohexanehexol, is a kind of important physiological function vitamin substance, as the isomer of glucose, widely exists in animals, plants and microorganisms, is one of the key factors to maintain the normal growth of animals and microorganisms. It plays an important role in biological membrane structure, not only one of the components of cell membrane phospholipid, participates in maintaining the dynamic balance of membrane lipid, but also the biosynthesis precursor of intracellular second messenger-phosphatidylinositol (PI), and participates in the regulation of PI, phosphatidylcholine (PC) and cardiolipin (CL) and other key phospholipid synthase expression. In addition, inositol also participates in calcium ion homeostasis regulation and cell signal transduction process, has irreplaceable role in maintaining normal cell function and body health.

[0003] The market demand for inositol continues to grow due to its wide application in many fields such as medicine, food and feed. In the field of medicine, inositol has been used to assist in the treatment of cancer, respiratory distress syndrome and various gynecological diseases; in the feed industry, it is added to animal diets as a growth promoter, with both nutritional and health functions; in the food field, it is mainly used as a nutritional enhancer and is widely used in health products, functional beverages and infant formula milk powder.

[0004] The current industrial production of inositol is still mainly based on the traditional pressurized hydrolysis method. With the progress of science and technology and the improvement of environmental protection requirements, researchers have developed new preparation processes including atmospheric catalytic hydrolysis, enzymatic hydrolysis, etc. However, the existing methods (including hydrolysis method and in vitro enzyme synthesis method) generally have the problems of serious environmental pollution, complex process flow, high production cost, etc. Especially in the enzyme method route, the imbalance of catalytic reaction seriously restricts its industrialization application.

[0005] Therefore, developing a new inositol production process that is efficient, environmentally friendly and economically feasible has become a key issue that needs to be broken through in this field. By using cheap and readily available alternative raw materials to construct an innovative synthesis route, it is expected to fundamentally overcome the bottlenecks of existing production technologies and achieve efficient and green manufacturing of inositol. SUMMARY

[0006] The purpose of the present application is to provide a method for synthesizing inositol from maltose and hexametaphosphoric acid by using a multi-enzyme system. The method realizes the synthesis of inositol from low-value raw materials with high conversion rate, uses maltose as the initial raw material, and designs a multi-enzyme cascade catalytic reaction system to efficiently synthesize inositol.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides an enzyme composition comprising proteins or polypeptides having the following functions: A) maltose phosphorylase, B) polyphosphate glucose kinase, C) β-glucose phosphate mutase, D) glucose-6-phosphate-1-epimerase, E) inositol-3-phosphate synthase, and F) inositol monophosphatase.

[0009] Further, the enzyme composition comprises one of the following features: A) the maltose phosphorylase is derived from Enterococcus faecalis, B) the polyphosphate glucose kinase is derived from Thermobifida fusca, C) the beta-glucose phosphomutase is derived from Pyrococcus horikoshii, D) the glucose-6-phosphate-1-epimerase is derived from Salmonella typhimurium, E) the myo-inositol-3-phosphate synthase is derived from Tetrahymena thermophila, and F) the myo-inositol monophosphatase is derived from Thermotoga maritima.

[0010] Further, the enzyme composition comprises one of the following features: A) the maltose phosphorylase is derived from Enterococcus faecalis, B) the polyphosphate glucose kinase is derived from Thermobifida fusca, C) the beta-glucose phosphomutase is derived from Pyrococcus horikoshii, D) the glucose-6-phosphate-1-epimerase is derived from Salmonella typhimurium, E) the myo-inositol-3-phosphate synthase is derived from Tetrahymena thermophila, and F) the myo-inositol monophosphatase is derived from Thermotoga maritima.

[0011] In a second aspect, the present application provides a method for synthesizing myo-inositol by using a multi-enzyme cascade catalytic reaction system. The multi-enzyme cascade catalytic reaction system uses maltose as a substrate, uses partial hexaphosphoric acid as an auxiliary factor, and uses the enzyme composition of the first aspect to catalyze a reaction to synthesize myo-inositol.

[0012] Further, the catalytic reaction is performed in a 20-50 mM sodium phosphate buffer solution. The reaction system contains 50-200 mM maltose, 15 mM partial hexaphosphoric acid, a certain concentration of the enzyme composition, 5 mM MgCl2, the temperature of the reaction system is controlled at 50°C, the pH is controlled at 7.0, the rotation speed is 220 rpm, and the reaction time is 6-24 h.

[0013] In one embodiment of the present application, the yield of the target substance myo-inositol is 180.62%.

[0014] Compared with existing technologies, this solution has the following beneficial effects: This invention uses maltose as raw material, and under the catalytic action of a multi-enzyme reaction system constructed from six enzymes—maltose phosphorylase, polyphosphoglucokinase, β-glucose phosphate mutase, glucose-6-phosphate-1-epimerase, inositol-3-phosphate synthase, and inositol monophosphatase—the raw material maltose is completely converted into two intermediate products: glucose and β-glucose-1-phosphate, ultimately efficiently generating the target product inositol. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of a multi-enzyme cascade catalytic reaction system.

[0016] Figure 2 This is a schematic diagram of the structure of maltose.

[0017] Figure 3 This is a schematic diagram of the structure of inositol.

[0018] Figure 4 The liquid chromatography (LC) spectra of maltose, glucose, and inositol are shown. Note: The retention times for maltose (8.474 min), glucose (10.197 min), and inositol (13.038 min) are as follows. Detailed Implementation

[0019] definition

[0020] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0021] Throughout this specification and the appended claims, the terms "comprising" and "including," as well as variations thereof, shall be interpreted inclusively. That is, where the context permits, these terms are intended to express the possibility of including other elements or integers not specifically listed.

[0022] The article “a / an” is used in this text to refer to one / an or more than one / an (i.e., one / an or at least one / at least one of the grammatical objects of the article). For example, “an element / an element” can mean one element / an element or more than one element / an element. When a noun (e.g., compound, additive, etc.) is mentioned in the singular form, it is intended to include the plural form. Therefore, when referring to a specific part (e.g., “gene”), unless otherwise specified, it means “at least one” of the genes, e.g., “at least one gene”.

[0023] Unless otherwise expressly indicated, the various embodiments of the invention described herein can be combined in various ways.

[0024] The term "carbon source" refers to a source of carbon, preferably a compound or molecule containing carbon. Preferably, the carbon source is a carbohydrate, amino acid, or its derivative. In this document, a carbon source is understood as an organic compound composed of elements such as carbon, oxygen, and hydrogen.

[0025] The term "cell" refers to a eukaryotic or prokaryotic organism, preferably existing as a single cell. In this invention, the cell can be recombinant *Escherichia coli*. That is, the recombinant cell is selected from a cell population of a genus of *Escherichia coli*.

[0026] As used herein, the term “recombinant” / “engineered” (e.g., references to “recombinant E. coli,” “recombinant cell,” “recombinant microorganism,” and / or “recombinant strain”) can refer to a cell, microorganism, or strain containing nucleic acids as a result of one or more gene modifications. Simply put, a cell, microorganism, or strain contains different combinations of nucleic acids from one or more of its parents (any one of them). To construct recombinant cells, microorganisms, or strains, one or more recombinant DNA techniques and / or another one or more mutagenesis techniques can be used. For example, recombinant E. coli and / or recombinant E. coli cells may contain nucleic acids not present in the corresponding wild-type E. coli and / or cells, which have been introduced into the E. coli or E. coli cells using recombinant DNA techniques, or the absence of the nucleic acid in the wild-type E. coli and / or cells is the result of one or more mutations in the nucleic acid sequence (such as a gene encoding a wild-type polypeptide) present in the wild-type E. coli and / or E. coli cells (e.g., using recombinant DNA techniques or another mutagenesis technique such as UV irradiation). Furthermore, the term “recombinant” can suitably refer to, for example, cells, microorganisms, or strains from which nucleic acid sequences have been removed using recombinant DNA techniques.

[0027] In this paper, "recombinant E. coli containing or having a certain activity" is understood to mean that recombinant E. coli can contain one or more nucleic acid sequences encoding proteins having such activity. Therefore, recombinant E. coli is permitted to functionally express such proteins or enzymes.

[0028] The term "functional expression" refers to functional transcription in which the relevant nucleic acid sequence is present, allowing the nucleic acid sequence to actually be transcribed, for example, leading to protein synthesis.

[0029] As used herein with respect to proteins or peptides, the term "mutation" means that, compared to the wild-type or naturally occurring protein or peptide sequence, at least one amino acid has been substituted, inserted into, or deleted from the amino acid sequence. Amino acid substitutions, insertions, or deletions can be achieved, for example, by mutagenesis of the nucleic acids encoding those amino acids. Mutagenesis is a method well known in the art and includes, for example, site-directed mutagenesis by means of PCR or by oligonucleotide-mediated mutagenesis, as described in Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., vols. 1–3 (1989), published by Cold Spring Harbor Publishing.

[0030] As used herein with respect to genes, the term "mutation" means that, compared to a wild-type or naturally occurring nucleic acid sequence, at least one nucleotide in the nucleic acid sequence of a gene or its regulatory sequence has been replaced, inserted into, or deleted from the nucleic acid sequence by a different nucleotide. The substitution, insertion, or deletion of amino acids can be achieved, for example, via mutagenesis, resulting in transcription of a protein sequence having a qualitatively or quantitatively altered function, or the knockout of the gene. In the context of this invention, "altered gene" has the same meaning as a mutated gene.

[0031] As used herein, the term "gene" refers to a nucleic acid sequence of mRNA that can be transcribed and then translated into a protein. A gene that encodes a protein is one or more nucleic acid sequences that encode that protein.

[0032] As used herein, the term "nucleic acid" or "nucleotide" refers to a monomeric unit in a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer (i.e., a polynucleotide), and unless otherwise limited, encompasses known analogs that have the inherent properties of natural nucleotides because they hybridize with single-stranded nucleic acids (e.g., peptide nucleic acids) in a manner similar to naturally occurring nucleotides. For example, an enzyme defined by the nucleotide sequence encoding an enzyme includes (unless otherwise limited) a nucleotide sequence that hybridizes with a reference nucleotide sequence encoding the enzyme. A polynucleotide can be a natural or heterologous structure or a full-length or subsequence of a regulatory gene. Unless otherwise indicated, the term includes references to the specified sequence and its complementary sequence. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as contemplated herein. Furthermore, DNA or RNA containing rare bases (such as inosine) or modified bases (such as triphenylmethylated bases) (to name just two examples) is a polynucleotide as used herein. It will be understood that DNA and RNA have been modified in a wide variety of ways for many useful purposes known to those skilled in the art. As used herein, the term polynucleotide includes such chemical, enzymatic, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA specific to viruses and cells (especially simple and complex cells).

[0033] The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably in this article. An example of a nucleic acid sequence is a DNA sequence.

[0034] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers, for example, those containing amino acid residues as shown in the amino acid sequence. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. An essential property of such analogs of naturally occurring amino acids is that, when incorporated into a protein, the protein exhibits a specific reactivity to antibodies induced by proteins composed entirely of the same, but entirely, naturally occurring amino acids. The terms “polypeptide,” “peptide,” and “protein” also include modifications, including but not limited to glycosylation, lipid attachment, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0035] The term "enzyme" in this document refers to a protein that has a catalytic function. In cases where a protein catalyzes a biological reaction, the terms "protein" and "enzyme" may be used interchangeably herein. When referring to enzymes in the Enzyme Classification (EC), an enzyme class is a category in which an enzyme is classified or can be classified according to the enzyme nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which can be found at http: / / www.chem.qmul.ac.uk / iubmb / enzyme / . It is intended to include other suitable enzymes that are not yet (not yet) classified in the specified category but can be so classified.

[0036] If a protein or nucleic acid sequence (such as a gene) is referred to in this document by a reference accession number, unless otherwise specified, that number is specifically used to refer to a protein or nucleic acid sequence (gene) that can be found via www.ncbi.nlm.nih.gov / (available as of October 1, 2020).

[0037] Each nucleic acid sequence encoding a polypeptide in this paper also includes variants of any conserved modifications thereof. This includes, by reference to the genetic code, every possible silent variant of the nucleic acid. The term “conserved variant” applies to both amino acids and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conserved variant refers to those nucleic acids that encode the same amino acid sequence or a variant of a conserved modified amino acid sequence due to the degeneracy of the genetic code. The term “degeneracy of the genetic code” refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where the codon specifies alanine, the codon can be changed to any of the described corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are “silent variants” and represent a variant of a conserved modification.

[0038] As used herein, the term "functional homolog" (or simply "homology") of a gene having a specific sequence (e.g., "SEQ ID NO:X") refers to a polypeptide and / or amino acid sequence containing the specific sequence, or a nucleic acid sequence containing a polypeptide and / or amino acid sequence encoding the specific sequence, provided that one or more amino acids are mutated, substituted, deleted, added, and / or inserted, and that the polypeptide has (qualitatively) the same enzymatic function for substrate transformation.

[0039] As used herein, the term "functional homolog" (or simply "homolog") of a polynucleotide and / or nucleic acid sequence having a specific sequence (e.g., "SEQ ID NO:X") refers to a polynucleotide and / or nucleic acid sequence containing said specific sequence, provided that one or more nucleic acids are mutated, substituted, deleted, added, and / or inserted, and that the polynucleotide encodes a polypeptide sequence having (qualitatively) the same enzymatic function for substrate transformation. Regarding nucleic acid sequences, the term "functional homolog" is intended to include nucleic acid sequences that differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[0040] In this document, sequence identity is defined as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. Typically, sequence identity or similarity is compared over the entire length of the sequences being compared. In this art, “identity” also means the degree of sequence correlation between amino acid or nucleic acid sequences, as determined by matching strings of such sequences.

[0041] Amino acid or nucleotide sequences are said to be homologous when they exhibit a certain level of similarity. Two sequences being homologous indicates a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "identity percentage" or "similarity percentage," which are high or low, respectively. Although controversial, "homology level" or "homology percentage" is often used interchangeably to indicate "identity percentage" or "similarity percentage." Sequence comparisons and the determination of the identity percentage between two sequences can be accomplished using mathematical algorithms. Technicians will realize that several different computer programs can be used to align two sequences and determine their homology (Kruskal et al., "An overview of sequence comparison: Time warps, string edits, and macromolecules", (1983), Society for Industrial and Applied Mathematics (SIAM), Vol. 25, No. 2, pp. 201-237; and the handbook edited by D. Sankoff and J.B. Kruskal, "Time warps, string edits and macro molecules: the theory and practice of sequence comparison", (1983), pp. 1-44, published by Addison-Wesley Publishing Company, Massachusetts USA).

[0042] The Needleman and Wunsch algorithm can be used to align two sequences to determine the percentage of identity between the two amino acid sequences. (Needleman et al., "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins" (1970), J. Mol. Biol., Vol. 48, pp. 443-453). This algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or later, see Rice et al., "EMBOSS: The European Molecular Biology Open Software Suite", (2000), Trends in Genetics, Vol. 16, (6), pp. 276-277, http: / / emboss.bioinformatics.nl / ) was used. For protein sequences, EBLOSUM62 was used as the substitution matrix. For nucleotide sequences, EDNAFULL was used. Other matrices may be specified. Optional parameters for amino acid sequence alignment are a vacancy opening penalty of 10 and a vacancy expansion penalty of 0.5. Those skilled in the art will understand that all these different parameters will produce slightly different results, but the overall percentage of identity between the two sequences does not change significantly when different algorithms are used.

[0043] Homology or identity is the percentage of identical matches between two complete sequences across the total aligned region, including any vacancies or extensions. Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions in both sequences showing the same amino acid in the alignment divided by the total length of the alignment, including vacancies. Identity, as defined herein, is available from NEEDLE and is labeled "IDENTITY" in the program's output.

[0044] Homology or identity between two aligned sequences is calculated as follows: the number of positions in the alignment showing the same amino acid in both sequences is divided by the total length of the alignment after subtracting the total number of vacancies in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "longest-identity" in the program's output.

[0045] Variants of the nucleotide or amino acid sequences disclosed herein may also be defined as nucleotide or amino acid sequences having one or more mutations, substitutions, insertions, and / or deletions compared to the nucleotide or amino acid sequences specifically disclosed herein (e.g., in the sequence listing).

[0046] Optionally, when determining the degree of amino acid similarity, those skilled in the art may also consider so-called “conservative” amino acid substitutions, which will be clear to them. Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. In one embodiment, the group of conserved amino acid substitutions is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are variants in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted at its position. Preferably, the amino acid changes are conserved. In one embodiment, the conserved substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0047] The nucleotide sequences of the present invention can also be defined by their ability to partially hybridize with specific nucleotide sequences disclosed herein, respectively, under moderate hybridization conditions or preferably under stringent hybridization conditions. Stringent hybridization conditions are defined herein as conditions that allow nucleic acid sequences of at least about 25 nucleotides, preferably about 50, 75, or 100 nucleotides, and most preferably about 200 or more nucleotides, to hybridize at about 65°C in a solution containing about 1 M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to be washed at 65°C in a solution containing about 0.1 M or less salt (preferably 0.2x SSC or any other solution with equivalent ionic strength). Preferably, hybridization is performed overnight, i.e., at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with about 90% or higher sequence identity. In this document, moderate conditions are defined as conditions that allow nucleic acid sequences of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at about 45°C in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to wash at room temperature in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent ionic strength). Preferably, hybridization is performed overnight, i.e., at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with up to 50% sequence identity. Those skilled in the art will be able to modify these hybridization conditions to specifically identify sequences with identity varying between 50% and 90%.

[0048] "Expression" refers to the transcription of genes into structural RNA (rRNA, tRNA) or messenger RNA (mRNA), which is then translated into proteins.

[0049] "Overexpression" refers to the expression of a gene (corresponding nucleic acid sequence) in recombinant cells exceeding its expression in the corresponding wild-type cells. Such overexpression can be arranged, for example, by increasing the transcription frequency of one or more nucleic acid sequences, such as by operatively linking the nucleic acid sequence to a functional promoter in the recombinant cell; and / or by increasing the copy number of a nucleic acid sequence.

[0050] The term "upregulation" and its variations refer to the process by which cells increase the amount of cellular components, such as RNA or proteins. This upregulation can be in response to or caused by gene modification.

[0051] In this article, the term "pathway" or "metabolic pathway" is understood as a series of chemical reactions that build up and break down molecules in the cell.

[0052] Nucleic acid sequences (i.e., polynucleotides) or proteins (i.e., polypeptides) can be native or heterologous to the host cell's genome.

[0053] The terms "natural," "homologous," or "endogenous" in relation to a host cell mean that the nucleic acid sequence is indeed naturally present in the host cell's genome, or that the protein is naturally produced by that cell. The terms "natural," "homologous," and "endogenous" are used interchangeably in this document.

[0054] As used herein, “heterologous” or “exogenous” can refer to a nucleic acid sequence or a protein. For example, in relation to a host cell, “heterologous” can refer to a polynucleotide that is not naturally present in the genome of the host cell in this manner, or a polypeptide or protein that is not naturally produced by the cell in this manner. A heterologous nucleic acid sequence is a nucleic acid derived from a foreign species, or, if from the same species, substantially modified relative to its natural form in terms of composition and / or genomic loci through deliberate human intervention. For example, a promoter operatively linked to a natural structural gene is derived from a species different from the species from which the structural gene is derived, or, if from the same species, one or both are substantially modified relative to their original form. A heterologous protein can be derived from a foreign species, or, if from the same species, substantially modified relative to its original form through deliberate human intervention. That is, heterologous protein expression involves the expression of a protein that is not naturally expressed in the host cell in this manner. The term “heterologous expression” refers to the expression of a heterologous nucleic acid in a host cell. The expression of heterologous proteins in eukaryotic host cell systems (such as *Escherichia coli*) is well known to those skilled in the art. Polynucleotides containing nucleic acid sequences encoding genes for proteins or enzymes with specific activities can be expressed in such eukaryotic systems. In some embodiments, transformed / transfected cells can be used as expression systems for enzymes. The expression of heterologous proteins in *E. coli* is well known. *The Cold Spring Harbor Laboratory* is a recognized work describing various methods that can be used to express proteins in *E. coli*.

[0055] As used herein, a "promoter" is a DNA sequence that directs the transcription of a (structural) gene or other (partial) nucleic acid sequence. Appropriately, the promoter is located in the 5' region of the gene, near the transcription start site of the (structural) gene. The promoter sequence can be constitutive, inducible, or repressive. In one embodiment, no (external) inducer is required.

[0056] As used herein, the term "vector" includes references to autosomal expression vectors and integration vectors for integration into chromosomes.

[0057] The term "expression vector" refers to a linear or circular DNA molecule containing a segment encoding a target polypeptide, which is controlled (i.e., operatively linked to) additional nucleic acid segments that provide for its transcription. These additional segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both.

[0058] "Plasmid" refers to autonomously replicating extrachromosomal DNA that does not integrate into the genome of a microorganism and is usually circular in nature.

[0059] In this paper, "host cell" is understood to be a cell (such as an E. coli cell) that will be transformed with one or more nucleic acid sequences encoding one or more heterologous proteins to construct a transformed cell (also known as a recombinant cell). For example, the transformed cell may contain a vector and may support the replication and / or expression of the vector.

[0060] As used herein, “transformation” refers to the insertion of a foreign polynucleotide into a host cell, regardless of the method used for insertion, such as direct uptake, transduction, f-conjugation, or electroporation. The foreign polynucleotide may be maintained as a non-integrating vector (e.g., a plasmid) or alternatively integrated into the host cell genome.

[0061] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0062] Those skilled in the art will understand that the gene or its functional homologs disclosed herein comprise a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the exemplarily listed sequences, or having one or more mutated, substituted, inserted, and / or deleted amino acid sequences compared to the amino acid sequence it encodes.

[0063] Preferably, compared with the amino acid sequence encoded by a gene, the amino acid sequence encoded by any functional homolog of that gene has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 amino acid mutations, substitutions, insertions, and / or deletions.

[0064] Exemplarily, and not limitingly, the functional homolog of the EfMP gene shown in SEQ ID NO.2 comprises a nucleic acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any of those nucleic acid sequences, or its functional homolog comprises a nucleic acid sequence having one or more mutations, substitutions, insertions, and / or deletions when compared with any of those nucleic acid sequences; preferably, the nucleic acid sequence of any such functional homolog has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 nucleic acid mutations, substitutions, insertions, and / or deletions compared with such nucleic acid sequences. More preferably, the functional homolog of the EfMP gene comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the polypeptide shown by SEQ ID NO. 1.

[0065] Those skilled in the art will understand that other sequences include the above-described cases.

[0066] Unless otherwise specified herein, the terms and terminology used herein should be understood in accordance with the conventional knowledge and usage of those skilled in the art. Unless otherwise stated, the specific operational methods (including: preparation processes, experimental steps, detection methods, etc.) employed in this application utilize conventional techniques in the fields of biochemistry, cell biology, molecular biology, gene editing (e.g., recombinant DNA technology), zoology, and related areas. These techniques are well-described in existing literature. For details, please refer to Sam Brook et al., *Molecular Cloning: a Laboratory Manual*, 4th edition, Cold Spring Harbor Laboratory Press, 2012; Ausubel et al., *Current Protocols in Molecular Biology*, Wiley Online Press, updated irregularly; Kursad Turksen et al., *Embryonic StemCell Protocols*, 3rd edition, Springer Press, 2016; P. Nagarajan et al., *Essentials of Laboratory Animal Science: Principles and Practices*, Springer Press, 2021; and Jann Hau et al., *Handbook of Laboratory Animal Science: Essential Principles and Practices*, 4th edition, CRC Press, 2021.

[0067] The Escherichia coli DH5α competent cells in the following examples are products of Beijing Qingke Biotechnology Co., Ltd., product catalog number TSC01.

[0068] The carrier pBAD / HisB in the following embodiments is a product of Invitrogen, catalog number V430-01.

[0069] The maltose used in the following examples is a product of Aladdin, catalog number M104816.

[0070] The inositol used in the following examples is a product of Aladdin, catalog number I108336.

[0071] The glucose used in the following examples is a product of Sigma-Aldrich, catalog number G8270. Detailed Implementation

[0072] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. In the following examples, the quantitative experiments were all performed in triplicate, and the results were averaged. In the following examples, for chromatograms under the same conditions, the peak in the test sample with the same retention time (±0.1 min) as the standard can be identified as the target peak.

[0073] The liquid LB medium (pH 7.0) in the following examples contains 1 g / 100 mL NaCl, 1 g / 100 mL tryptone, 0.5 g / 100 mL yeast extract, and the remainder is water. The solid medium is obtained by adding agarose to the liquid medium.

[0074] The specific *Escherichia coli* strain used in the following examples may be one of *Escherichia coli* MG1655, BW25113, or MC02. *Escherichia coli* MG1655 (CGSC#: 6300) and BW25113 (CGSC#: 7636) are products of the Yale University Genetic Collection Center for *Escherichia coli* (CGSC). *Escherichia coli* MC02 is deposited at the China General Microbiological Culture Collection Center (CGMCC) (accession number CGMCC No. 34378).

[0075] The primer sequences involved in the following examples are shown in Table 1 below.

[0076] Table 1 shows the primer names and primer nucleotide sequences in the examples.

[0077]

[0078] Example 1 - Construction of recombinant plasmids expressing various enzymes in a multi-enzyme system

[0079] I. Obtaining the six gene fragments

[0080] Using the artificially synthesized sequence containing SEQ ID NO.1 (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, PCR amplification was performed using primers EfMP-F and EfMP-R to obtain the PCR product, namely the gene sequence encoding maltose phosphorylase EfMP (shown as SEQ ID NO.2 in the sequence listing).

[0081] Using the artificially synthesized sequence containing SEQ ID NO.3 (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, PCR amplification was performed using primers TfPPGK-F and TfPPGK-R to obtain the PCR product, namely the gene sequence encoding polyphosphoglucosinolate kinase TfPPGK (shown as SEQ ID NO.4 in the sequence listing).

[0082] Using the artificially synthesized sequence containing SEQ ID NO.5 (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, PCR amplification was performed using primers PhBPGM-F and PhBPGM-R to obtain the PCR product, namely the gene sequence encoding β-glucose phosphate mutase PhBPGM (shown as SEQ ID NO.6 in the sequence listing).

[0083] Using the artificially synthesized sequence containing SEQ ID NO.7 (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, PCR amplification was performed using primers StG6PE-F and StG6PE-R to obtain the PCR product, namely the gene sequence encoding glucose-6-phosphate-1-epimerase StG6PE (shown as SEQ ID NO.8 in the sequence listing).

[0084] Using the artificially synthesized sequence containing SEQ ID NO.9 (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, PCR amplification was performed using primers TtIPS-F and TtIPS-R to obtain the PCR product, namely the gene sequence encoding inositol-3-phosphate synthase TtIPS (shown as SEQ ID NO.10 in the sequence listing).

[0085] Using the artificially synthesized sequence containing SEQ ID NO.11 (synthesized by Beijing Qingke Biotechnology Co., Ltd.) as a template, PCR amplification was performed using primers TmIMP-F and TmIMP-R to obtain the PCR product, namely the gene sequence encoding inositol monophosphatase TmIMP (shown as SEQ ID NO.12 in the sequence listing).

[0086] PCR products were detected by 1% agarose gel electrophoresis. The PCR products were approximately 2300bp, 800bp, 600bp, 800bp, 1600bp, and 800bp in size, which matched the target fragments and were named EfMP, TfPPGK, PhBPGM, StG6PE, TtIPS, and TmIMP. The six gene fragments were then recovered from the gel.

[0087] II. Construction of Six Recombinant Carriers

[0088] The pBAD / HisB vector was double-digested with XhoI and SpeI, and the vector fragment (approximately 3500 bp) was recovered. The EfMP, TfPPGK, PhBPGM, StG6PE, TtIPS, and TmIMP gene fragments recovered in step 1 were ligated to the recovered vector fragment using the Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. NatMethods 2009, 6:343-345.) to obtain the ligation product. The ligation product was transformed into DH5α competent cells and plated on LB agar plates containing streptomycin. Incubate overnight at 37℃, select single clones to extract plasmids, and perform sequencing verification. The correctly sequenced vectors are named pBAD / HisB-EfMP, pBAD / HisB-TfPPGK, pBAD / HisB-PhBPGM, pBAD / HisB-StG6PE, pBAD / HisB-TtIPS, and pBAD / HisB-TmIMP (abbreviated as pEfMP, pTfPPGK, pPhBPGM, pStG6PE, pTtIPS, and pTmIMP).

[0089] Example 2 - Construction of recombinant expression strains for each enzyme in a multi-enzyme system

[0090] Expression vectors pEfMP, pTfPPGK, pPhBPGM, pStG6PE, pTtIPS, and pTmIMP (constructed in Example 1) were chemically transformed into *E. coli* MC02. Positive clones were screened on LB agar plates containing streptomycin (50 μg / mL) to obtain the corresponding recombinant strains. Specifically, the recombinant strain obtained by transforming *E. coli* MC02 with expression vector pEfMP was named S101; the recombinant strain obtained by transforming *E. coli* MC02 with expression vector pTfPPGK was named S102; the recombinant strain obtained by transforming *E. coli* MC02 with expression vector pPhBPGM was named S103; the recombinant strain obtained by transforming *E. coli* MC02 with expression vector pStG6PE was named S104; the recombinant strain obtained by transforming *E. coli* MC02 with expression vector pTtIPS was named S105; and the recombinant strain obtained by transforming *E. coli* MC02 with expression vector pTtIPS was named S106.

[0091] Example 3 - Protein expression of each enzyme in a multi-enzyme system and preparation of crude enzyme solution

[0092] Protein expression

[0093] Each of the following strains (S101, S102, S103, S104, S105, and S106) was used as a recombinant bacterium, and the following experiments were performed simultaneously: The recombinant bacteria (the recombinant expression strains of the six enzymes constructed in Example 2) were streaked onto LB agar plates containing 1.5 g / 100 mL of streptomycin (containing 50 μg / mL of streptomycin) and incubated at 37°C for 12 h. Single colonies were picked and inoculated into liquid LB medium containing 50 μg / mL of streptomycin, and cultured at 37°C with shaking for 10–12 h at 220 rpm to obtain an overnight culture. The overnight culture was then inoculated into LB liquid medium at a volume percentage of 1%, and cultured at 37°C with shaking at 220 rpm until the OD600nm reached approximately 0.6–0.8 (approximately 2–3 h). An inducer (arabinose, final concentration 2 g / L) was then added, and the culture was incubated at 25°C with shaking for 12–16 h to obtain the induced recombinant Escherichia coli. The recombinant Escherichia coli cultured after induction was centrifuged at 4℃ and 4500rpm for 10 min to collect the bacterial cells.

[0094] Preparation of crude enzyme solution

[0095] Resuspend each 2g of wet bacterial cells collected in step one in 20mL of phosphate buffer (pH 7.0) and heat-treat at 80℃ for 5-10min. Centrifuge the treated solution and collect the supernatant to obtain the corresponding enzyme solutions: maltose phosphorylase (EfMP) solution I, polyphosphoglucose kinase (TfPPGK) solution II, β-glucose phosphate mutase (PhBPGM) solution III, glucose-6-phosphate-1-epimerase (StG6PE) solution IV, inositol-3-phosphate synthase (TtIPS) solution V, and inositol monophosphatase (TmIMP) solution VI. Mix enzyme solutions I through VI in a volume ratio of 2:1:1:1:2:1 to obtain a mixed enzyme solution.

[0096] Example 4 - Construction of a multi-enzyme cascade catalytic reaction system for the synthesis of inositol from maltose

[0097] The multi-enzyme cascade catalytic reaction system was constructed using a 10 mL reaction volume containing 50 mM maltose, 50 mM sodium phosphate (pH 7.0), 15 mM unsymmetrical hexaphosphate, 5 mM MgCl2, 200 μL maltose phosphorylase (EfMP) enzyme solution I, 100 μL polyphosphate glucokinase (TfPPGK) enzyme solution II, 100 μL β-glucose phosphate mutase (PhBPGM) enzyme solution III, 100 μL glucose-6-phosphate-1-epimerase (StG6PE) enzyme solution IV, 200 μL inositol-3-phosphate synthase (TtIPS) enzyme solution V, and 100 μL inositol monophosphatase (TmIMP) enzyme solution VI. The reaction was carried out in a constant-temperature shaker at 50 °C and 200 rpm for 24 h. The pH of the system was adjusted to 7.0 using NaOH. Samples were taken during the process for HPLC analysis to monitor the reaction progress. After the reaction is complete, NaOH is added to terminate the reaction. The reaction solution is diluted 10 times with distilled water, and the concentration of inositol and residual maltose is detected by HPLC. The conversion rate is calculated according to the following formula (Formula 1).

[0098]

[0099] Chromatographic conditions: Bio-Rad Aminex HPX-87C Column (300 × 7.8 mm); mobile phase: ultrapure water; flow rate: 0.6 mL / min; column temperature: 80℃; injection volume: 10 μL; refractive index detector (RID). HPLC chromatograms of maltose, glucose, and inositol standards are shown below. Figure 4 As shown. Example 5 - Optimization of substrate addition in multi-enzyme systems

[0100] The amount of maltose substrate added to the reaction system in Example 4 was changed to 50 mM, 100 mM, and 200 mM, respectively, while other conditions remained unchanged. After the reaction was completed, the inositol yield was determined by HPLC.

[0101] The inositol content and conversion rate in the catalytic system are shown in Table 2.

[0102] Table 2. Inositol content and conversion rate in the catalytic system

[0103]

[0104] The results showed that the optimal addition amount of maltose substrate was 50 mM, under which the inositol yield reached 90.31 ± 3.75 mM and the conversion rate reached 180.62 ± 0.38%. This indicates that the proposed method can achieve high-efficiency whole-cell catalytic synthesis of inositol using maltose as a raw material.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An enzyme composition comprising a protein or polypeptide having the following functions: A) maltose phosphorylase, B) polyphosphoglucose kinase, C) β-glucose phosphate mutase, D) glucose-6-phosphate-1-epimerase, E) inositol-3-phosphate synthase, F) inositol monophosphatase.

2. The enzyme composition according to claim 1, wherein the enzyme composition comprises one of the following characteristics: A) maltose phosphorylase is derived from Enterococcus faecalis, B) polyphosphoglucose kinase is derived from Thermobifida fusca, C) β-glucose phosphate mutase is derived from Pyrococcus horikoshii, D) glucose-6-phosphate-1-epimerase is derived from Salmonella typhimurium, E) inositol-3-phosphate synthase is derived from Tetrahymena thermophila, and F) inositol monophosphatase is derived from Thermotoga maritima.

3. The enzyme composition according to claims 1 to 2, wherein the enzyme composition comprises one of the following features: A) the amino acid sequence of maltose phosphorylase comprises the amino acid sequence shown in SEQ ID NO:1; B) the amino acid sequence of polyphosphoglucose kinase comprises the amino acid sequence shown in SEQ ID NO:3; C) the amino acid sequence of β-glucose phosphate mutase comprises the amino acid sequence shown in SEQ ID NO:5; D) the amino acid sequence of glucose-6-phosphate-1-epimerase comprises the amino acid sequence shown in SEQ ID NO:7; E) the amino acid sequence of inositol-3-phosphate synthase comprises the amino acid sequence shown in SEQ ID NO:9; and F) the amino acid sequence of inositol monophosphatase comprises the amino acid sequence shown in SEQ ID NO:

11.

4. A multi-enzyme cascade catalytic reaction system for synthesizing inositol, wherein the multi-enzyme cascade catalytic reaction system uses maltose as a substrate and unsymmetrical hexaphosphate as a cofactor, and utilizes the enzyme composition described in claims 1-3 to carry out the catalytic reaction to synthesize inositol.

5. Based on the multi-enzyme cascade reaction system of claim 4, further, the catalytic reaction is carried out in a 20-50 mM sodium phosphate buffer solution, the reaction system contains 50-200 mM maltose, 15 mM metahexaphosphate, a certain concentration of the enzyme composition of claims 1-4, the reaction system also contains 5 mM MgCl2, the temperature of the reaction system is controlled at 50℃, the pH is controlled at 7.0, the rotation speed is 220 rpm, and the reaction time is 6-24 h.

6. The method for synthesizing inositol using a multi-enzyme cascade catalytic reaction system according to any one of claims 1 to 5.

7. The application of the multi-enzyme cascade catalytic reaction system according to any one of claims 1 to 6 in the production of inositol.

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