Method for preparing inositol

Abstract

The invention discloses a method for preparing inositol, particularly relates to a method for converting glucose into inositol through in-vitro multi-enzyme catalysis, and belongs to the field of enzyme catalysis preparation of inositol. According to the invention, the phosphoglucose synthase capable of realizing phosphorylation of glucose by using inorganic phosphate is screened, and the inositol can be generated through conversion by taking glucose as a substrate, so that a novel method for synthesizing the inositol through enzyme catalytic conversion is provided. In addition, the invention further provides hexokinase capable of improving the yield of inositol, and the conversion efficiency of glucose is greatly improved. Furthermore, the invention provides a multi-enzyme molecular machine containing the glucose phosphate synthase and / or hexokinase, the multi-enzyme molecular machine is applied to preparation of inositol, and the reaction steps are simple, green and pollution-free.

Description

Technical Field

[0001] The invention relates to a method for preparing inositol, in particular to a method for converting glucose into inositol by in vitro multi-enzyme catalysis, and belongs to the field of enzyme catalysis production of inositol. Background Art

[0002] Inositol, also known as cyclohexanehexol, is a water-soluble vitamin B. Inositol is an essential substance for the growth of humans, animals and microorganisms, and is widely used in medicine, food, feed and other industries. In recent years, thanks to the continuous development of the downstream market, the demand for inositol has also been increasing. The demand for inositol in China has been increasing year by year, from 1,654 tons in 2015 to 2,502.4 tons in 2021. With the in-depth research on inositol, the changes in the international economic situation and the improvement of people's living standards, the scale of my country's inositol market has continued to grow. It is expected that the scale of China's inositol market will continue to rise in the future.

[0003] At present, the production of inositol is mainly obtained by high temperature and pressure hydrolysis of phytic acid (inositol hexaphosphate). The material requirements of this process equipment are strict. Since the ion exchange resin needs to be regenerated regularly, a large amount of acidic or alkaline solution is discharged into the environment, causing serious environmental pollution. Patent document CN106148425A discloses a method for producing inositol by in vitro multi-enzyme catalysis of starch and starch derivatives. This method can achieve the preparation of inositol by at least 4 steps of reaction. In order to improve the utilization rate of substrate starch and starch derivatives, it is usually necessary to increase 1-3 steps of reaction, such as adding isoamylase, pullulanase, polyphosphate glucose synthase, etc., the reaction steps are relatively cumbersome, and the conversion rate is still low. Summary of the invention

[0004] Problem that the invention aims to solve

[0005] The current enzymatic method for preparing inositol has a low conversion rate, and the enzyme used in the enzymatic conversion method has a small selectivity, which is not conducive to the preparation and application of inositol.

[0006] Solutions for solving problems

[0007] Based on the existing problems, the inventors screened out phosphoglucose synthase, which can use inorganic phosphate to phosphorylate glucose, thus providing a new method for synthesizing inositol by enzyme-catalyzed conversion, with simple and pollution-free synthesis steps. In addition, the inventors also screened out hexokinase, which can increase the yield of inositol, greatly improving the conversion efficiency of glucose.

[0008] The technical solution of the present invention is as follows:

[0009] [1] A method for preparing inositol, wherein glucose or its derivatives are used as substrates in a reaction system containing phosphate groups, and an enzyme catalyst is used to catalyze the substrate to produce inositol, wherein the enzyme catalyst comprises phosphoglucose synthase and / or hexokinase with an EC number of EC 2.7.1.1.

[0010] [2] The method according to [1], wherein the source of the phosphoglucose synthase comprises Clostridium thermocellum, Acidothermus cellulolyticus and / or Thermomonospora curvata;

[0011] Optionally, the source of the hexokinase comprises Sulfurisphaera tokodaii, Ogataea angusta, Thermus caldophilus and / or Thermus thermophilus.

[0012] [3] The method according to [1] or [2], wherein:

[0013] The phosphoglucose synthase is selected from any one of the following (a1) to (a3):

[0014] (a1) comprising a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1;

[0015] (a2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate;

[0016] (a3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2);

[0017] The hexokinase is selected from any one of the following (b1) to (b3):

[0018] (b1) comprising a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4;

[0019] (b2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate;

[0020] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0021] [4] The method according to any one of [1] to [3], wherein the phosphate groups in the reaction system are derived from inorganic phosphate and / or adenine nucleoside phosphate.

[0022] [5] The method according to any one of [1] to [4], wherein the enzyme catalyst further comprises an inositol-3-phosphate synthase with an EC number of EC 5.5.1.4 and an inositol monophosphatase with an EC number of EC 3.1.3.25;

[0023] Optionally, the source of the inositol 3-phosphate synthase includes Archaeoglobus fulgidus, Archaeoglobus profundus, Thermotoga maritima, Thermococcus kodakarensis and / or Pyrococcus furiosus;

[0024] Optionally, the source of the inositol monophosphatase comprises Thermotoga maritima, Methanocaldococcusjannaschii and / or Mycolicibacterium smegmatis.

[0025] [6] The method according to [5], wherein the inositol-3-phosphate synthase is selected from any one of the following (c1) to (c3):

[0026] (c1) comprising a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1;

[0027] (c2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose 6-phosphate into inositol 1-phosphate;

[0028] (c3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (c1) or (c2);

[0029] The inositol monophosphatase is selected from any one of the following (d1) to (d3):

[0030] (d1) comprising a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86;

[0031] (d2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86, in which one or more amino acids are substituted, deleted or added, and having the activity of converting inositol 1-phosphate into inositol;

[0032] (d3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (d1) or (d2).

[0033] [7] The method according to any one of [1] to [6], wherein the reaction system comprises an ATP regeneration system;

[0034] Optionally, regeneration of ATP is achieved enzymatically;

[0035] Preferably, the enzymatic method utilizes polyphosphate kinase with an EC number of EC 2.7.4.1 to achieve ATP regeneration in a regeneration system containing polyphosphate;

[0036] Optionally, the source of the polyphosphate kinase includes Rhodobacter sphaeroides, Thermosynechococcus vestitus and / or Mycobacterium tuberculosis, preferably Thermosynechococcus vestitus;

[0037] Optionally, the polyphosphate kinase derived from Thermosynechococcus vestitus is selected from any one of the following (e1) to (e1):

[0038] (e1) comprising a polypeptide as shown in Uniprot ID: Q8DMA8;

[0039] (e2) a polypeptide having an amino acid sequence of the polypeptide shown in Uniprot ID: Q8DMA8, in which one or more amino acids are substituted, deleted or added, and having the activity of converting polyphosphate and ADP into ATP;

[0040] (e3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (e1) or (e2);

[0041] Optionally, the polyphosphoric acid is provided by a polyphosphate, and optionally, the polyphosphate comprises sodium pyrophosphate, sodium hexametaphosphate and / or sodium tripolyphosphate.

[0042] [8] The method according to any one of [1] to [7], wherein the enzyme catalyst is in the form of pure enzyme, crude enzyme, cell culture, cell lysate or whole cells; optionally, the pure enzyme or crude enzyme is in free or immobilized form, and the whole cells are in free or immobilized form.

[0043] [9] A method according to any one of [1] to [8], wherein:

[0044] The reaction system contains magnesium ions and reacts at 0-100° C.;

[0045] Optionally, the method further comprises the step of separating and purifying the inositol from the reaction system.

[0046]

[10] A multi-enzyme molecular machine comprising: a first module comprising a first enzyme element, and, optionally, a second module comprising a second enzyme element and a non-enzyme element;

[0047] The first enzyme element in the first module comprises phosphoglucose synthase and / or hexokinase with EC number EC 2.7.1.1, and, optionally, inositol-3-phosphate synthase with EC number EC 5.5.1.4 and inositol monophosphatase with EC number EC 3.1.3.25.

[0048]

[11] The multi-enzyme molecular machine according to

[10] , wherein the source of the phosphoglucose synthase includes Clostridium thermocellum, Acidothermus cellulolyticus and / or Thermomonosporacurvata;

[0049] Optionally, the source of the hexokinase comprises Sulfurisphaera tokodaii, Ogataea angusta, Thermus caldophilus and / or Thermus thermophilus;

[0050] Optionally, the source of the inositol-3-phosphate synthase includes Archaeoglobus fulgidus, Archaeoglobus profundus, Thermotoga maritima, Thermococcus kodakarensis and / or Pyrococcus furiosus;

[0051] Optionally, the source of the inositol monophosphatase comprises Thermotoga maritima, Methanocaldococcusjannaschii and / or Mycolicibacterium smegmatis.

[0052]

[12] The multi-enzyme molecular machine according to

[10] or

[11] , wherein

[0053] The phosphoglucose synthase is selected from any one of the following (a1) to (a3):

[0054] (a1) comprising a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1;

[0055] (a2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate;

[0056] (a3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2);

[0057] The hexokinase is selected from any one of the following (b1) to (b3):

[0058] (b1) comprising a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4;

[0059] (b2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate;

[0060] (b3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2);

[0061] The inositol-3-phosphate synthase is selected from any one of the following (c1) to (c3):

[0062] (c1) comprising a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1;

[0063] (c2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose 6-phosphate into inositol 1-phosphate;

[0064] (c3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (c1) or (c2);

[0065] The inositol monophosphatase is selected from any one of the following (d1) to (d3):

[0066] (d1) comprising a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86;

[0067] (d2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86, in which one or more amino acids are substituted, deleted or added, and having the activity of converting inositol 1-phosphate into inositol;

[0068] (d3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (d1) or (d2).

[0069] 13. The multi-enzyme molecular machine according to any one of

[10] to

[12] , wherein the second module comprises an ATP regeneration module;

[0070] The second enzyme element comprises a polyphosphate kinase having an EC number of EC2.7.4.1, and the non-enzyme element comprises ADP;

[0071] The source of the polyphosphate kinase includes Rhodobacter sphaeroides, Thermosynechococcus vestitus and / or Mycobacterium tuberculosis, preferably Thermosynechococcus vestitus;

[0072] Optionally, the polyphosphate kinase derived from Thermosynechococcus vestitus is selected from any one of the following (e1) to (e1):

[0073] (e1) comprising a polypeptide as shown in Uniprot ID: Q8DMA8;

[0074] (e2) a polypeptide having an amino acid sequence of the polypeptide shown in Uniprot ID: Q8DMA8, in which one or more amino acids are substituted, deleted or added, and having the activity of converting polyphosphate and ADP into ATP;

[0075] (e3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (e1) or (e2).

[0076]

[14] . The multi-enzyme molecular machine according to any one of

[10] to

[13] , wherein the enzyme element is in the form of pure enzyme, crude enzyme, cell culture, cell lysate or whole cell; optionally, the pure enzyme or crude enzyme is in free or immobilized form, and the whole cell is in free or immobilized form.

[0077]

[15] . Use of the multi-enzyme molecular machine described in any one of

[10] to

[14] in the preparation of inositol.

[0078] Effects of the Invention

[0079] 1. The preparation method of inositol provided by the present invention only requires three steps of reaction to realize the synthesis of glucose to inositol, and the reaction steps are simple, green and pollution-free.

[0080] 2. In some embodiments of the present invention, the phosphoglucose synthase with new functions can be used to achieve the synthesis of inositol without using cofactors such as ATP.

[0081] 3. In some embodiments of the present invention, inositol can be obtained at a higher yield, and the yield of inositol can be as high as 100%.

[0082] 4. In some embodiments of the present invention, the multi-enzyme molecular machine is applied to the preparation of inositol, with simple preparation steps and high glucose conversion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The pathway for the production of inositol using glucose and inorganic phosphate as substrates is shown. Pi refers to inorganic phosphate, PGS refers to phosphoglucose synthase, IPS refers to inositol-3-phosphate synthase, and IMP refers to inositol monophosphatase.

[0084] Figure 2 The pathway for the production of inositol using glucose and ATP as substrates is shown. Pi refers to inorganic phosphate, HK refers to hexokinase, IPS refers to inositol-3-phosphate synthase, and IMP refers to inositol monophosphatase.

[0085] Figure 3The pathway for preparing inositol from glucose, ATP and polyphosphate as substrates is shown. Pi refers to inorganic phosphate, HK refers to hexokinase, IPS refers to inositol-3-phosphate synthase, IMP refers to inositol monophosphatase, and PolyPn-1 refers to polyphosphate.

[0086] Figure 4 A comparison of the relative activities of phosphoglucose synthases from different sources is shown.

[0087] Figure 5 The liquid phase spectrum of preparing inositol using glucose as substrate and coupled with ATP regeneration system is shown. DETAILED DESCRIPTION

[0088] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0089] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0090] In the present invention, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined with each other to form a new technical solution.

[0091] In the present invention, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0092] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0093] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0094] The use of "a", "an", "an" or similar expressions to describe the components and technical features of the present invention is merely for the convenience of expression and to provide a general meaning to the scope of the present invention. Therefore, such description should be understood to include one or at least one, and the singular also includes the plural, unless it is obvious that it refers to another meaning.

[0095] In this document, "or their combination" means "or any combination thereof", "any one", "any one" and "any one" means "any one", "any one" and "any one".

[0096] The terms "preferably" and "preferred" as used herein do not limit the scope of the claimed invention, or imply that certain features are critical, necessary, or even important to the structure or function of the claimed invention. Instead, these terms are merely intended to emphasize alternative or additional features that can or cannot be used in a particular embodiment of the invention.

[0097] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0098] As used in the claims and description, the words "comprising," "having," "including," "having," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0099] As used herein, the term "about" means that a value includes the standard deviation of error of the device or method used to determine the value. Exemplarily, the standard deviation is generally within the range of 20-30% of the original value.

[0100] Although the disclosure supports the definition of the term "or" as only alternatives and "and / or", the term "or" in the claims means "and / or" unless it is explicitly stated that it is only alternatives or the alternatives are mutually exclusive. In this specification, when the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0101] When used in the claims or specification, a selected / optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0102] As used in the present disclosure, the terms "polypeptide", "enzyme", "polypeptide or enzyme" or "polypeptide / enzyme" have the same meaning and are interchangeable in the present disclosure. The aforementioned terms refer to a polymer composed of a plurality of amino acids through peptide bonds, which may or may not contain modifications such as phosphate groups and formyl groups.

[0103] The terms "transformation, transfection, and transduction" in this disclosure have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0104] The term "enzyme-catalyzed reaction" means a chemical reaction that is carried out under the action of a biological catalyst - an enzyme.

[0105] The term "in vitro synthetic enzymatic biosystem" is based on the principle of simplifying the complex. It uses multiple natural enzymes, artificial enzymes, and (biomimetic) coenzymes to reconstruct in vitro artificial metabolic pathways, break away from the limitations of self-reproduction of organisms, surpass the limits of cell synthesis, and achieve important biochemical synthesis. It is one of the core platform technologies of biomanufacturing. In vitro multi-enzyme molecular machines follow the designed multi-enzyme catalytic pathway, rationally optimize and adapt several purified or partially purified enzyme elements, and efficiently convert specific substrates into target compounds in vitro.

[0106] As used in the present invention, the term "suitable reaction conditions" refers to those conditions in an enzyme-catalyzed reaction system, such as the range of enzyme dosage, substrate dosage, temperature, pH, buffer, cofactors, etc., under which the enzyme-catalyzed reaction of the present invention can convert glucose into inositol. Some exemplary "suitable reaction conditions" are provided herein.

[0107] As used herein, the term "dosage", such as in "enzyme dosing" or "substrate dosing", refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0108] The term "phosphoglucose synthase (PGS)" refers to an enzyme that is capable of phosphorylating glucose (e.g., converting glucose to glucose 6-phosphate) using a compound containing a phosphate group, such as inorganic phosphate, and catalyzes the following reaction:

[0109]

[0110] The term "polyphosphate kinase" refers to an enzyme that utilizes the substrate polyphosphate to interconvert ATP and ADP, thereby achieving cyclic regeneration of ATP.

[0111] The term "polyphosphate" refers to any salt containing several POP bonds generated by corner sharing of phosphate (PO4) tetrahedrons, thereby forming a long chain. n" is used synonymously, where n represents the average chain length of the number of phosphate residues, e.g. PolyP 25 Refers to polyphosphates with about 25 phosphate residues and PolyP 14 Refers to a polyphosphate having about 14 phosphate residues.

[0112] <Detailed description of the invention>

[0113] Method for preparing inositol

[0114] According to some embodiments of the present invention, a method for preparing inositol by enzymatic conversion of glucose using glucose as a substrate is provided. In some embodiments of the present invention, a method for preparing inositol is provided, wherein glucose or a derivative thereof is used as a substrate in a reaction system containing a phosphate group, and an enzyme catalyst is used to catalyze the substrate to generate inositol, wherein the enzyme catalyst comprises phosphoglucose synthase and / or a hexokinase with an EC number of EC 2.7.1.1.

[0115] In some embodiments, the method for preparing inositol comprises: in a reaction system containing a phosphate group, using phosphate glucose synthase (PGS) and / or hexokinase (HK) to convert glucose into glucose 6-phosphate; then using inositol-3-phosphate synthase (IPS) to convert glucose 6-phosphate into inositol 1-phosphate; and finally using inositol monophosphatase (IMP). That is, a multi-enzyme catalytic reaction system is constructed by multiple enzymes to convert glucose into inositol.

[0116] In some exemplary embodiments, the method for preparing inositol comprises any one of (I) to (III):

[0117] (I) in a reaction system containing glucose and inorganic phosphate, using phosphoglucose synthase (PGS), inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) to catalyze glucose to produce inositol;

[0118] (II) In a system containing glucose and ATP, hexokinase (HK), inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) catalyze glucose to produce inositol;

[0119] (III) In a system containing glucose, inorganic phosphate and ATP, glucose is converted into inositol using phosphoglucose synthase (PGS), hexokinase (HK), inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP).

[0120] In some specific embodiments, the phosphoglucose synthase refers to an enzyme that can utilize a compound containing a phosphate group, such as an inorganic phosphate, to phosphorylate glucose, and its source includes but is not limited to Clostridiumthermocellum, Acidothermus cellulolyticus and / or Thermomonospora curvata; illustratively, it can be selected from any one of the following (a1) to (a3):

[0121] (a1) comprising a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1;

[0122] (a2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate in one step;

[0123] (a3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2).

[0124] In some specific embodiments, the EC number of the hexokinase is EC 2.7.1.1, and its source includes but is not limited to Sulfurisphaera tokodaii, Ogataea angusta, Thermus caldophilus and / or Thermus thermophilus; illustratively, it can be selected from any one of the following (b1) to (b3):

[0125] (b1) comprising a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4;

[0126] (b2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate in one step;

[0127] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0128] In some specific embodiments, the EC number of the inositol-3-phosphate synthase is EC5.5.1.4, and its source includes but is not limited to Archaeoglobus fulgidus, Archaeoglobus profundus, Thermotogamaritima MSB8, Thermococcus kodakarensis KOD1 and / or Pyrococcus furiosus; illustratively, it can be selected from any one of the following (c1) to (c3):

[0129] (c1) comprising a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1;

[0130] (c2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose 6-phosphate into inositol 1-phosphate in one step;

[0131] (c3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (c1) or (c2).

[0132] In some specific embodiments, the EC number of the inositol monophosphatase is EC3.1.3.25, and its source includes but is not limited to Thermotoga maritima, Methanocaldococcus jannaschii and / or Mycolicibacterium smegmatis; illustratively, it can be selected from any one of the following (d1) to (d3):

[0133] (d1) comprising a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86;

[0134] (d2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86, in which one or more amino acids are substituted, deleted or added, and having the activity of converting inositol 1-phosphate into inositol in one step;

[0135] (d3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (d1) or (d2).

[0136] In the present invention, there is no particular limitation on the amount and ratio of the enzymes used.

[0137] In some optional embodiments, the ratio of enzyme units used in the method of preparing inositol of the present invention is 1:1:1 (PGS:IPS:IMP). To optimize the yield of inositol, these ratios can be adjusted in any combination of numbers. For example, ratios of 5:1:1, 5:5:1, etc. can be used. To increase the efficiency of inositol production, a particular enzyme can be present in an amount of about 2×, 3×, 4×, 5×, etc.

[0138] In other optional embodiments, the ratio of enzyme units used in the method for preparing inositol of the present invention is 1:1:1 (HK:IPS:IMP). In order to optimize the yield of inositol, these ratios can be adjusted in any combination of numbers. For example, 3:1:1, 1:1:3, etc. ratios can be used. In order to improve the production efficiency of inositol, a specific enzyme can be present in an amount of about 2×, 3×, 4×, 5×, etc.

[0139] In other optional embodiments, the ratio of enzyme units used in the method for preparing inositol of the present invention is 1:1:1:1 (PGS:HK:IPS:IMP). In order to optimize the yield of inositol, these ratios can be adjusted in any combination of numbers. For example, a ratio of 5:1:1:1 can be used. In order to improve the production efficiency of inositol, a specific enzyme can be present in an amount of about 2×, 3×, 4×, 5×, etc.

[0140] Furthermore, in order to reduce production costs, in some embodiments of the present invention, in the step of catalyzing the conversion of glucose into glucose 6-phosphate, an ATP regeneration system is also included, that is, any ATP regeneration method known in the art can be used for recycling. Preferably, ATP regeneration can be performed by an enzymatic method, illustratively, polyphosphate kinase (PPK, EC 2.7.4.1) is used to regenerate ATP using cheap and readily available polyphosphate as a substrate, thereby achieving ATP recycling.

[0141] In some embodiments of the present invention, the polyphosphate kinase can be replaced by any enzyme having the function of converting polyphosphate and ADP into ATP, or a mutant enzyme having the same function obtained by protein engineering. For example, the source of the polyphosphate kinase includes but is not limited to: Rhodobacter sphaeroides, Thermosynechococcus vestitus, Mycobacterium tuberculosis, etc.; preferably, the polyphosphate kinase derived from Thermosynechococcus vestitus; illustratively, it can be selected from any one of the following (e1) to (e2):

[0142] (e1) comprising a polypeptide as shown in Uniprot ID: Q8DMA8;

[0143] (e2) a polypeptide having an amino acid sequence of the polypeptide shown in Uniprot ID: Q8DMA8, in which one or more amino acids are substituted, deleted or added, and having the activity of converting polyphosphate and ADP into ATP;

[0144] (e3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (e1) or (e2).

[0145] In some specific embodiments, the polyphosphate includes sodium pyrophosphate, sodium hexametaphosphate, sodium tripolyphosphate, etc., and commercially available products containing the above substances, such as Graham' salt, can be used as they are.

[0146] In some embodiments of the present invention, a salt containing magnesium ions is included in the reaction system for preparing inositol. The salt containing magnesium ions includes at least one of magnesium chloride, magnesium sulfate, etc., and commercial products containing the above substances can be used as they are.

[0147] In some embodiments of the present invention, the inorganic phosphate includes at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc., and commercially available products containing the above substances can be used as they are.

[0148] In the present invention, any enzyme in the enzyme-catalyzed reaction system for preparing inositol can be replaced by any enzyme with equivalent function, or can be a mutant enzyme with equivalent function obtained by protein engineering.

[0149] In the present invention, any enzyme in the enzyme-catalyzed reaction system for preparing inositol may be used in various forms, including but not limited to purified or partially purified enzymes, microorganisms expressing the enzymes, cultures of the microorganisms, or combinations thereof.

[0150] In the present invention, enzymes with various functions in the enzyme-catalyzed reaction system for preparing inositol can be used independently in various forms, or can be co-expressed and used, for example, expressed on different expression vectors and simultaneously transferred into Escherichia coli for use, for example, used in the form of fusion proteins or enzyme complexes.

[0151] In the present invention, the enzymes of various functions in the enzyme-catalyzed reaction system for preparing inositol can be used directly in the form of pure enzymes (i.e., free enzymes), or the pure enzymes can be immobilized (i.e., immobilized enzymes) to maintain stability and recyclability; or, whole-cell microorganisms or cultures of whole-cell microorganisms (i.e., fermentation products) containing the enzymes of various functions can be used directly, or the whole cells can be immobilized (i.e., immobilized cells) to maintain stability and recyclability. In some specific embodiments of the present invention, in order to obtain a fast reaction rate, the whole cells can be subjected to a permeability treatment, illustratively, using a heat treatment method to promote cell membrane permeability.

[0152] In the method for preparing inositol of the present invention, an enzyme catalytic reaction system is established at a certain temperature to carry out the reaction. The appropriate temperature depends on the following factors: for example, the properties and amounts of enzymes with various functions in the enzyme catalytic reaction system and the amount of substrate glucose.

[0153] In some embodiments, the method for preparing inositol is carried out at a temperature of 0 to 100° C. In some preferred embodiments, the method for preparing inositol is carried out at a temperature of 25 to 80° C. In some more preferred embodiments, the method for preparing inositol is carried out at a temperature of 45 to 70° C., 50 to 70° C.

[0154] In the method for preparing inositol of the present invention, an enzyme catalytic reaction system is established at a certain pH to carry out the reaction. The appropriate pH depends on the following factors: for example, the properties and amounts of enzymes with various functions in the enzyme catalytic reaction system and the amount of substrate glucose.

[0155] In some embodiments, the method for preparing inositol is carried out at least pH 5.0, at least pH 5.5, at least pH 6.0, at least pH 6.5, at least pH 7.0, at least pH 7.5, at least pH 8.0, at least pH 8.5, at least pH 9.0, at least pH 9.5. In some preferred embodiments, the method for preparing inositol is carried out at pH 6.0 to pH 9.0. In some more preferred embodiments, the method for preparing inositol is carried out at pH 6.5 to pH 8.0.

[0156] In the method for preparing inositol of the present invention, an enzyme catalytic reaction system is established in a certain buffer to carry out the reaction. Suitable buffer and buffer concentration depend on factors such as the pH of the reaction, the properties and amounts of enzymes of various functions in the enzyme catalytic reaction system, the amount of substrate glucose, and the amounts of magnesium ions and inorganic phosphates and / or polyphosphates.

[0157] In some embodiments of the present invention, the buffer of the method for preparing inositol can be phosphate buffer, Tris-HCl buffer, HEPES buffer. In some embodiments of the present invention, the concentration of the buffer of the method for preparing inositol can be 0-200 mM.

[0158] In the method for preparing inositol of the present invention, an enzyme catalytic reaction system is set up to react for a period of time. The appropriate reaction time depends on factors such as the properties and amounts of enzymes of various functions in the enzyme catalytic reaction system and the amount of substrate glucose.

[0159] In some embodiments of the present invention, the reaction time of the method for preparing inositol is no more than 96 hours, no more than 72 hours, no more than 60 hours, no more than 48 hours, no more than 36 hours, no more than 24 hours, no more than 12 hours, no more than 10 hours, no more than 8 hours, no more than 6 hours, no more than 4 hours, or no more than 2 hours; illustratively, the reaction time is 12 to 96 hours.

[0160] In some preferred embodiments of the present invention, the combination of temperature, pH, buffer and / or reaction time is effective to ensure a higher yield of inositol.

[0161] In some optional embodiments, the step of synthesizing inositol further includes the step of separating and purifying the inositol from the reaction solution.

[0162] Multienzyme molecular machine

[0163] According to some embodiments of the present invention, there is provided a multi-enzyme molecular machine comprising: a first module comprising a first enzyme element, and, optionally, a second module comprising a second enzyme element and a non-enzyme element;

[0164] The first enzyme element in the first module comprises phosphoglucose synthase and / or hexokinase with EC number EC 2.7.1.1, and, optionally, inositol-3-phosphate synthase with EC number EC 5.5.1.4 and inositol monophosphatase with EC number EC 3.1.3.25.

[0165] In the multi-enzyme molecular machine of the present invention, the source of the phosphoglucose synthase includes but is not limited to Clostridium thermocellum, Acidothermus cellulolyticus and / or Thermomonosporacurvata; illustratively, it can be selected from any one of the following (a1) to (a3):

[0166] (a1) comprising a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1;

[0167] (a2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate in one step;

[0168] (a3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2).

[0169] In some specific embodiments, the EC number of the hexokinase is EC 2.7.1.1, and its source includes but is not limited to Sulfurisphaera tokodaii, Ogataea angusta, Thermus caldophilus and / or Thermus thermophilus; illustratively, it can be selected from any one of the following (b1) to (b3):

[0170] (b1) comprising a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4;

[0171] (b2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate in one step;

[0172] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0173] In some specific embodiments, the EC number of the inositol-3-phosphate synthase is EC 5.5.1.4, and its source includes but is not limited to Archaeoglobus fulgidus, Archaeoglobus profundus, Thermotogamaritima MSB8, Thermococcus kodakarensis KOD1 and / or Pyrococcus furiosus; illustratively, it can be selected from any one of the following (c1) to (c3):

[0174] (c1) comprising a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1;

[0175] (c2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose 6-phosphate into inositol 1-phosphate in one step;

[0176] (c3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (c1) or (c2).

[0177] In some specific embodiments, the EC number of the inositol monophosphatase is EC3.1.3.25, and its source includes but is not limited to Thermotoga maritima, Methanocaldococcus jannaschii and / or Mycolicibacterium smegmatis; illustratively, it can be selected from any one of the following (d1) to (d3):

[0178] (d1) comprising a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86;

[0179] (d2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86, in which one or more amino acids are substituted, deleted or added, and having the activity of converting inositol 1-phosphate into inositol in one step;

[0180] (d3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (d1) or (d2).

[0181] In some optional embodiments, the second module in the multi-enzyme molecular machine includes an ATP regeneration module; the second enzyme element includes a polyphosphate kinase with an EC number of EC2.7.4.1, and the non-enzyme element includes ADP. The polyphosphate kinase can be replaced by any enzyme that has the function of converting polyphosphate and ADP into ATP, or it can be a mutant enzyme with equivalent function obtained by protein engineering. For example, the source of the polyphosphate kinase includes but is not limited to: Rhodobacter sphaeroides, Thermosynechococcus vestitus, Mycobacteriumtuberculosis, etc.; preferably, it is a polyphosphate kinase derived from Thermosynechococcus vestitus; illustratively, it can be selected from any one of the following (e1) to (e1):

[0182] (e1) comprising a polypeptide as shown in Uniprot ID: Q8DMA8;

[0183] (e2) a polypeptide having an amino acid sequence of the polypeptide shown in Uniprot ID: Q8DMA8, in which one or more amino acids are substituted, deleted or added, and having the activity of converting polyphosphate and ADP into ATP;

[0184] (e3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (e1) or (e2).

[0185] The technical means and effects adopted by the present invention are further described, and the technical scheme of the present invention is further illustrated by specific embodiments below. However, it should be understood that the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical scheme of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0186] Example

[0187] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0188] The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0189] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0190] Materials and Methods

[0191] Drugs and materials

[0192] Unless otherwise noted, all drugs were of analytical grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Sinopharm (Shanghai, China). E. coli TOP10 and DH5α (Thermo Fisher Scientific, Waltham, MA, USA) were used for DNA manipulation and plasmid amplification. E. coli BL21 (DE3) (Invitrogen Life Sciences, Carlsbad, CA, USA) was used for recombinant protein expression.

[0193] Preparation and purification of plasmids and recombinant proteins

[0194] Those skilled in the art can prepare recombinant engineered bacteria using technical means known in the art according to their actual needs, and can refer to "Molecular Cloning Laboratory Guide (3rd Edition)" (Science Press). Those skilled in the art can culture engineered microbial cells to produce the enzyme. Guidelines and procedures for the production of recombinant enzymes by microbial cells can be found in the following publications, such as "Handbook of Fermentation and Biochemical Engineering: Principles, Process Design and Instruments" (2nd Edition, Henry C. Vogel and Celeste L. Todaro, Noyes Publications 1997) and "Principles of Fermentation Technology" (2nd Edition, PF Stanbury et.al., Butterworth Heineman, 2003).

[0195] The vectors, plasmids, and hosts used in the experiment are all conventional vectors, plasmids, and host series used for expression of bacteria (such as Escherichia coli), such as PET series vectors and plasmids, and BL21 series host bacteria; the culture medium used is a culture medium for conventional bacteria (such as Escherichia coli) engineering bacteria, such as LB culture medium; the culture method used is a conventional bacteria (such as Escherichia coli) engineering bacteria culture method.

[0196] Exemplarily, the preparation method of pET plasmid carrying the corresponding protein encoding gene is as follows: the corresponding genes are amplified from their corresponding genomes or obtained by gene synthesis. Through the simple cloning technology based on extended overlap extension PCR (POE-PCR) (You, C., X.-Z. Zhang and Y.-HP Zhang (2012). "Simple Cloning: direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis." Appl. Environ. Microbiol. 78: 1593-1595.), they are inserted into the pET20b plasmid to construct a plasmid containing the nucleotide sequence encoding the corresponding target protein.

[0197] The plasmid containing the nucleotide sequence encoding the corresponding target protein was transformed into E. coli BL21 (DE3) competent cells, and a single colony of overnight culture was picked and added to a 5mL LB test tube containing the corresponding resistance, and cultured overnight at 37℃ constant temperature shaker 200rpm. According to the inoculum of 1% (v / v), it was inoculated into a triangular flask containing the corresponding resistance and 200mL LB medium, and expanded in a 37℃ constant temperature shaker 200rpm. When OD 600 When the p-value reaches 0.8-1.0, add the inducer IPTG to a final concentration of 0.1 mM, and culture at 200 rpm in a 16°C constant temperature shaker for 16-18 hours to express the target protein and obtain a cell culture fluid. The cell culture fluid is collected by centrifugation at 4°C, and the collected cells are washed once with a 0.9% (w / v) sodium chloride solution. The cell pellet is resuspended in a 50 mM sodium phosphate buffer (pH 7.0) buffer solution and lysed by ultrasound. After centrifugation, the target protein in the supernatant is purified by Ni-NTA affinity chromatography.

[0198] Enzymes used and activity assays

[0199] The phosphoglucose synthase that can be used in the embodiments of the present invention can be derived from Clostridium thermocellum (Uniprot ID: A3DC21), from Clostridium thermocellum (Uniprot ID: A3DJZ0), from Acidothermus cellulolyticus (Uniprot ID: A0LR15), and from Thermomonospora curvata (Uniprot ID: D1A2R1).

[0200] The activity of phosphoglucose synthase was determined by enzymatic determination of the content of glucose 6-phosphate in the product:

[0201] In a 1mL reaction system, 50mM sodium phosphate buffer (pH 7.2), 1000mM glucose, 5mM MgSO4 and an appropriate amount of phosphoglucose synthase were reacted at 50°C. Control reactions without enzymes and without substrate glucose were set up. After the reaction, the content of glucose 6-phosphate in the product was determined by enzymatic method. The determination method was as follows: in a 100μL reaction system, 100mM HEPES buffer, 5mM MgSO4, 2mM NAD + , 10U / mL glucose 6-phosphate dehydrogenase and 10μL of the sample to be tested or glucose 6-phosphate (standard), react at room temperature for 30min, and measure the absorbance at 340nm. Draw a standard curve with glucose 6-phosphate as the standard, and then calculate the content of glucose 6-phosphate in the sample. The experimental results are as follows Figure 4 The experimental results showed that phosphoglucose synthase A3DC21, A3DJZ0, A0LR15, and D1A2R1 all have the activity of catalyzing the production of glucose 6-phosphate from glucose and phosphate.

[0202] Detection of Inositol

[0203] 1. The amount of inositol produced was detected by high performance liquid chromatography. The HPLC detection conditions were as follows: the chromatographic column was Bio-Rad87H; the mobile phase was 5 mM H2SO4, the flow rate was 0.6 mL / min; the column temperature was 60°C; the detector was a differential refractometer; and the injection volume was 20 μL.

[0204] 2. Detection of inositol production by enzymatic method: inositol and NAD + NADH is generated under the action of inositol 2-dehydrogenase (EC 1.1.1.18), and NADH has a specific absorption peak at a wavelength of 340 nm. Therefore, the concentration of inositol can be characterized by detecting the increase in the absorbance value at 340 nm.

[0205] Example 1

[0206] In a 1mL reaction system, including 50mM sodium phosphate buffer (pH 7.2), 1000mM glucose, 5mM MgSO4, 1U / mL phosphoglucose synthase (from Acidothermus cellulolyticus, Uniprot ID: A0LR15), 1U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480) and 1U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), after reacting at 50℃ for 24h, samples were taken and inositol production was detected by enzymatic method. The amount of inositol produced was 2.1mM.

[0207] Example 2

[0208] In a 1mL reaction system, including 50mM sodium phosphate buffer (pH 7.2), 1000mM glucose, 5mMMgSO4, 5U / mL phosphoglucose synthase (from Thermomonospora curvata, Uniprot ID: D1A2R1), 1U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480) and 1U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), after reacting at 50℃ for 24h, samples were taken and inositol production was detected by enzymatic method. The amount of inositol produced was 2.5mM.

[0209] Example 3

[0210] In a 1mL reaction system, including 50mM sodium phosphate buffer (pH 7.2), 1000mM glucose, 5mMMgSO4, 5U / mL phosphoglucose synthase (from Clostridium thermocellum, Uniprot ID: A3DJZ0), 1U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480) and 1U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), after reacting at 60℃ for 24h, samples were taken and inositol production was detected by enzymatic method. The amount of inositol produced was 3.2mM.

[0211] Example 4

[0212] In a 1mL reaction system, including 50mM sodium phosphate buffer (pH 7.2), 500mM glucose, 5mMMgSO4, 5U / mL phosphoglucose synthase (from Clostridium thermocellum, Uniprot ID: A3DJZ0), 5U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480) and 1U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), after reacting at 60℃ for 24h, samples were taken and inositol production was detected by enzymatic method. The amount of inositol produced was 4.5mM.

[0213] Example 5

[0214] In a 1 mL reaction system, 50 mM Tris-HCl buffer (pH 7.2), 100 mM glucose, 5 mM MgSO4, 100 mM ATP, 1 U / mL hexokinase (from Sulfurisphaera tokodaii, Uniprot ID: Q96Y14), 1 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480) and 1 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832) were included. After reacting at 70°C for 24 hours, samples were taken and inositol generation was detected by HPLC. HPLC results showed that the yield of inositol was 100 mM, and the yield of inositol reached 100%.

[0215] Example 6

[0216] A 1 mL reaction system contains 50 mM Tris-HCl buffer (pH 7.2), 10 mM glucose, 5 mM MgSO4, 2 mM ATP, 5 mM sodium hexametaphosphate, 1 U / mL hexokinase (from Sulfurisphaera tokodaii, Uniprot ID: Q96Y14), 1 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480), 1 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), and 1 U / mL polyphosphate kinase (from Thermosynechococcus vestitus, Uniprot ID: Q8DMA8). After reacting at 50°C for 12 hours, samples were taken and inositol production was detected by HPLC. The test results are as follows: Figure 5 As shown, HPLC results showed that the yield of inositol was 9.6 mM, and the yield of inositol reached 96%.

[0217] Example 7

[0218] A 1 mL reaction system contains 50 mM Tris-HCl buffer (pH 7.2), 100 mM glucose, 35 mM MgSO4, 2 mM ATP, 35 mM sodium hexametaphosphate, 1 U / mL hexokinase (from Sulfurisphaera tokodaii, Uniprot ID: Q96Y14), 1 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480), 1 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), and 1 U / mL polyphosphate kinase (from Thermosynechococcus vestitus, Uniprot ID: Q8DMA8). After reacting at 50°C for 24 hours, samples were taken and inositol production was detected by HPLC. HPLC results showed that the yield of inositol was 95 mM, and the yield of inositol reached 95%.

[0219] Example 8

[0220] A 1 mL reaction system contains 50 mM Tris-HCl buffer (pH 7.2), 500 mM glucose, 170 mM MgSO4, 2 mM ATP, 170 mM sodium hexametaphosphate, 5 U / mL hexokinase (from Sulfurisphaera tokodaii, Uniprot ID: Q96Y14), 5 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480), 5 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), and 5 U / mL polyphosphate kinase (from Thermosynechococcus vestitus, Uniprot ID: Q8DMA8). After reacting at 50°C for 24 hours, samples were taken and inositol production was detected by HPLC. HPLC results showed that the yield of inositol was 450 mM, and the yield of inositol reached 90%.

[0221] Example 9

[0222] A 1 mL reaction system contains 50 mM Tris-HCl buffer (pH 7.2), 500 mM glucose, 170 mM MgSO4, 2 mM ATP, 170 mM sodium hexametaphosphate, 5 U / mL hexokinase (from Sulfurisphaera tokodaii, Uniprot ID: Q96Y14), 5 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480), 5 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), and 5 U / mL polyphosphate kinase (from Thermosynechococcus vestitus, Uniprot ID: Q8DMA8). After reacting at 65°C for 72 hours, samples were taken and inositol production was detected by HPLC. HPLC results showed that the yield of inositol was 460 mM, and the yield of inositol reached 92%.

[0223] Example 10: Production of inositol using glucose, ATP and polyphosphate as substrates

[0224] A 1 mL reaction system contains 20 mM sodium phosphate buffer (pH 6.5), 500 mM glucose, 170 mM MgSO4, 2 mM ATP, 170 mM sodium hexametaphosphate, 5 U / mL hexokinase (from Sulfurisphaera tokodaii, Uniprot ID: Q96Y14), 5 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480), 5 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), and 5 U / mL polyphosphate kinase (from Thermosynechococcus vestitus, Uniprot ID: Q8DMA8). After reacting at 55°C for 96 hours, samples were taken and inositol production was detected by HPLC. HPLC results showed that the yield of inositol was 430 mM, and the yield of inositol reached 86%.

[0225] Embodiment 11

[0226] In a 1 mL reaction system, 20 mM sodium phosphate buffer (pH 7.2), 50 mM glucose, 5 mM MgSO4, 50 mM ATP, 0.5 U / mL phosphoglucose synthase (from Clostridium thermocellum, Uniprot ID: A3DJZ0), 1 U / mL hexokinase (from Sulfurisphaera tokodaii, Uniprot ID: Q96Y14), 1 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480) and 1 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832) were included. After reacting at 60°C for 24 hours, samples were taken and inositol generation was detected by HPLC. HPLC results showed that the yield of inositol was 30 mM, and the yield of inositol reached 60%.

[0227] Example 12

[0228] A 1 mL reaction system contained 20 mM sodium phosphate buffer (pH 7.2), 100 mM glucose, 40 mM MgSO4, 2 mM ATP, 40 mM sodium hexametaphosphate, 0.5 U / mL phosphoglucose synthase (from Acidothermus cellulolyticus, Uniprot ID: A0LR15), 5 U / mL hexokinase (from Sulfuris phaeratokodaii, Uniprot ID: Q96Y14), 5 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480) and 1 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832) and 1 U / mL polyphosphate kinase (from Thermosynechococcus vestitus, Uniprot ID: Q8DMA8). After reacting at 50°C for 12 h, samples were taken and the production of inositol was detected by HPLC. HPLC results showed that the yield of inositol was 70 mM, and the yield of inositol reached 70%.

[0229] Example 13

[0230] A 1 mL reaction system contained 20 mM phosphate buffer (pH 7.2), 500 mM glucose, 150 mM MgSO4, 2 mM ATP, 150 mM sodium hexametaphosphate, 0.1 U / mL phosphoglucose synthase (from Acidothermus cellulolyticus, Uniprot ID: A0LR15), 5 U / mL hexokinase (from Sulfuris phaeratokodaii, Uniprot ID: Q96Y14), 5 U / mL inositol-3-phosphate synthase (from Archaeoglobus fulgidus, Uniprot ID: O28480), 5 U / mL inositol monophosphatase (from Thermotoga maritima, Uniprot ID: O33832), and 5 U / mL polyphosphate kinase (from Thermosynechococcus vestitus, Uniprot ID: Q8DMA8). After reacting at 65 °C for 72 h, samples were taken and the production of inositol was detected by HPLC. HPLC results showed that the yield of inositol was 450 mM, and the yield of inositol reached 90%.

[0231] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0232] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing inositol, characterized in that, In a reaction system containing a phosphate group, glucose or its derivative is used as a substrate, and an enzyme catalyst is used to catalyze the substrate to generate inositol. The enzyme catalyst includes phosphoglucose synthase and / or hexokinase with an EC number of EC 2.7.1.

1.

2. The method according to claim 1, characterized in that: The source of the phosphoglucose synthase includes Clostridium thermocellum, Acidothermus cellulolyticus and / or Thermomonosporacurvata; Optionally, the source of the hexokinase comprises Sulfurisphaera tokodaii, Ogataea angusta, Thermus caldophilus and / or Thermus thermophilus.

3. The method according to claim 1 or 2, characterized in that: The phosphoglucose synthase is selected from any one of the following (a1) to (a3): (a1) comprising a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1; (a2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate; (a3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2); The hexokinase is selected from any one of the following (b1) to (b3): (b1) comprising a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4; (b2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate; (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

4. The method according to any one of claims 1 to 3, characterized in that: The phosphate groups in the reaction system are derived from inorganic phosphate and / or adenine nucleoside phosphate.

5. The method according to any one of claims 1 to 4, characterized in that: The enzyme catalyst also includes inositol-3-phosphate synthase with an EC number of EC 5.5.1.4 and inositol monophosphatase with an EC number of EC 3.1.3.25; Optionally, the source of the inositol 3-phosphate synthase includes Archaeoglobus fulgidus, Archaeoglobus profundus, Thermotoga maritima, Thermococcus kodakarensis and / or Pyrococcus furiosus; Optionally, the source of the inositol monophosphatase comprises Thermotoga maritima, Methanocaldococcusjannaschii and / or Mycolicibacterium smegmatis.

6. The method according to claim 5, characterized in that The inositol-3-phosphate synthase is selected from any one of the following (c1) to (c3): (c1) comprising a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1; (c2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose 6-phosphate into inositol 1-phosphate; (c3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (c1) or (c2); The inositol monophosphatase is selected from any one of the following (d1) to (d3): (d1) comprising a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86; (d2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86, in which one or more amino acids are substituted, deleted or added, and having the activity of converting inositol 1-phosphate into inositol; (d3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (d1) or (d2).

7. The method according to any one of claims 1 to 6, characterized in that: The reaction system comprises an ATP regeneration system; Optionally, regeneration of ATP is achieved enzymatically; Preferably, the enzymatic method is to use polyphosphate kinase with EC number EC 2.7.4.1 to achieve ATP regeneration in a regeneration system containing polyphosphate; Optionally, the source of the polyphosphate kinase includes Rhodobacter sphaeroides, Thermosynechococcus vestitus and / or Mycobacterium tuberculosis; Optionally, the polyphosphoric acid is provided by a polyphosphate, and optionally, the polyphosphate comprises sodium pyrophosphate, sodium hexametaphosphate and / or sodium tripolyphosphate.

8. The method according to any one of claims 1 to 7, characterized in that: The enzyme catalyst is in the form of pure enzyme, crude enzyme, cell culture, cell lysate or whole cell; optionally, the pure enzyme or crude enzyme is in free or immobilized form, and the whole cell is in free or immobilized form.

9. The method according to any one of claims 1 to 8, characterized in that: The reaction system contains magnesium ions and reacts at 0-100° C.; Optionally, the method further comprises the step of separating and purifying the inositol from the reaction system.

10. A multi-enzyme molecular machine, comprising: a first module comprising a first enzymatic element and, optionally, a second module comprising a second enzymatic element and a non-enzymatic element; The first enzyme element in the first module comprises phosphoglucose synthase and / or hexokinase with EC number EC 2.7.1.1, and, optionally, inositol-3-phosphate synthase with EC number EC 5.5.1.4 and inositol monophosphatase with EC number EC 3.1.3.

25.

11. The multi-enzyme molecular machine according to claim 10, characterized in that: The source of the phosphoglucose synthase includes Clostridium thermocellum, Acidothermus cellulolyticus and / or Thermomonosporacurvata; Optionally, the source of the hexokinase comprises Sulfurisphaera tokodaii, Ogataea angusta, Thermus caldophilus and / or Thermus thermophilus; Optionally, the source of the inositol 3-phosphate synthase includes Archaeoglobus fulgidus, Archaeoglobus profundus, Thermotoga maritima, Thermococcus kodakarensis and / or Pyrococcus furiosus; Optionally, the source of the inositol monophosphatase comprises Thermotoga maritima, Methanocaldococcusjannaschii and / or Mycolicibacterium smegmatis.

12. The multi-enzyme molecular machine according to claim 10 or 11, characterized in that: The phosphoglucose synthase is selected from any one of the following (a1) to (a3): (a1) comprising a polypeptide as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1; (a2) a polypeptide having an amino acid sequence as shown in any one of Uniprot IDs: A3DC21, A3DJZ0, A0LR15 or D1A2R1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate; (a3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2); The hexokinase is selected from any one of the following (b1) to (b3): (b1) comprising a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4; (b2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: Q96Y14, Q96VP5, Q5RLG0 or Q5SLJ4, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose or its derivatives into glucose 6-phosphate; (b3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2); The inositol-3-phosphate synthase is selected from any one of the following (c1) to (c3): (c1) comprising a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1; (c2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O28480, D2RGT5, Q9X1D6 or Q5JDB1, in which one or more amino acids are substituted, deleted or added, and having the activity of converting glucose 6-phosphate into inositol 1-phosphate; (c3) a polypeptide encoded by a polynucleotide encoding the polypeptide shown in (c1) or (c2); The inositol-3-phosphate synthase is selected from any one of the following (d1) to (d3): (d1) comprising a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86; (d2) a polypeptide having an amino acid sequence of a polypeptide as shown in any one of Uniprot IDs: O33832, Q57573 or A0QX86, in which one or more amino acids are substituted, deleted or added, and having the activity of converting inositol 1-phosphate into inositol; (d3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (d1) or (d2).

13. The multi-enzyme molecular machine according to any one of claims 10 to 12, characterized in that: The second module includes an ATP regeneration module; The second enzymatic element comprises a polyphosphate kinase having an EC number of EC 2.7.4.1, and the non-enzymatic element comprises ADP.

14. The multi-enzyme molecular machine according to any one of claims 10 to 13, characterized in that The enzyme element is in the form of pure enzyme, crude enzyme, cell culture, cell lysate or whole cell; optionally, the pure enzyme or crude enzyme is in free or immobilized form, and the whole cell is in free or immobilized form.

15. Use of the multi-enzyme molecular machine according to any one of claims 10 to 14 in the preparation of inositol.

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