In-vitro biotransformation synthesis system of trehalose 6-phosphoric acid and application thereof

Abstract

The invention provides an in-vitro biotransformation synthesis system of trehalose 6-phosphoric acid and application of the in-vitro biotransformation synthesis system, and belongs to the technical field of biological catalysis. According to the method for synthesizing the trehalose 6-phosphoric acid through in-vitro biotransformation, the maltose and the polyphosphate which are low in price and easy to obtain are used as raw materials to synthesize the trehalose 6-phosphoric acid, expensive cofactors such as ATP and UTP do not need to be added in the reaction process, the production cost of the trehalose 6-phosphoric acid is reduced, and the method has the advantages of being low in cost, low in pollution and high in yield.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to an in vitro biotransformation synthesis system of trehalose 6-phosphate and application thereof. Background Art

[0002] Trehalose 6-phosphate (T6P) is an important physiological metabolite with extensive applications in biology, biochemistry, and the food industry. As a key intermediate of trehalose in plants, T6P participates in regulating plant growth and development. T6P has multiple functions. First, it is a signaling molecule involved in regulating plant nutrient and carbon metabolism. T6P modulates plant metabolic pathways, such as enhancing photosynthesis, thereby influencing plant growth and development. Under stressful conditions, T6P can promote plant resistance to drought, cold, and disease, maintaining normal plant growth. Second, T6P promotes sucrose metabolism in plants. When T6P levels increase, the plant's ability to utilize sucrose also improves. Therefore, T6P can be considered an indicator of sucrose status in plants. In addition, trehalose 6-phosphate also has the function of regulating plant starch metabolism, promoting flowering, embryogenesis, budding and branching, and can be called a new type of plant hormone used to promote crop yield. Studies have shown that trehalose 6-phosphate can be applied to crops through spraying and other methods to increase crop yield and improve quality, and has great application prospects in agricultural production. However, due to factors such as production technology, the production cost of trehalose 6-phosphate is relatively high, and industrial production has not yet been achieved. Therefore, it is necessary to develop a low-cost, efficient method for synthesizing trehalose 6-phosphate to meet the demand for trehalose 6-phosphate.

[0003] In 1998, Junko Doi et al. synthesized trehalose 6-phosphate by yeast fermentation (Bioscience, Biotechnology, and Biochemistry, 1998, 62(4): 735-9.), this system uses glucose as the substrate, and ATP and ATP react to form G6P through glucokinase (GK); α-phosphoglucomutase (α-PGM) catalyzes G6P to form α-glucose 1-phosphate (α-G1P); simultaneously, another substrate UMP reacts with ATP to form urinary UTP under the action of nucleoside monophosphate kinase (NMK) and nucleoside diphosphate kinase (NDK); α-G1P and UTP react with UTP to form UDP-glucose (UDPG) under the catalysis of UTP-monosaccharide 1-phosphate uridylyltransferase (USP); and trehalose 6-phosphate synthase (TPS) catalyzes G6P and UDPG to synthesize T6P. This technology uses a large amount of ATP and UMP as substrates, which is expensive, the T6P yield is only 11%, and the yeast fermentation regulation is complex.

[0004] In comparison, the in vitro enzyme-catalyzed method is simpler and more efficient. It is urgent to develop a new method that does not use expensive raw materials such as ATP and UTP, but only uses cheap and readily available raw materials, to construct a low-cost, low-pollution, high-yield, and suitable for large-scale production of trehalose 6-phosphate. Summary of the Invention

[0005] Based on various problems existing in the current methods for producing trehalose 6-phosphate, a new method for producing trehalose 6-phosphate by multi-enzyme catalysis in vitro is provided, which is efficient, environmentally friendly and low-cost.

[0006] To address the problems existing in the prior art, the present invention provides a method for preparing trehalose 6-phosphate using maltose and polyphosphate as raw materials through in vitro bioconversion. The present invention utilizes an in vitro three-enzyme cascade catalytic reaction system to efficiently synthesize trehalose 6-phosphate, eliminating the need for expensive cofactors such as ATP and UTP for energy supply and recycling. This method achieves high conversion rates of the raw materials and high product yields at low cost, making it suitable for large-scale production.

[0007] The specific technical solutions are as follows:

[0008] [1]. A method for synthesizing trehalose 6-phosphate, wherein the method uses maltose and polyphosphate as substrates to synthesize trehalose 6-phosphate;

[0009] The method comprises the following steps:

[0010] The first step is to catalyze maltose into glucose and β-glucose 1-phosphate;

[0011] The second step reaction: converting glucose and polyphosphate into glucose 6-phosphate;

[0012] The third step reaction: converting β-glucose 1-phosphate and glucose 6-phosphate into trehalose 6-phosphate;

[0013] In the first step, maltose phosphorylase catalyzes the production of glucose and β-glucose 1-phosphate.

[0014] In the second step, polyphosphate glucokinase catalyzes the reaction of polyphosphate and glucose to generate glucose 6-phosphate.

[0015] In the third step, trehalose 6-phosphate is generated by catalysis of trehalose 6-phosphate phosphorylase;

[0016] The maltose phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.1; the polyphosphate glucokinase comprises: the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.3; the trehalose 6-phosphate phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.5;

[0017] and / or,

[0018] The maltose phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.7; the polyphosphate glucokinase comprises: the amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.9; the trehalose 6-phosphate phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.5.

[0019] [2] The method according to [1], wherein the reaction system further comprises a buffer solution, wherein the buffer solution comprises a phosphate buffer solution, and the pH of the phosphate buffer solution is 6.5 to 8.0.

[0020] [3] The method according to [1] or [2], wherein the concentration of the substrate maltose is not less than 5 g / L, preferably 10-100 g / L.

[0021] [4] The method according to any one of [1] to [3], wherein the polyphosphate comprises a polyphosphate composed of 3, 6, 12, 24, 48 or 100 phosphoric acid molecules;

[0022] [5] The method according to any one of [1] to [4], wherein the polyphosphate comprises a sodium salt, a potassium salt and / or an ammonium salt.

[0023] [6] The method according to any one of [1] to [5], wherein the added amount of maltose phosphorylase, polyphosphate glucokinase, and trehalose 6-phosphate phosphorylase is 0.1-10 U / mL, respectively.

[0024] [7] The method according to [6], wherein the amount of budose phosphorylase added is 1-5 U / mL, the amount of polyphosphate glucokinase added is 1-5 U / mL, and the amount of trehalose 6-phosphate phosphorylase added is 2-10 U / mL.

[0025] [8] The method according to any one of [1] to [7], wherein the reaction system further contains 1 to 50 mM magnesium ions and the reaction temperature is 25 to 40°C.

[0026] The present invention provides a novel method utilizing in vitro biotransformation through pathway design, wherein trehalose 6-phosphate is prepared from inexpensive maltose and polyphosphate. No cofactors such as ATP and UTP need to be added during the reaction process, and the obtained trehalose 6-phosphate has a high yield and high content. High-purity trehalose 6-phosphate can be obtained by simply removing proteins and salt ions. The purification process is simple, which can reduce the production cost of trehalose 6-phosphate. The preparation process is environmentally friendly and has the advantages of low cost, low pollution, and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a pathway for the preparation of trehalose 6-phosphate from maltose and polyphosphate catalyzed by a three-enzyme system.

[0028] Figure 2The following are SDS-PAGE images of the enzymes in the three-enzyme system. M represents a protein molecular weight marker. Lane 1 shows maltose phosphorylase MP from Enterococcus faecalis; lane 2 shows maltose phosphorylase MP from Bacillus subtilis 168; lane 3 shows polyphosphate glucokinase PPGK from Mycobacterium tuberculosis; lane 4 shows polyphosphate glucokinase PPGK from Thermobifida fusca YX; and lane 5 shows trehalose 6-phosphate phosphorylase TrePP from Lactococcus lactis subsp. lactis Il1403.

[0029] Figure 3A This is the standard curve of trehalose 6-phosphate standard.

[0030] Figure 3B This is the ion chromatogram of trehalose 6-phosphate standard. DETAILED DESCRIPTION

[0031] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0032] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0033] Unless otherwise stated, the units used in this specification are 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.

[0034] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0035] References throughout this specification to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the particular 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 be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0036] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0037] In this specification, a numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0038] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0039] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0040] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the corresponding fields and routine procedures. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, Joseph Frank et al. "Molecular Cloning: A Laboratory Manual." (2001). (hereinafter referred to as "Sambrook"). In addition, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0041] As used herein, the terms "polypeptide," "enzyme," "polypeptide or enzyme," or "polypeptide / enzyme" have the same meaning and are interchangeable in this disclosure. The aforementioned terms refer to a polymer composed of a plurality of amino acids linked by peptide bonds, which may or may not contain modifications such as phosphate groups and formyl groups.

[0042] In the present invention, sequence analysis software is generally used to measure the sequence similarity of polypeptides. Protein analysis software has used similarity metrics assigned to various substitutions, deletions and other modifications including conservative amino acid substitutions to match similar sequences. For example, GCG software contains such as GAP and BESTFIT programs, which can be used under default parameters to determine sequence homology or sequence identity between closely related polypeptides (such as between homologous polypeptides from different organism species or between wild-type protein and its mutant protein). See, for example, GCG version 6.1. FASTA can also be used to compare polypeptide sequences under default or recommended parameters; The program in GCG version 6.1 FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlapping region between the query sequence and the search sequence. When the sequence of the present invention is compared with a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0043] As used herein, the terms "transformation," "transfection," and "transduction" have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. Such methods include any method for introducing nucleic acid into cells, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0044] The term "enzyme-catalyzed reaction" refers to a chemical reaction that is carried out under the action of a biological catalyst - an enzyme.

[0045] As used herein, the term "suitable reaction conditions" refers to those conditions in an enzyme-catalyzed reaction system, such as ranges of enzyme dosage, substrate dosage, temperature, pH, buffer, cofactors, etc., under which the enzyme-catalyzed reaction of the present invention is capable of converting maltose to trehalose 6-phosphate. Some exemplary "suitable reaction conditions" are provided herein.

[0046] As used herein, the term "dosage," such as in "enzyme dosage" or "substrate dosage," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0047] As used herein, the term "polyphosphate" refers to any salt containing several POP bonds generated by corner sharing of phosphate (PO4) tetrahedrons, thereby forming long chains. n " is used synonymously, where n represents the average chain length in terms 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.

[0048] As used herein, "conversion" may also refer to the enzymatic conversion (or bioconversion) of a substrate(s) to the corresponding product(s). "Percent conversion" refers to the percentage of substrate that is converted to product under specified conditions over a period of time. Thus, the "enzyme activity" or "activity" of an aminoglycosylation polypeptide can be expressed as the "percent conversion" of substrate to product over a specific period of time.

[0049] As used herein, "culturing" refers to growing a microbial cell population under any suitable conditions (e.g., using a liquid, gel, or solid culture medium), including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, and various culture conditions such as temperature, time, and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0050] The EC number, or EC number, is a classification system for enzymes developed by the Enzyme Commission (EC). It is based on the chemical reaction each enzyme catalyzes. This classification system also provides a suggested name for each enzyme, hence the name "EC Nomenclature."

[0051] In the present invention, any enzyme in the enzyme-catalyzed reaction system for preparing trehalose 6-phosphate can 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.

[0052] In the present invention, the various functional enzymes in the enzyme-catalyzed reaction system for preparing trehalose 6-phosphate can be used directly as pure enzymes (i.e., free enzymes), or the pure enzymes can be immobilized (i.e., immobilized enzymes) to maintain stability and recyclability. Alternatively, whole-cell microorganisms or cultures of whole-cell microorganisms (i.e., fermentation products) containing the various functional enzymes 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 rapid reaction rate, the whole cells can be permeabilized. For example, heat treatment is used to promote cell membrane permeability.

[0053] In the present invention, all enzymes useful in the present invention may be wild-type enzymes or enzymes that have undergone certain genetic modifications, wherein the genetic modifications have enhanced enzyme properties, such as activity, substrate specificity, and thermostability. Enzymes derived from wild-type enzymes through genetic engineering techniques that possess desired properties are referred to as "mutants," "mutant derivatives," or "mutants derived from the corresponding wild-type enzymes." Enzymes useful in practicing the present invention include "mutants" with these enhanced properties.

[0054] In the method for preparing trehalose 6-phosphate of the present invention, an enzyme-catalyzed reaction system is established at a certain temperature to carry out the reaction. The appropriate temperature depends on factors such as the properties and amounts of the various functional enzymes in the enzyme-catalyzed reaction system and the amount of the substrate maltose.

[0055] Based on the existing problems, the present invention provides a method for synthesizing trehalose 6-phosphate, which uses maltose and polyphosphate as substrates and converts maltose and polyphosphate into trehalose 6-phosphate in a three-enzyme cascade catalytic reaction system.

[0056] In some embodiments, the three-enzyme cascade catalytic reaction system is composed of three enzymes: maltosephosphorylase (MP), polyphosphate glucokinase (PPGK), and trehalose 6-phosphate phosphorylase (Trehalose 6-phosphate phosphorylase, TrePP), and the reaction process is as follows: Figure 1 shown.

[0057] In some specific embodiments, MP uses maltose as a substrate and catalyzes the hydrolysis of maltose into glucose and β-glucose 1-phosphate (β-G1P); the polyphosphate-dependent PPGK uses glucose and polyphosphate as substrates and catalyzes glucose and polyphosphate to produce glucose 6-phosphate (G6P); TrePP uses β-G1P and G6P as substrates and catalyzes the synthesis of the target products trehalose 6-phosphate and inorganic phosphate from the two substrates.

[0058] In some specific embodiments, the three-enzyme cascade catalytic reaction system does not require ATP to provide energy, and the phosphate radical provided by the inorganic polyphosphate participates in the catalytic reaction.

[0059] In some embodiments, the MP is selected from maltose phosphorylase with an EC number of EC 2.4.1.8; optionally, the source of the MP includes but is not limited to Escherichia coli, Bacillus sp., Enterococcus faecalis, Lactobacillus acidophilus, Levilactobacillus brevis, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, Thermococcus litoralis, Thermobifida fusca and / or Sulfolobus tokodaii.

[0060] In some preferred embodiments, the MP is derived from Enterococcus faecalis or Bacillus subtilis 168.

[0061] In some preferred embodiments, the MP comprises the amino acid sequence set forth in SEQ ID NO. 1 or the amino acid sequence set forth in SEQ ID NO. 7, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, or at least 99% or higher homology to the amino acid sequence set forth in SEQ ID NO. 1 or SEQ ID NO. 7. Exemplarily, the nucleotide sequences encoding the MP are set forth in SEQ ID NO. 2 and SEQ ID NO. 8.

[0062] In some embodiments, the PPGK is selected from polyphosphate glucokinase with EC number EC 2.7.1.63; optionally, the source of the PPGK includes but is not limited to Mycobacterium tuberculosis, Arthrobacter sp., Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotogamaritima, Thermococcus litoralis, Thermobifida fusca, Sulfolobus tokodaii), Streptomyces murinus, and / or Bifidobacterium adolescentis

[0063] In some preferred embodiments, the PPGK is derived from Mycobacterium tuberculosis or Thermobifida fusca YX.

[0064] In some preferred embodiments, the PPGK comprises the amino acid sequence shown in SEQ ID NO. 3 or the amino acid sequence shown in SEQ ID NO. 9, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, or at least 99% or higher homology to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 9. Exemplarily, the nucleotide sequence encoding the PPGK is shown in SEQ ID NO. 4 or SEQ ID NO. 10.

[0065] In some embodiments, the TrePP is selected from trehalose 6-phosphate phosphorylase with EC number EC 2.4.1.216; optionally, the TrePP source includes but is not limited to Callinectes sapidus, Carnobacterium sp., Lactiplantibacillus pentosus, Lactococcus lactis, Weissella ceti, Pediococcus pentosaceus, Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus litoralis and / or Sulfolobus tokodaii.

[0066] In some preferred embodiments, the TrePP is derived from Lactococcus lactis subsp. lactis, more preferably, the TrePP is derived from Lactococcus lactis subsp. Lactis I11403.

[0067] In some preferred embodiments, the TrePP comprises the amino acid sequence shown in SEQ ID NO. 5, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, or at least 99% or higher homology to the amino acid sequence shown in SEQ ID NO. 5. Exemplarily, the nucleotide sequence encoding TrePP is shown in SEQ ID NO. 6.

[0068] Furthermore, the three-enzyme cascade catalytic reaction is carried out in a buffer solution.

[0069] In some preferred embodiments, the buffer solution includes phosphate buffer, Tris-HCl buffer, HEPES buffer, etc. Preferably, the buffer is phosphate buffer.

[0070] In some more preferred embodiments, the phosphate buffer is 5-300 mM sodium phosphate buffer, preferably 10-150 mM sodium phosphate buffer, and more preferably, the pH of the sodium phosphate buffer is 6.5-8.0.

[0071] In some embodiments of the present invention, the methods for synthesizing trehalose 6-phosphate described herein can recycle inorganic phosphate, and / or at least one step of the method for synthesizing trehalose 6-phosphate includes an energetically favorable chemical reaction. In particular, the inorganic phosphate produced by TrePP's catalytic conversion of β-G1P and G6P to trehalose 6-phosphate can assist the MP-catalyzed reaction.

[0072] In some specific embodiments, the concentration of the substrate maltose is not less than 5 g / L, preferably 10-100 g / L.

[0073] In some specific embodiments, the added amounts of maltose phosphorylase, polyphosphate glucokinase, and trehalose 6-phosphate phosphorylase are 0.1-10 U / mL, respectively, preferably 1-5 U / mL of maltose phosphorylase, 1-5 U / mL of polyphosphate glucokinase, and 2-10 U / mL of trehalose 6-phosphate phosphorylase.

[0074] In some specific embodiments, the polyphosphate comprises a polyphosphate composed of 3, 6, 12, 24, 48, or 100 phosphoric acid molecules; the polyphosphate comprises a sodium salt, a potassium salt, and / or an ammonium salt. Exemplarily, the polyphosphate is sodium hexametaphosphate, and the concentration is 5 to 300 mM, preferably 15 to 150 mM.

[0075] Furthermore, the reaction system further contains magnesium ions, which can be provided by magnesium chloride and / or magnesium sulfate, for example, one or a mixture of MgCl2 and MgSO4. In some exemplary embodiments, the magnesium ions are provided by MgSO4 at a concentration of 1 to 50 mM, preferably 1 to 25 mM.

[0076] In some embodiments, the present invention is carried out at a temperature range of about 20°C to about 90°C, a pH range of about 5.0 to about 8.0, and / or for about 0.5 h to about 72 h. For example, the present invention is carried out at 25-60°C and a pH range of 6.5-8.0.

[0077] In some embodiments, the steps of the method for preparing trehalose 6-phosphate are carried out in one bioreactor. Alternatively, the catalytic reactions of the intermediates and final products of the present invention can also be carried out in a plurality of bioreactors arranged in series.

[0078] Example

[0079] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0080] Example 1: Construction of recombinant expression strains of relevant enzymes in the three-enzyme system

[0081] Maltose phosphorylase MP can be derived from Enterococcus faecalis, with a Uniprot accession number of Q836Y7; maltose phosphorylase MP can also be derived from Bacillus subtilis 168, with a Uniprot accession number of O06993;

[0082] The polyphosphate glucokinase PPGK may be derived from Mycobacterium tuberculosis, with the Uniprot accession number being P9WIN1; the polyphosphate glucokinase PPGK may also be derived from Thermobifida fusca, with the Uniprot accession number being Q47NX5;

[0083] Trehalose 6-phosphate phosphorylase TrePP can be derived from Lactococcus lactis subsp. lactis Il1403, with the Uniprot accession number being Q9CID5.

[0084] The gene sequences encoding the above enzymes were synthesized by a gene synthesis company and ligated into the pET20b plasmid to construct the corresponding expression plasmids. The constructed plasmids were transformed into competent E. coli BL21(DE3) cells. After selection on plates containing ampicillin, strains carrying the above recombinant plasmids were obtained. The specific procedure was as follows: E. coli BL21(DE3) competent cells were removed from a -80°C freezer and slowly thawed on ice. Immediately, 5 μL of the recombinant plasmid was added, gently mixed, and placed on ice for 30 minutes. The cells were then heat-shocked in a 42°C water bath for 90 seconds, followed by an ice bath for 2 minutes. Under sterile conditions, 500 μL of LB liquid medium was added, and the cells were incubated at 37°C at 220 rpm for 50 minutes to allow the cells to recover. The cells were then evenly plated onto solid LB medium containing ampicillin (working concentration: 100 μg / mL). The plates were sealed with parafilm and incubated upside down in a 37°C incubator overnight.

[0085] Example 2: Protein expression and purification of each enzyme in the three-enzyme system

[0086] From the LB solid plate, the monoclonal strains of the above transformants were picked and inoculated into 1L shake flasks containing 200 mL LB liquid medium (containing ampicillin antibiotic at a working concentration of 100 μg / mL) for overnight culture. The culture was expanded at 37°C and 220 rpm until the OD 600 The pH value was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. The culture was continued at 16°C for 16-20 h. The cells were collected by centrifugation at 5000 rpm for 15 min at 4°C, and the fermentation / shake flask fermentation supernatant was removed. An equal volume of 0.9% NaCl solution was added to resuspend the cell pellet, and the cell pellet was centrifuged at 4°C and 5000 rpm for 15 min. The supernatant was removed. Lysis buffer (100 mM phosphate buffer (pH 7.0), 0.3 M NaCl) was added to resuspend the cell pellet so that the concentration of the cell resuspension reached OD 600 The cell suspension is about 50, and the cell weight is crushed by a high-pressure homogenizer or ultrasonic disruptor. After lysis, the supernatant is collected by centrifugation and filtered with a 0.45 μm filter head. The expressed recombinant protein is affinity adsorbed by a nickel ion affinity chromatography column pre-equilibrated with lysis buffer. After loading, 5 column volumes are washed with lysis buffer containing 10 mM imidazole to remove impurities, and then the target protein is eluted with lysis buffer containing 100 mM imidazole. The eluate is replaced with 100 mM phosphate buffer (pH 7.0) in a short time to remove imidazole. After ultrafiltration, a high-concentration protein is obtained for storage. The concentration of the target protein is determined by the Bradford method (Coomassie blue staining method), and the purity is detected by protein gel electrophoresis ( Figure 2Protein samples are generally stored by adding glycerol to a final concentration of 5%, rapidly freezing at -80°C, and storing in aliquots.

[0087] Example 3: Quantitative detection of trehalose 6-phosphate

[0088] To detect and quantify the target product, high-performance anion exchange chromatography (HPLC) using a Dionex Ion PacTMAS11-HC Analytical column (4 mm id × 250 mm; Thermo Fisher Scientific) was used to determine the content of trehalose 6-phosphate. The injection volume of the test sample was 10 μL.

[0089] The retention time of trehalose 6-phosphate standard (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number T339557) was determined to be 8.287 min by high performance anion exchange chromatography. Trehalose 6-phosphate standard was prepared into a 0.1 g / L mother liquor solution, which was diluted to 0.001 g / L, 0.005 g / L, 0.01 g / L, and 0.05 g / L standard solutions. The product was quantified and the molar conversion rate was analyzed using the standard curve ( Figure 3A and Figure 3B ).

[0090] The calculation formula of the molar conversion rate of trehalose 6-phosphate is the ratio of the molar concentration of trehalose 6-phosphate in the final reaction system to the molar concentration of maltose in the initial reaction system×100%.

[0091] Example 4: Construction of a three-enzyme cascade catalytic reaction system to catalyze the synthesis of trehalose 6-phosphate from maltose

[0092] In this example, the selected maltose phosphorylase MP was derived from Bacillus subtilis 168, with a Uniprot accession number of O06993; the polyphosphate glucokinase PPGK was derived from Thermobifida fusca YX, with a Uniprot accession number of Q47NX5; and the trehalose 6-phosphate phosphorylase TrePP was derived from Lactococcus lactis subsp. lactis Il1403, with a Uniprot accession number of Q9CID5. The corresponding enzymes were transformed and expressed as described in Examples 1 and 2, and used in the construction and reaction of a three-enzyme cascade catalytic reaction system.

[0093] A 5 ml reaction system contained 50 mM phosphate buffer (pH 7.5), 15 mM Mg 2+The reaction mixture contained 15 mM sodium hexametaphosphate, 10 g / L maltose (29.2 mM molar concentration, 342 g / mol maltose), 2 U / mL maltose phosphorylase (MP), 2 U / mL polyphosphate glucokinase (PPGK), and 2 U / mL trehalose 6-phosphate phosphorylase (TrePP). The reaction was carried out in a thermoshaker at 37°C and 200 rpm, with samples taken every 30 minutes for approximately 2 hours. The reaction was terminated by heating at 100°C for 10 minutes, followed by protein precipitation and centrifugation at 12,000 rpm. The supernatant was then collected for chromatographic analysis. The results showed a yield of 12.07 g / L trehalose 6-phosphate at a molar concentration of 28.6 mM (422 g / mol trehalose 6-phosphate), with a molar conversion of 97.9%.

[0094] Example 5: Construction of a three-enzyme cascade catalytic reaction system to catalyze the synthesis of trehalose 6-phosphate from maltose

[0095] In this example, maltose phosphorylase MP, derived from Enterococcus faecalis and with Uniprot accession number Q836Y7, polyphosphate glucokinase PPGK, derived from Mycobacterium tuberculosis and with Uniprot accession number P9WIN1, and trehalose 6-phosphate phosphorylase TrePP, derived from Lactococcus lactis subsp. lactis Il1403 and with Uniprot accession number Q9CID5, can be selected. The corresponding enzymes were transformed and expressed as described in Examples 1 and 2, and used in the construction and reaction of a three-enzyme cascade catalytic reaction system.

[0096] A 5 ml reaction system contained 50 mM phosphate buffer (pH 7.5), 15 mM Mg 2+The reaction mixture contained 15 mM sodium hexametaphosphate, 10 g / L maltose (29.2 mM molar concentration, 342 g / mol maltose), 2 U / mL maltose phosphorylase (MP), 2 U / mL polyphosphate glucokinase (PPGK), and 2 U / mL trehalose 6-phosphate phosphorylase (TrePP). The reaction was carried out in a thermoshaker at 37°C and 200 rpm, with samples taken every 30 minutes for approximately 2 hours. The reaction was terminated by heating at 100°C for 10 minutes, followed by protein precipitation and centrifugation at 12,000 rpm. The supernatant was then collected for chromatographic analysis. The supernatant showed a new peak at a retention time of 8.287 minutes, consistent with that of the standard, indicating the formation of trehalose 6-phosphate in the reaction solution. The results showed that the yield of trehalose 6-phosphate was 11.78 g / L, the molar concentration was 27.9 mM (the molecular weight of trehalose 6-phosphate was 422 g / mol), and the molar conversion rate of trehalose 6-phosphate was 95.5%.

[0097] SEQ ID NO.1: MP amino acid sequence, Bacillus subtilis 168, Uniprot ID: 006993

[0098] MINQRLFEIDEWKIKTNTFNKEHTRLLESLTSLANGYMGVRGNFEEGYSGDSHQGTYIAGVWFPDKTRVGWWKNGYPEYFGKVINAMNFMGIGLYVDGEKIDLHQNPIELFEVELNMKEGILRRSAVVRIQDKTVRIRSERFLSLAVKELCAIHYEAECLTGDAVITLVPYLDGNVANEDSNYQEQFWQEEAKGADSHSGHLAAKTIENPFGTPRFTVLAAMANETEGFVHESFKTTEMYVENRYSYQTKASLKKFVIVTTSRDFREEELLSKAKELLADVVENGYEDAKRRHTDRWKERWAKADIEIKGDEELQQGIRYNIFQLFSTYYGGDARLNIGPKGFTGEKYGGAAYWDTEAYAVPMYLATAEPEVTKNLLLYRYHQLEAAKRNAAKLGMKGALYPMVTFTGDECHNEWEITFEEIHRNGAICYAIYNYINYTGDRNYMEEYGIDVLVAVSRFWADRVHFSKRKNKYMIHGVTGPNEYENNVNNNWYTNVIAAWTLEYTLQSLESISAEKRRHLDVQEVELEVWREIIQHMYYPFSEELQIFVQHDTFLDKDLQTVDELDPAERPLYQNWSWDKILRSNFIKQADVLQGIYLFNDRFTMEEKRRNFEFYEPMTVHESSLSPSVHAILAAELKLEKKALELYKRTARLDLDNYNHDTEEGLHITSMTGSWLAIVHGFAGMRTANETLSFAPFLPKEWDEYSFNINYRNRLINVTVDEKRVIFELVKGEPLHMNVYEEPVVLQGRCERRTPNE

[0099] SEQ ID NO.2: MP nucleotide sequence, Bacillus subtilis 168

[0100]

[0101] SEQ ID NO.3: PPGK amino acid sequence, Thermobifida fusca YX, Uniprot accession number Q47NX5

[0102] MASRGRVGLGIDIGGSGIKGAPVDLDRGTFVVDRVKIATPQPATPEAVAAVVAEIVTAFADDVPQDAPLGVTFPAVIQHGVARSAANVDRSWIGTNVEELLSAVTGRRVLVVNDADAAAMAEHRYGAASGV DGVVLLTTLGTGIGTAVLVDGVLLPNTEFGHLEIDGYDAETTRASASAKERENLSYKEWAEERLQRYYSVIEDLLWPDLIVVGGGVSRKADKFLPHLRLRAPIVPAKLRNTAGIVGAAVLAAERLGGDRVSA

[0103] SEQ ID NO.4: PPGK nucleotide sequence, Thermobifida fusca YX

[0104] Atggcatctcggggacgggtcgggctggggattgacatcgggggaagcgggatcaaaggcgcccctgtggacttggaccggggaacgttcgtggtggaccgggtcaagatcgctactccgcagcccgcaacccctgaggcggtggctgcggtggtggcggagatagtcaccgcgttcgccgacgatgtgccgcaggatgcaccgttgggggtgacgtttcccgcggtgatccagcacggggtggcgcgcagcgccgccaacgtggaccgctcgtggatcggcaccaacgtcgaggagctgctgtctgcggtgacggggcggcgggtgctggtggtcaacgacgctgacgccgcagcgatggcggagcaccgctacggcgctgcctcaggcgtcgacggggtggtgctgttgactactttgggtaccggtattggtacggcggtgctagtggacggggtgctgctccccaacacggagttcgggcacttggagatcgacggctacgacgctgagacccgggcctctgctagcgctaaggagcgcgagaacctctcctacaaggagtgggctgaggagcggctgcagcgctactactcggtgatcgaggatttgctgtggccggacttgatcgtggtgggcggcggggtcagccgcaaggcggacaagtttttgccgcatctccgcttgcgcgcgccgatcgtgccggcgaagttgcgcaataccgcggggatcgtgggtgcggccgtgctggccgcggagcggctggggggtgaccgggtc tctgcctga

[0105] SEQ ID NO.5:Amino acid sequence of TrePP, Lactococcus lactis subsp. lactis Il1403, Uniprot ID: Q9CID5

[0106] MTEKDWIIQYDKKEVGKRSYGQESLMSLGNGYLGLRGAPLWSTCSDNHYPGLYVAGVFNRTSTEVAGHDVINEDMVNWPNPQLIKVYIDGELVDFEASVEKQATIDFKNALQIERYQVKLAKGNLTLVTTKFVDPINFHDFGFVGEIIADFSCKLRIETFTDGSVLNQNVERYRAFDSKEFEVTKISKGLLVAKTRTSEIELAIASKSFLNGLAFPKIDSENDEILAEAIEIDLQKNQEVQFDKTIVIASSYESKNPVEFVLTELSATSVSKIQENNTNYWEKVWSDADIVIESDHEDLQRMVRMNIFHIRQAAQHGANQFLDASVGSRGLTGEGYRGHIFWDEIFVLPYYAANEPETARDLLLYRINRLTAAQENAKVDGEKGAMFPWQSGLIGDEQSQFVHLNTVNNEWEPDNSRRQRHVSLAIVYNLWIYSQLTEDESILTDGGLDLIIETTKFWLNKAELGDDGRYHIDGVMGPDEYHEAYPGQEGGICDNAYTNLMLTWQLNWLTELSEKGFEIPKELLEKAQKVRKKLYLDIDENGVIAQYAKYFELKEVDFAAYEAKYGDIHRIDRLMKAEGISPDEYQVAKQADTLMLIYNLGQEHVTKLVKQLAYELPENWLKVNRDYYLARTVHGSTTSRPVFAGIDVKLGDFDEALDFLITAIGSDYYDIQGGTTAEGVHIGVMGETLEVIQNEFAGLSLREGQFAIAPYLPKSWTKLKFNQIFRGTKVEILIENGQLLLTASADLLTKVYDDEVQLKAGVQTKFDLK

[0107] SEQ ID NO.6: TrePP nucleotide sequence, Lactococcus lactis subsp. lactis Il1403

[0108]

[0109] SEQ ID NO.7: MP amino acid sequence, Enterococcus faecalis, Uniprot ID: Q836Y7

[0110] MKQIKRLFQIDPWKIRTTHLDKENLRLQESLTSIGNGYMGMRGNFEEHYSGDHHQGTYLAGVWYPDKTRVGWWKNGYPEYFGKVINAINFIAMDLQIDGQTIDLATTPYEDFSLELDMQNGVLSRQFTIQTPKNKVRFSFERFLSLEKKEAAYIHLTIEMLEGTGTITLHSKLDGDVQNEDSNYEEHFWEERAIETQETLGFVTTKTIPNNFEIERFTVTAGMRHFIDGASVVPTYTQQPLALTAELTVSLNEGETTAITKEVLVVTSRDVPETQQITRVNELFAEMTTLYPEAKAGQAAAWAKRWQLADVVIEGDDEAQQGIRFNLFQLFSTYYGEDDRLNIGPKGFTGEKYGGATYWDTEAYAVPLYLALAKPEVTKNLLKYRHNQLPQAIHNAQQQGLKGALYPMVTFTGVECHNEWEITFEEIHRNGAIAYAIYNYVNYTGDEDYLKDAGLEVLVAIARFWADRVHFSQRHKQYMIHGVTGPNEYENNINNNWYTNTIAAWVLRYTRESYLKFQEETTLKIADDELAKWADIVENMYFPVDNELGIFVQHDTFLDKDLMPVSDLPLSELPLNQHWSWDKILRSCFIKQADVLQGIYFFNDAFSLEEKRRNFNFYEPMTVHESSLSPSIHAVLAAELGMEEKAVEMYQRTARLDLDNYNNDTEDGLHITSMTGSWLAIVQGFAQMKTDHQQLKFAPFLPATWTAYSFHINYRNRLLFVEVAADQVAFTLLDGPAIPLTVYDQKYTLKDRLVLPIRKEEVHV

[0111] SEQ ID NO.8: MP nucleotide sequence, Enterococcus faecalis

[0112]

[0113] SEQ ID NO.9: PPGK amino acid sequence, Mycobacterium tuberculosis, Uniprot ID: P9WIN1

[0114] MTSTGPETSETPGATTQRHGFGIDVGGSGIKGGIVDLDTGQLIGDRIKLLTPQPATPLAVAKTIAEVVNGFGWRGPLGVTYPGVVTHGVVRTAANVDKSWIGTNARDTIGAELGGQQVTILNDADAAGLAET RYGAGKNNPGLVVLLTFGTGISAVIHNGTLIPNTEFGHLEVGGKEAEERAASSVKEKNDWTYPKWAKQVIRVLIAIENAIWPDLFIAGGGISRKADKWVPLLENRTPVVPAALQNTAGIVGAAMASVADTTH

[0115] SEQ ID NO.10: PPGK nucleotide sequence, Mycobacterium tuberculosis

[0116] atgaccagcaccggccccgagacgtccgaaacaccgggtgccacgacacagcgtcatggcttcggcatcgacgtcggcggcagcggcatcaagggcgga atcgtcgacttggacaccggccagctgatcggcgaccggatcaagctgctgaccccgcaaccggccactccgttggcggtcgccaaaaccatcgccgagg tcgtcaacggtttcggctggcggggtccgctgggggtgacctatcccggcgtcgtcactcacggcgtcgtccggaccgcggctaacgtggacaagtcctg gatagggaccaacgcacgcgacactatcggcgccgagctgggcggtcagcaggtcaccatcctcaacgacgctgatgccgccgggctggccgagacacgc tacggggccggcaagaacaaccctggcttagtggtactgctcacattcggaaccgggatcgggtccgcggtcatccacaacgggacgttgatacccaac accgagttcggacatcttgaggtcggcggcaaggaagcggaggaaagggccgcctcctcggtaaaggaaaagaacgactggacctatccaaagtgggcca agcaggtgatacgcgtgctcatcgccatcgagaacgcgatctggcctgacctgttcatcgccggcggcggcatcagccgcaaggccgacaaatgggtgcc gctactggaaaaccgcacaccagtagtgcccgcggccctgcagaacaccgccggaattgtcggtgcggccatggcctctgtcgcagatacgacgcactga

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for synthesizing trehalose 6-phosphate, characterized in that: The method uses maltose and polyphosphate as substrates to synthesize trehalose 6-phosphate; The method comprises the following steps: The first step is to catalyze maltose into glucose and β-glucose 1-phosphate; The second step reaction: converting glucose and polyphosphate into glucose 6-phosphate; The third step reaction: converting β-glucose 1-phosphate and glucose 6-phosphate into trehalose 6-phosphate; In the first step, maltose phosphorylase catalyzes the production of glucose and β-glucose 1-phosphate. In the second step, polyphosphate glucokinase catalyzes the reaction of polyphosphate and glucose to generate glucose 6-phosphate. In the third step, trehalose 6-phosphate is generated by catalysis of trehalose 6-phosphate phosphorylase; The maltose phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.1; the polyphosphate glucokinase comprises: the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.3; the trehalose 6-phosphate phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.5; and / or, The maltose phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.7; the polyphosphate glucokinase comprises: the amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.9; the trehalose 6-phosphate phosphorylase comprises: the amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 97% or higher homology to the amino acid sequence shown in SEQ ID NO.

5.

2. The method according to claim 1, characterized in that The reaction system further comprises a buffer solution, which comprises a phosphate buffer solution, and the pH of the phosphate buffer solution is 6.5-8.

0.

3. The method according to claim 1 or 2, characterized in that The concentration of substrate maltose is not less than 5 g / L.

4. The method according to claim 3, characterized in that The polyphosphate comprises a polyphosphate polymerized from 3, 6, 12, 24, 48 or 100 phosphoric acid molecules.

5. The method according to claim 4, characterized in that The polyphosphates include sodium salts, potassium salts and / or ammonium salts.

6. The method according to claim 4 or 5, characterized in that The added amounts of the maltose phosphorylase, polyphosphate glucokinase, and trehalose 6-phosphate phosphorylase are 0.1-10 U / mL, respectively.

7. The method according to claim 4 or 5, characterized in that The reaction system also contains 1-50 mM magnesium ions; the reaction temperature is 25-40°C.

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