In-vitro biosynthesis system of trehalose 6-phosphate and application thereof

Abstract

The application provides an in-vitro biosynthesis system of trehalose 6-phosphate and application thereof, and belongs to the technical field of biological catalysis. The method for synthesizing trehalose 6-phosphate in-vitro provided by the application uses cheap and readily available maltose and polyphosphate as raw materials to synthesize trehalose 6-phosphate, and does not need to add expensive cofactors such as ATP and UTP in the reaction process, thereby reducing the production cost of trehalose 6-phosphate and having the advantages of low cost, low pollution and high yield.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological catalysis, and particularly relates to an in-vitro biosynthesis system of trehalose 6-phosphate and application thereof. BACKGROUND

[0002] Trehalose 6-phosphate (T6P) is an important physiological metabolite with wide application value in the fields of biology, biochemistry and food industry. T6P is a key intermediate product of trehalose in plants, and participates in the regulation of plant growth and development. T6P has multiple functions. Firstly, it is a signal molecule that participates in the regulation of plant nutrient metabolism and carbon metabolism. T6P can regulate plant metabolic pathways such as photosynthesis, thereby affecting plant growth and development. Under stress conditions, T6P can promote the drought resistance, cold resistance and disease resistance of plants, and maintain the normal growth of plants. Secondly, T6P can promote the sucrose metabolism of plants. When the content of T6P increases, the ability of plants to utilize sucrose also increases, so T6P can be regarded as an indicator of the sucrose status in plants. In addition, T6P also has the functions of regulating plant starch metabolism, promoting flowering, embryogenesis, budding and branching, and can be regarded as a new type of plant hormone for promoting crop yield. Studies have shown that the application of T6P to crops through spraying and other methods can achieve crop yield increase and quality improvement, and has a high application prospect in agricultural production. However, due to factors such as production process, the production cost of T6P is high, and industrialized production has not been realized. Therefore, it is necessary to develop a low-cost and efficient method for synthesizing T6P to meet the demand for T6P.

[0003] In 1998, Junko Doi et al. synthesized trehalose 6-phosphate by yeast fermentation (Bioscience, Biotechnology, and Biochemistry, 1998, 62(4): 735-9.). The system uses glucose as a substrate, and glucokinase (GK) catalyzes the reaction of ATP and G6P; alpha-phosphoglucomutase (alpha-PGM) catalyzes the reaction of G6P and alpha-G1P; another substrate UMP is converted into UTP under the action of nucleoside monophosphate kinase (NMK) and nucleoside diphosphate kinase (NDK); alpha-G1P and UTP are catalyzed by UTP-monosaccharide 1-phosphate uridylyltransferase (USP) to generate UDPG; trehalose 6-phosphate synthase (TPS) catalyzes the reaction of G6P and UDPG to generate T6P. The technology uses a large amount of ATP and UMP as substrates, which are expensive, and the yield of T6P is only 11%. The yeast fermentation regulation is complex.

[0004] In contrast, the in vitro enzyme-catalyzed method is simpler and more efficient, and a new method for producing trehalose 6-phosphate with low cost, low pollution, high yield and suitable for large-scale production is urgently needed. SUMMARY

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

[0006] In order to solve the problems existing in the prior art, the purpose of the present application is to provide a method for preparing trehalose 6-phosphate by in vitro biological transformation using maltose and polyphosphate as raw materials. The present application designs an in vitro three-enzyme cascade catalytic reaction system to efficiently synthesize trehalose 6-phosphate, which does not need expensive cofactors ATP and UTP to supply energy and recycle, realizes high conversion rate of raw materials, high yield of products, low cost and 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 synthesizes trehalose 6-phosphate using maltose and polyphosphate as substrates;

[0009] The method comprises the following steps:

[0010] The first step reaction: catalyzing 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 reaction, maltose phosphorylase is used to catalyze the generation of glucose and β-glucose 1-phosphate;

[0014] In the second step reaction, polyphosphate glucose kinase is used to catalyze polyphosphate and glucose to generate glucose 6-phosphate;

[0015] In the third step reaction, trehalose 6-phosphate phosphorylase is used to catalyze the generation of trehalose 6-phosphate;

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

[0017] and / or,

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

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

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

[0021] [4]. The method according to any one of [1]-[3], wherein the polyphosphate comprises a polyphosphate polymerized by 3, 6, 12, 24, 48 or 100 phosphate molecules.

[0022] [5]. The method according to any one of [1]-[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]-[5], wherein the addition amount of the maltose phosphorylase, the polyphosphate glucose kinase and the trehalose 6-phosphate phosphorylase is 0.1-10 U / mL, respectively.

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

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

[0026] The present application provides a novel method for preparing trehalose 6-phosphate by in vitro biotransformation, using cheap maltose and polyphosphate, without adding cofactors such as ATP and UTP in the reaction process, and the obtained trehalose 6-phosphate has high yield and high content, and high-purity trehalose 6-phosphate can be obtained by simply removing protein and salt ions, the purification process is simple, the production cost of trehalose 6-phosphate can be reduced, the preparation process is environmentally friendly, and the method has the advantages of low cost, low pollution and high yield. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The figure is a three-enzyme system catalyzing the preparation of trehalose 6-phosphate from maltose and polyphosphate.

[0028] Figure 2SDS-PAGE of each enzyme in the three-enzyme system. M is a protein molecular weight marker; lane 1 is maltose phosphorylase MP derived from Enterococcus faecalis; lane 2 is maltose phosphorylase MP derived from Bacillus subtilis 168; lane 3 is polyphosphate glucose kinase PPGK derived from Mycobacterium tuberculosis; lane 4 is polyphosphate glucose kinase PPGK derived from Thermobifida fusca YX; and lane 5 is trehalose 6-phosphate phosphatase TrePP derived from Lactococcus lactis subsp. lactis Il1403.

[0029] Figure 3A Standard curve of trehalose 6-phosphate standard.

[0030] Figure 3B Ion chromatogram of trehalose 6-phosphate standard. DETAILED DESCRIPTION

[0031] Various illustrative embodiments, features and aspects of the present application are explained below in detail. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0032] In addition, for the purpose of convenience and brevity, detailed descriptions of well-known methods, apparatus, materials and steps are omitted so as not to obscure the teachings of the present application. It should be noted that the use of particular

[0033] Unless specifically stated otherwise, units used in the specification are in accordance with international standard units, and the numerical values, numerical ranges, appearing in the present application should be understood to include the systematic errors that are inevitable in industrial production.

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

[0035] In this specification, references to "some embodiments," "other embodiments," "exemplary embodiments," etc., indicate that the described features, items, etc. are among numerous possible specific combinations and embodiments of the disclosure. However, just because an embodiment is described does not mean that such embodiment is necessarily included in the disclosure, and / or the disclosure can include other embodiments not necessarily described below. In addition, reference to certain features

[0036] In this specification, "optional" or "optionally" means that the subsequently described event or circumstance can or can 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 "numerical value A ~ numerical value B" means a range including the end point values A, B.

[0038] As used herein, the term "and / or" encompasses all combinations of the items linked by the term. As used herein, the term "and / or" encompasses all combinations of the items linked by the term. As used herein, the term "and / or" encompasses all combinations of the items linked by the term. For example, "A and / or B" covers "A", "B", and "A and B". For example, "A, B, and / or C" covers "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0039] The word "comprising" is used herein to mean that a protein or nucleic acid can consist of the sequence recited, or can have additional amino acids or nucleotides at either or both ends of the protein or nucleic acid, but still have the activity described in the present disclosure. Furthermore, it is clear to a person skilled in the art that the methionine encoded by the start codon at the N-terminus of a polypeptide is in some practical cases (e.g. when expressed in a particular expression system) retained, but does not materially affect the function of the polypeptide. Therefore, in the description and claims of the present application, when a specific polypeptide amino acid sequence is described, this also encompasses sequences comprising the methionine encoded by the start codon at the N-terminus, even though it can not comprise the methionine, and correspondingly, the nucleotide sequence encoding it can comprise the start codon, and vice versa.

[0040] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art unless otherwise indicated. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory procedures steps used herein are terms and procedures widely used in the respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present application are well known to those skilled in the art and are described more fully in Sambrook, Joseph Frank et al. “Molecular Cloning: A Laboratory Manual.” (2001). (hereinafter “Sambrook”). Also, for better understanding of the present application, the definitions and explanations of the related 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 the present disclosure. The aforementioned terms refer to a polymer composed of many amino acids linked by peptide bonds, which can or can not contain modifications such as phosphoryl and formyl groups.

[0042] In the present application, sequence analysis software is generally used to measure the sequence similarity of polypeptides. Protein analysis software uses similarity measures specified to various substitutions, deletions, and other modifications including conservative amino acid substitutions to match similar sequences. For example, the GCG software contains programs such as GAP and BESTFIT, which can be used under default parameters to determine the sequence homology or sequence identity between closely related polypeptides, for example, between homologous polypeptides from different organism species or between a wild-type protein and its mutein. See, e.g., GCG Version 6.1. Polypeptide sequences can also be compared using FASTA under default or recommended parameters; programs in GCG Version 6.1 FASTA (e.g., FASTA2 and FASTA3) provide alignments and percent sequence identities of the best overlap between the query and search sequences. When comparing a sequence of the present application to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. 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] The term "transformation, transfection, transduction" in the present invention has the meaning commonly understood by those skilled in the art, i.e. the process of introducing foreign DNA into a host. The methods of transformation, transfection, transduction include any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate (CaP04) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0044] The term "enzymatic reaction" means a chemical reaction that proceeds 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, e.g. ranges of enzyme loading, substrate loading, temperature, pH, buffers, 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 "loading", such as in "enzyme loading" or "substrate loading", refers to the concentration or amount of a component in a reaction mixture at the start of a reaction.

[0047] As used herein, the term "polyphosphate" refers to any salt that contains several P-O-P bonds created by corner sharing of phosphate (P04) tetrahedra, forming long chains. The term "PolyP n " is used synonymously, where n represents the average chain length of phosphate residues, e.g. PolyP 25 refers to a polyphosphate having about 25 phosphate residues and PolyP 14 refers to a polyphosphate having about 14 phosphate residues.

[0048] As used herein, "conversion" can also refer to the enzymatic conversion (or biotransformation) of a substrate (or substrates) to a corresponding product (or products). The "percent conversion" refers to the percentage of substrate that is converted to product over a period of time under specified conditions. Thus, the "enzyme activity" or "activity" of a hexosamine synthase polypeptide can be expressed as the "percent conversion" of substrate to product over a particular time period.

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

[0050] EC number or EC number is a set of classification system for enzymes made by Enzyme Commission, which is based on the chemical reaction catalyzed by each enzyme. This classification system also gives a recommended name for each enzyme, so it is also called Enzyme Commission nomenclature.

[0051] In the present application, the form of use of any one of the enzymes in the enzyme-catalyzed reaction system for preparing trehalose 6-phosphate can be various, 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 application, the enzymes with various functions in the enzyme-catalyzed reaction system for preparing trehalose 6-phosphate can be directly used 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 the whole-cell microorganisms containing the enzymes with various functions or the cultures of whole-cell microorganisms (i.e. fermentation products) can be directly used, or the whole cells can be immobilized (i.e. immobilized cells) to maintain stability and recyclability. In some specific embodiments of the present application, in order to obtain a rapid reaction rate, the whole cells can be subjected to permeability treatment, exemplarily, heat treatment is used to promote cell membrane permeability.

[0053] In the present application, all the enzymes that can be used in the present application can be wild-type enzymes or enzymes subjected to certain genetic modifications, which means that the enzymes have improved properties, such as activity, for example, substrate specificity, for example, thermal stability, etc. The enzymes with desired properties derived from wild-type enzymes by genetic engineering technology are referred to as "mutants", "mutant derivatives" or "mutants from corresponding wild-type enzymes". In the present application, the enzymes used to implement the present application include these "mutants" with improved properties.

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

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

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

[0057] In some specific embodiments, MP catalyzes the hydrolysis of maltose into glucose and β-glucose 1-phosphate (β-G1P) using maltose as the substrate; PPGK catalyzes the generation of glucose 6-phosphate (G6P) from glucose and polyphosphate using glucose and polyphosphate as the substrates; and TrePP catalyzes the synthesis of trehalose 6-phosphate and inorganic phosphate from β-G1P and G6P.

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

[0059] In some embodiments, the MP is selected from maltose phosphorylase with EC number 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 an amino acid sequence as set forth in SEQ ID NO. 1 or as set forth in SEQ ID NO. 7, or an amino acid sequence having at least 95%, at least 96%, at least 97%, or at least 99% or more homology to the amino acid sequence as set forth in SEQ ID NO. 1 or as set forth in SEQ ID NO. 7. Illustratively, the nucleotide sequence encoding the MP is set forth in SEQ ID NO. 2 and SEQ ID NO. 8.

[0062] In some embodiments, the PPGK is selected from the group consisting of polyphosphate glucose kinase of EC number EC 2.7.1.63; optionally, the PPGK source includes, but is not limited to, Mycobacterium tuberculosis, Arthrobacter sp., Hungateiclostridium thermocellum, Thermus thermophilus, Pyrococcus sp., Pyrococcus horikoshii, Pyrococcus furiosus, Thermococcus barophilus, Thermococcus kodakarensis, Thermotoga maritima, 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 an amino acid sequence as set forth in SEQ ID NO. 3 or as set forth in SEQ ID NO. 9, or an amino acid sequence having at least 95%, at least 96%, at least 97%, or at least 99% or more homology to the amino acid sequence as set forth in SEQ ID NO. 3 or as set forth in SEQ ID NO. 9. Illustratively, a nucleotide sequence encoding PPGK is set forth in SEQ ID NO. 4 or as set forth in 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 Il1403.

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

[0068] Further, the three-enzyme cascade catalyzes the reaction in a buffer solution.

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

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

[0071] In some embodiments of the present application, the method for synthesizing trehalose 6-phosphate can recycle inorganic phosphate, and / or at least one step of the method for synthesizing trehalose 6-phosphate comprises an energy-favorable chemical reaction. In particular, the inorganic phosphate produced when TrePP catalyzes the reaction of β-G1P and G6P to form trehalose 6-phosphate can assist the catalytic reaction of MP.

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

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

[0074] In some specific embodiments, the polyphosphate comprises a polyphosphate polymerized from 3, 6, 12, 24, 48, or 100 phosphate molecules; and the polyphosphate comprises a sodium salt, a potassium salt, and / or an ammonium salt. Illustratively, the polyphosphate is sodium hexametaphosphate at a concentration of 5-300 mM, and preferably 15-150 mM.

[0075] Further, the reaction system further comprises magnesium ions, which can be provided by magnesium chloride and / or magnesium sulfate, such as one or a mixture of MgCl2and MgSO4. In some illustrative embodiments, the magnesium ions are provided by MgSO4at a concentration of 1-50 mM, and preferably 1-25 mM.

[0076] In some embodiments, the present application 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 a time period of about 0.5 h to about 72 h. For example, the present application is carried out at 25~60°C, pH 6.5~8.0.

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

[0078] Examples

[0079] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. The specific conditions not indicated in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by purchase.

[0080] Example 1: Construction of recombinant enzyme expression strain of related enzymes in three-enzyme system

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

[0082] The polyphosphate glucose kinase PPGK can be derived from Mycobacterium tuberculosis, with Uniprot accession number P9WIN1; the polyphosphate glucose kinase PPGK can also be derived from Thermobifida fusca, with Uniprot accession number Q47NX5;

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

[0084] The coding gene sequence of the above enzyme was synthesized by a gene synthesis company and ligated to a pET20b plasmid to construct a corresponding expression plasmid. The constructed plasmid was transformed into E. coli BL21 (DE3) competent cells, and a strain carrying the above recombinant plasmid was obtained through plate screening containing ampicillin. The specific process is as follows: the E. coli BL21 (DE3) competent cells were taken out from the-80°C refrigerator and placed on ice to thaw slowly, then 5 μL of the recombinant plasmid was immediately added, mixed gently and placed on ice for 30 min, then placed in a 42°C water bath for 90 s, and then placed in an ice bath for 2 min. 500 μL of LB liquid medium was added under sterile conditions, and the bacteria were cultured in a 37°C, 220 rpm shaker for 50 min to recover, then evenly coated on LB solid medium containing ampicillin (working concentration 100 μg / mL), the plate was sealed with a sealing film, and inverted in a 37°C incubator for overnight culture.

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

[0086] The single clone strains of the above transformants were picked from the LB solid plate and inoculated into a 1L flask containing 200 mL of LB liquid medium (containing a working concentration of 100 μg / mL ampicillin) for overnight culture, and expanded at 37°C, 220 rpm to OD 600 0.6-0.8, and 0.1 mM IPTG was added, and the culture was continued at 16°C for 16-20 h. The bacteria were centrifuged at 5000 rpm, 15 min, 4°C to collect the bacterial pellets, and the fermentation / shaking flask fermentation supernatant was removed. An equal volume of 0.9% NaCl solution was added to resuspend the bacterial pellets, which were centrifuged at 4°C, 5000 rpm for 15 min, and the centrifugal supernatant was removed. The bacterial pellets were resuspended with lysis buffer (100 mM phosphate buffer (pH 7.0), 0.3 M NaCl) to make the concentration of the bacterial resuspension OD 600 about 50, and the bacterial resuspension was broken using a high-pressure homogenizer or an ultrasonic disrupter. The supernatant was collected after lysis and filtered with a 0.45 μm filter, and the expressed recombinant protein was subjected to affinity adsorption by a nickel ion affinity chromatography column pre-equilibrated with lysis buffer. After loading, the column was washed with 5 column volumes of lysis buffer containing 10 mM imidazole to remove impurities, and the target protein was eluted with lysis buffer containing 100 mM imidazole. The eluate was replaced with 100 mM phosphate buffer (pH 7.0) in a short time to remove imidazole, and high-concentration protein was obtained after ultrafiltration for storage. The concentration of the target protein was determined by the Bradford method (Coomassie blue staining method), and the purity was detected by protein gel electrophoresis (SDS-PAGE) Figure 2). The protein samples were stored by adding glycerol to a final concentration of 5%, quick-freezing at -80 °C and storing in aliquots.

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

[0088] For the detection and quantification of the target product, high-performance anion exchange chromatography and a Dionex Ion Pac™ AS11-HC Analytical column (4 mm i.d. x 250 mm; Thermo Fisher Scientific) were used to detect the content of trehalose 6-phosphate. The test sample injection volume was 10 μL.

[0089] The retention time of the trehalose 6-phosphate standard (Shanghai Aladdin Biochem Technology Co., Ltd., Catalog No. T339557) was 8.287 min, and the trehalose 6-phosphate standard was prepared into a 0.1 g / L stock solution, which was diluted into 0.001 g / L, 0.005 g / L, 0.01 g / L, and 0.05 g / L standard solutions. The standard curve was used for quantitative analysis of the product and molar conversion rate (Y = 0.0002X + 0.0001, R2= 0.9999). 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 x 100%.

[0091] Example 4: Construction of a three-enzyme cascade catalytic reaction system for synthesizing trehalose 6-phosphate from maltose

[0092] In this embodiment, the maltose phosphorylase MP was derived from Bacillus subtilis 168, with Uniprot accession number O06993; the polyphosphate glucose kinase PPGK was derived from Thermobifida fusca YX, with Uniprot accession number Q47NX5; and the trehalose 6-phosphate phosphorylase TrePP was derived from Lactococcus lactis subsp. lactis Il1403, with Uniprot accession number Q9CID5. The corresponding enzymes were prepared by transformation and expression according to the methods of Example 1 and Example 2, and were used for the construction and reaction of the three-enzyme cascade catalytic reaction system.

[0093] In a 5-milliliter reaction system, 50 mM phosphate buffer (pH 7.5), 15 mM Mg 2+, 10 g / L maltose (molar concentration 29.2 mM, molecular weight of maltose 342 g / mol), 2 U / mL of maltose phosphorylase MP, 2 U / mL of polyphosphate glucokinase PPGK, and 2 U / mL of trehalose 6-phosphate phosphorylase TrePP. The reaction was carried out in a constant temperature shaker at a reaction temperature of 37°C and a rotation speed of 200 rpm, and samples were taken every 30 min. The reaction was carried out for about 2 h, and after the reaction was completed, the reaction was terminated by heating at 100°C for 10 min, and the protein was precipitated. After centrifugation at a rotation speed of 12000 rpm, the supernatant was used for chromatographic detection. The results showed that the yield of trehalose 6-phosphate was 12.07 g / L, the molar concentration was 28.6 mM (molecular weight of trehalose 6-phosphate 422 g / mol), and the molar conversion rate of trehalose 6-phosphate was 97.9%.

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

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

[0096] In a 5-milliliter reaction system, 50 mM phosphate buffer (pH 7.5), 15 mM Mg 2+, 15 mM sodium hexametaphosphate, 10 g / L maltose (molar concentration 29.2 mM, molecular weight of maltose 342 g / mol), the amount of maltose phosphorylase MP is 2 U / mL, the amount of polyphosphate glucose kinase PPGK is 2 U / mL, the amount of trehalose 6-phosphate phosphorylase TrePP is 2 U / mL. The reaction was carried out in a constant temperature shaker with a reaction temperature of 37℃ and a rotation speed of 200 rpm, and samples were taken every 30 min, the reaction was carried out for about 2 h, after the reaction was completed, the reaction was terminated by heating at 100℃ for 10 min, and the protein was precipitated, then centrifuged at a rotation speed of 12000 rpm, and the supernatant was taken for chromatographic detection. The supernatant after the reaction was detected, and the results showed that a new chromatographic peak consistent with the standard was generated at a retention time of 8.287 min, indicating that trehalose 6-phosphate was generated in the reaction solution. The results show that the yield of trehalose 6-phosphate is 11.78 g / L, the molar concentration is 27.9 mM (molecular weight of trehalose 6-phosphate 422 g / mol), and the molar conversion rate of trehalose 6-phosphate is 95.5%.

[0097] SEQ ID NO. 1: MP amino acid sequence, Bacillus subtilis 168, Uniprot ID: O06993

[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] MASRGRVGLGIDIGGSGIKGAPVDLDRGTFVVDRVKIATPQPATPEAVAAVVAEIVTAF ADDVPQDAPLGVTFPAVIQHGVARSAANVDRSWIGTNVEELLSAVTGRRVLVVNDADAA AMEHRYGAASGVDGVVLLTTLGTGIGTAVLVDGVLLPNTEFGHLEIDGYDAETRASAS AKERENLSYKEWAEERLQRYYSVIEDLLWPDLIVVGGGVSRKADKFLPHLRLRAPIVPA KLRNTAGIVGAAVLAAERLGGDRVSA

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

[0104] Atggcatctcggggacgggtcgggctggggattgacatcgggggaagcgggatcaaaggcgcccctgtggacttggaccggggaacgttcgtggtggaccgggtcaagatcgctactccgcagcccgcaacccctgaggcggtggctgcggtggtggcggagatagtcaccgcgttcgccgacgatgtgccgcaggatgcaccgttgggggtgacgtttcccgcggtgatccagcacggggtggcgcgcagcgccgccaacgtggaccgctcgtggatcggcaccaacgtcgaggagctgctgtctgcggtgacggggcggcgggtgctggtggtcaacgacgctgacgccgcagcgatggcggagcaccgctacggcgctgcctcaggcgtcgacggggtggtgctgttgactactttgggtaccggtattggtacggcggtgctagtggacggggtgctgctccccaacacggagttcgggcacttggagatcgacggctacgacgctgagacccgggcctctgctagcgctaaggagcgcgagaacctctcctacaaggagtgggctgaggagcggctgcagcgctactactcggtgatcgaggatttgctgtggccggacttgatcgtggtgggcggcggggtcagccgcaaggcggacaagtttttgccgcatctccgcttgcgcgcgccgatcgtgccggcgaagttgcgcaataccgcggggatcgtgggtgcggccgtgctggccgcggagcggctggggggtgaccgggtctctgcctga

[0105] SEQ ID NO. 5: TrePP amino acid sequence, 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] MTSTGPETSETPGATTQRHGFGIDVGGSGIKGGIVDLDTGQLIGDRIKLLTPQPATPLAVAKTIAEVVNGFGWRGPLGVTYPGVVTHGVVRTAANVDKSWIGTNARDTIGAELGGQQVTILNDADAAGLAETRYGAGKNNPGLVVLLTFGTGIGSAVIHNGTLIPNTEFGHLEVGGKEAEERAASSVKEKNDWTYPKWAKQVIRVLIAIENAIWPDLFIAGGGISRKADKWVPLLENRTPVVPAALQNTAGIVGAAMASVADTTH

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

[0116] atgaccagcaccggccccgagacgtccgaaacaccgggtgccacgacacagcgtcatggcttcggcatcgacgtcggcggcagcggcatcaagggcggaatcgtcgacttggacaccggccagctgatcggcgaccggatcaagctgctgaccccgcaaccggccactccgttggcggtcgccaaaaccatcgccgaggtcgtcaacggtttcggctggcggggtccgctgggggtgacctatcccggcgtcgtcactcacggcgtcgtccggaccgcggctaacgtggacaagtcctggatagggaccaacgcacgcgacactatcggcgccgagctgggcggtcagcaggtcaccatcctcaacgacgctgatgccgccgggctggccgagacacgctacggggccggcaagaacaaccctggcttagtggtactgctcacattcggaaccgggatcgggtccgcggtcatccacaacgggacgttgatacccaacaccgagttcggacatcttgaggtcggcggcaaggaagcggaggaaagggccgcctcctcggtaaaggaaaagaacgactggacctatccaaagtgggccaagcaggtgatacgcgtgctcatcgccatcgagaacgcgatctggcctgacctgttcatcgccggcggcggcatcagccgcaaggccgacaaatgggtgccgctactggaaaaccgcacaccagtagtgcccgcggccctgcagaacaccgccggaattgtcggtgcggccatggcctctgtcgcagatacgacgcactga

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application 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 make equivalent replacements for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of synthesizing trehalose 6-phosphate, characterized by, The method synthesizes trehalose 6-phosphate by using maltose and polyphosphate as substrates. The method comprises the following steps: The first step reaction: catalyzing maltose into glucose and beta-glucose 1-phosphate; The second step reaction: converting glucose and polyphosphate into glucose 6-phosphate; The third step reaction: converting beta-glucose 1-phosphate and glucose 6-phosphate into trehalose 6-phosphate; In the first step reaction, maltose phosphorylase is used to catalyze the generation of glucose and beta-glucose 1-phosphate; In the second step reaction, polyphosphate glucose kinase is used to catalyze polyphosphate and glucose to generate glucose 6-phosphate; In the third step reaction, trehalose 6-phosphate phosphorylase is used to catalyze the generation of trehalose 6-phosphate; The amino acid sequence of the maltose phosphorylase is shown in SEQ ID NO. 1; the amino acid sequence of the polyphosphate glucose kinase is shown in SEQ ID NO. 3; and the amino acid sequence of the trehalose 6-phosphate phosphorylase is shown in SEQ ID NO.

5. And / or, The amino acid sequence of the maltose phosphorylase is shown in SEQ ID NO. 7; the amino acid sequence of the polyphosphate glucose kinase is shown in SEQ ID NO. 9; and the amino acid sequence of the trehalose 6-phosphate phosphorylase is shown in SEQ ID NO.

5.

2. The method of claim 1, wherein, The reaction system further comprises a buffer, and the buffer comprises a phosphate buffer, and the pH of the phosphate buffer is 6.5-8.

0.

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

4. The method of claim 3, wherein, The polyphosphate comprises polyphosphate polymerized by 3, 6, 12, 24, 48 or 100 phosphate molecules.

5. The method of claim 4, wherein, The polyphosphate comprises sodium salt, potassium salt and / or ammonium salt.

6. The method according to claim 4 or 5, characterized in that, The addition amount of the maltose phosphorylase, the polyphosphate glucose kinase and the trehalose 6-phosphate phosphorylase is respectively 0.1-10 U / mL.

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

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