Method for efficient catalysis of sucrose to provide udp-glucose and applications thereof

Abstract

The present application provides an enzyme composition comprising sucrose phosphatase, UTP glucose-1-phosphate uridylyltransferase, polyphosphate kinase and the like, or a group of recombinant engineering bacteria expressing the above enzymes. By using the above enzyme composition or the recombinant engineering bacteria, UDP-glucose and its derivatives can be synthesized using sucrose as a raw material, which has application prospects in the field of biological manufacturing.

Description

Technical Field

[0001] This invention relates to the design of catalytic pathways, the construction of gene-edited microbial strains, and their applications in the field of biocatalysis. Specifically, it relates to an enzyme composition or recombinant engineered bacteria for catalyzing the synthesis of UDP-glucose and its derivatives from sucrose in a multi-enzyme system, and their applications. Background Technology

[0002] Natural products, possessing a range of biological activities such as antimalarial, antitumor, anti-inflammatory, and anti-aging properties, are frequently used in the production of pharmaceuticals, cosmetics, and nutritional supplements. Among traditional Chinese medicines, paclitaxel from Taxus chinensis, artemisinin from Artemisia annua stems and leaves, and rhodioloside from Rhodiola rosea all exhibit excellent pharmacological activity. However, these natural products often suffer from poor stability, low water solubility, and low bioavailability. To address this issue, methods such as glycosylation, methylation, and acylation of natural substrates are commonly employed, with glycosylation being the most widespread. Glycoside compounds formed through glycosylation reactions can enhance the biological activity of natural products, thereby improving their stability, water solubility, and bioavailability. They have found widespread application in the production of drug leads, cosmetics, nutrients, and sweeteners. Glycosylation is a crucial method for improving the physical properties and biological activity of natural products. The essential glycosyl donor for glycosylation is generally the UDP-glycosyl donor, with UDP-glucose (UDPG) being one of the most important glycosyl donors for the biosynthesis of glycosides, oligosaccharides, polysaccharides, and glycoproteins. Currently, the main methods for producing UDPG are chemical synthesis and biosynthesis. Chemical synthesis involves coupling uridine monophosphate or uridine diphosphate with a glycosyl phosphate; however, this method suffers from long reaction times, low synthesis efficiency, high cost, and low stereoselectivity, thus hindering industrial development.

[0003] Traditional biosynthetic methods include whole-cell catalysis and enzymatic synthesis. These typically involve using glucose or sucrose to provide glucose-1-phosphate (G1P), which is then catalyzed by UDP-glucose-1-phosphate transferase (GalU) to generate UDPG. However, current technologies suffer from low catalytic efficiency, byproduct accumulation, interference with cell growth, and generally low product yields. Based on this research, there is an urgent need in this field for a more efficient catalytic pathway that utilizes sucrose to provide UDPG, addressing the issues of byproduct accumulation and insufficient UDPG supply. Summary of the Invention

[0004] The problem this technical solution aims to solve is how to efficiently provide UDPG using sucrose as a substrate, thereby reducing byproducts, relieving enzyme inhibition, fully utilizing sucrose, and increasing the yield of the final product.

[0005] To address the aforementioned technical problems, in a first aspect, the present invention provides an enzyme composition comprising enzyme composition I, enzyme composition II, and enzyme composition III. Enzyme composition I comprises a protein or polypeptide having the following functions: A) sucrose phosphatase, B) glucose phosphokinase, C) glucose-6-phosphate isomerase, and D) glucose phosphomutase. Enzyme composition II comprises a protein or polypeptide having the following functions: E) UTP glucose-1-phosphate uridine transferase, and F) glycosyltransferase. Enzyme composition III comprises a protein or polypeptide having the following functions: G) polyphosphokinase, H) pyrophosphatase, and I) nucleoside diphosphate kinase.

[0006] Furthermore, A) sucrose phosphatase can be derived from *Bifidobacterium adolescentis*, B) glucose phosphokinase can be derived from *Thermobifida fusca*, C) glucose-6-phosphate isomerase can be derived from *Escherichia coli*, D) glucose phosphomutase can be derived from *Thermococcus kodakarensis*, E) UTP glucose-1-phosphate uridine transferase can be derived from *Escherichia coli*, F) glycosyltransferase can be derived from *Bacillus subtilis*, G) polyphosphokinase can be derived from *Deinococcus radiodurans*, H) pyrophosphatase can be derived from *Methanococcus jannaschii*, and I) nucleoside diphosphate kinase can be derived from *Escherichia coli*.

[0007] Furthermore, the amino acid sequence of A) sucrose phosphatase comprises the amino acid sequence shown in SEQ ID NO:2, B) glucose phosphokinase comprises the amino acid sequence shown in SEQ ID NO:4, C) glucose-6-phosphate isomerase comprises the amino acid sequence shown in SEQ ID NO:6, D) glucose phosphomutase comprises the amino acid sequence shown in SEQ ID NO:8, E) UTP glucose-1-phosphate uridine transferase comprises the amino acid sequence shown in SEQ ID NO:10, F) glycosyltransferase comprises the amino acid sequence shown in SEQ ID NO:12, G) polyphosphokinase comprises the amino acid sequence shown in SEQ ID NO:14, H) pyrophosphatase comprises the amino acid sequence shown in SEQ ID NO:16, and I) nucleoside diphosphate kinase comprises the amino acid sequence shown in SEQ ID NO:18.

[0008] Alternatively or alternatively, the amino acid sequences of A)-I) above may also be modified as shown in any of the following ways: SEQ ID:2, SEQ ID:4, SEQ ID:6, SEQ ID:8, SEQ ID:10, SEQ ID:12, SEQ ID:14, SEQ ID:16, and SEQ ID:18.

[0009] (i1) A protein with the same function is obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0010] (i2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus.

[0011] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0012] Furthermore, A)-I) can all be obtained by conventional methods, such as prokaryotic expression, cell disruption, or protein purification. Even further, cell disruption can be achieved through ultrasonic disruption or high-pressure homogenization. Still further, protein purification can be performed using nickel column affinity chromatography.

[0013] Furthermore, the recipient bacterium for prokaryotic expression in this invention is preferably Escherichia coli; more preferably Escherichia coli MC02.

[0014] Secondly, the present invention provides a method for synthesizing rhodioloside. The method comprises the following reaction system: (a1) the enzyme composition described in the first aspect; (a2) sucrose; (a3) ​​tyrosol; and (a4) water.

[0015] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0016] Furthermore, after the reaction is completed, the supernatant of the reaction system is collected to obtain rhodioloside.

[0017] Further, the enzyme composition as described in any one of claims 1 to 3, or at least one enzyme in the enzyme composition, is reacted, and the supernatant collected by centrifugation after the reaction is completed is the rhodioloside solution.

[0018] Thirdly, the present invention provides a method for synthesizing salicin. The method comprises the following reaction system: (a1) the enzyme composition described in the first aspect; (a2) sucrose; (a5) salicylol; and (a4) water.

[0019] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0020] Furthermore, after the reaction is completed, the supernatant of the reaction system is collected to obtain salicin.

[0021] Fourthly, the present invention provides a group of recombinant engineered bacteria, including: I) recombinant Escherichia coli I, comprising an introduced or enhanced expression of a gene encoding A) sucrose phosphatase, B) glucose phosphokinase, C) glucose-6-phosphate isomerase, and D) glucose phosphomutase; II) recombinant Escherichia coli II, comprising an introduced or enhanced expression of a gene encoding E) UTP glucose-1-phosphate uridine transferase and F) glycosyltransferase; III) recombinant Escherichia coli III, comprising an introduced or enhanced expression of a gene encoding G) polyphosphokinase, H) pyrophosphatase, and I) nucleoside diphosphate kinase.

[0022] Furthermore, A) sucrose phosphatase can be derived from *Bifidobacterium adolescentis*, B) glucose phosphokinase can be derived from *Thermobifida fusca*, C) glucose-6-phosphate isomerase can be derived from *Escherichia coli*, D) glucose phosphomutase can be derived from *Thermococcus kodakarensis*, E) UTP glucose-1-phosphate uridine transferase can be derived from *Escherichia coli*, F) glycosyltransferase can be derived from *Bacillus subtilis*, G) polyphosphokinase can be derived from *Deinococcus radiodurans*, H) pyrophosphatase can be derived from *Methanococcus jannaschii*, and I) nucleoside diphosphate kinase can be derived from *Escherichia coli*.

[0023] Furthermore, the amino acid sequence of A) sucrose phosphatase comprises the amino acid sequence shown in SEQ ID NO:2, B) glucose phosphokinase comprises the amino acid sequence shown in SEQ ID NO:4, C) glucose-6-phosphate isomerase comprises the amino acid sequence shown in SEQ ID NO:6, D) glucose phosphomutase comprises the amino acid sequence shown in SEQ ID NO:8, E) UTP glucose-1-phosphate uridine transferase comprises the amino acid sequence shown in SEQ ID NO:2, F) glycosyltransferase comprises the amino acid sequence shown in SEQ ID NO:4, G) polyphosphokinase comprises the amino acid sequence shown in SEQ ID NO:6, H) pyrophosphatase comprises the amino acid sequence shown in SEQ ID NO:8, and I) nucleoside diphosphate kinase comprises the amino acid sequence shown in SEQ ID NO:8.

[0024] Alternatively or alternatively, the amino acid sequences of A)-I) above may also be modified as shown in any of the following ways: SEQ ID:2, SEQ ID:4, SEQ ID:6, SEQ ID:8, SEQ ID:10, SEQ ID:12, SEQ ID:14, SEQ ID:16, and SEQ ID:18.

[0025] (j1) A protein with the same function is obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0026] (j2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus.

[0027] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0028] Furthermore, in the recombinant engineered bacteria, the encoding gene of A)-I) can be introduced into the recipient bacteria in the form of a recombinant vector. Even further, a vector promoter may be optionally included.

[0029] Furthermore, the recipient bacteria of the recombinant engineered bacteria in this invention are preferably Escherichia coli; more preferably Escherichia coli MC02.

[0030] Fifthly, the present invention provides a method for synthesizing rhodioloside. The method comprises the following reaction system: (a1) the recombinant engineered bacteria described in the fourth aspect; (a2) sucrose; (a3) ​​tyrosol; and (a4) water.

[0031] Furthermore, the recombinant engineered bacteria are in the form of cells or cell fragments. Even further, the cell fragments can be obtained by ultrasonic disruption or high-pressure homogenization of cells.

[0032] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0033] Furthermore, after the reaction is completed, the supernatant of the reaction system is collected to obtain rhodioloside.

[0034] In a sixth aspect, the present invention provides a method for synthesizing salicin. The method comprises the following reaction system: (a1) the recombinant engineered bacteria described in the fourth aspect; (a2) sucrose; (a5) salicylol; and (a4) water.

[0035] Furthermore, the recombinant engineered bacteria are in the form of cells or cell fragments. Even further, the cell fragments can be obtained by ultrasonic disruption or high-pressure homogenization of cells.

[0036] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0037] Furthermore, after the reaction is completed, the supernatant of the reaction system is collected to obtain salicin.

[0038] In a seventh aspect, the present invention provides the use of an enzyme composition or recombinant engineered bacteria according to any one of the first to sixth aspects in the production of UDP-glucose, UDP-glucose derivatives, rhodioloside, and salicin.

[0039] According to the above aspects of the present invention, the method of the present invention uses sucrose as a raw material and carries out a catalytic reaction through an enzyme composition or recombinant engineered bacteria, which can efficiently synthesize UDP-glucose and its derivatives, which is beneficial for industrial application. Attached Figure Description

[0040] Figure 1 The liquid chromatography chromatograms of sucrose and fructose standards are shown. The peak elution time for sucrose is 8.60 min, and for fructose it is 12.90 min.

[0041] Figure 2 The liquid chromatography chromatograms of tyrosol and rhodioloside standards are shown. The peak time for tyrosol is 12.00 min, and the peak time for rhodioloside is 9.16 min.

[0042] Figure 3 The liquid chromatography chromatograms of salicylol and salicin standards are shown. The peak time for salicylol is 12.50 min, and for salicin it is 8.50 min. Detailed Implementation

[0043] definition

[0044] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0045] Throughout this specification and the appended claims, the terms "comprising" and "including," as well as variations thereof, shall be interpreted inclusively. That is, where the context permits, these terms are intended to express the possibility of including other elements or integers not specifically listed.

[0046] The article “a / an” is used in this text to refer to one / an or more than one / an (i.e., one / an or at least one / at least one of the grammatical objects of the article). For example, “an element / an element” can mean one element / an element or more than one element / an element. When a noun (e.g., compound, additive, etc.) is mentioned in the singular form, it is intended to include the plural form. Therefore, when referring to a specific part (e.g., “gene”), unless otherwise specified, it means “at least one” of the genes, e.g., “at least one gene”.

[0047] Unless otherwise expressly indicated, the various embodiments of the invention described herein can be combined in various ways.

[0048] The term "carbon source" refers to a source of carbon, preferably a compound or molecule containing carbon. Preferably, the carbon source is a carbohydrate, amino acid, or its derivative. In this document, a carbon source is understood as an organic compound composed of elements such as carbon, oxygen, and hydrogen.

[0049] The term "cell" refers to a eukaryotic or prokaryotic organism, preferably existing as a single cell. In this invention, the cell can be recombinant *Escherichia coli*. That is, the recombinant cell is selected from a cell population of a genus of *Escherichia coli*.

[0050] As used herein, the term “recombinant” / “engineered” (e.g., references to “recombinant E. coli,” “recombinant cell,” “recombinant microorganism,” and / or “recombinant strain”) can refer to a cell, microorganism, or strain containing nucleic acids as a result of one or more gene modifications. Simply put, a cell, microorganism, or strain contains different combinations of nucleic acids from one or more of its parents (any one of them). To construct recombinant cells, microorganisms, or strains, one or more recombinant DNA techniques and / or another one or more mutagenesis techniques can be used. For example, recombinant E. coli and / or recombinant E. coli cells may contain nucleic acids not present in the corresponding wild-type E. coli and / or cells, which have been introduced into the E. coli or E. coli cells using recombinant DNA techniques, or the absence of the nucleic acid in the wild-type E. coli and / or cells is the result of one or more mutations in the nucleic acid sequence (such as a gene encoding a wild-type polypeptide) present in the wild-type E. coli and / or E. coli cells (e.g., using recombinant DNA techniques or another mutagenesis technique such as UV irradiation). Furthermore, the term “recombinant” can suitably refer to, for example, cells, microorganisms, or strains from which nucleic acid sequences have been removed using recombinant DNA techniques.

[0051] In this paper, "recombinant E. coli containing or having a certain activity" is understood to mean that recombinant E. coli can contain one or more nucleic acid sequences encoding proteins having such activity. Therefore, recombinant E. coli is permitted to functionally express such proteins or enzymes.

[0052] The term "functional expression" refers to functional transcription in which the relevant nucleic acid sequence is present, allowing the nucleic acid sequence to actually be transcribed, for example, leading to protein synthesis.

[0053] As used herein with respect to proteins or peptides, the term "mutation" means that, compared to the wild-type or naturally occurring protein or peptide sequence, at least one amino acid has been substituted, inserted into, or deleted from the amino acid sequence. Amino acid substitutions, insertions, or deletions can be achieved, for example, by mutagenesis of the nucleic acids encoding those amino acids. Mutagenesis is a method well known in the art and includes, for example, site-directed mutagenesis by means of PCR or by oligonucleotide-mediated mutagenesis, as described in Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., vols. 1–3 (1989), published by Cold Spring Harbor Publishing.

[0054] As used herein with respect to genes, the term "mutation" means that, compared to a wild-type or naturally occurring nucleic acid sequence, at least one nucleotide in the nucleic acid sequence of a gene or its regulatory sequence has been replaced, inserted into, or deleted from the nucleic acid sequence by a different nucleotide. The substitution, insertion, or deletion of amino acids can be achieved, for example, via mutagenesis, resulting in transcription of a protein sequence having a qualitatively or quantitatively altered function, or the knockout of the gene. In the context of this invention, "altered gene" has the same meaning as a mutated gene.

[0055] As used herein, the term "gene" refers to a nucleic acid sequence of mRNA that can be transcribed and then translated into a protein. A gene that encodes a protein is one or more nucleic acid sequences that encode that protein.

[0056] As used herein, the term "nucleic acid" or "nucleotide" refers to a monomeric unit in a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer (i.e., a polynucleotide), and unless otherwise limited, encompasses known analogs that have the inherent properties of natural nucleotides because they hybridize with single-stranded nucleic acids (e.g., peptide nucleic acids) in a manner similar to naturally occurring nucleotides. For example, an enzyme defined by the nucleotide sequence encoding an enzyme includes (unless otherwise limited) a nucleotide sequence that hybridizes with a reference nucleotide sequence encoding the enzyme. A polynucleotide can be a natural or heterologous structure or a full-length or subsequence of a regulatory gene. Unless otherwise indicated, the term includes references to the specified sequence and its complementary sequence. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as contemplated herein. Furthermore, DNA or RNA containing rare bases (such as inosine) or modified bases (such as triphenylmethylated bases) (to name just two examples) is a polynucleotide as used herein. It will be understood that DNA and RNA have been modified in a wide variety of ways for many useful purposes known to those skilled in the art. As used herein, the term polynucleotide includes such chemical, enzymatic, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA specific to viruses and cells (especially simple and complex cells).

[0057] The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably in this article. An example of a nucleic acid sequence is a DNA sequence.

[0058] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers, for example, those containing amino acid residues as shown in the amino acid sequence. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. An essential property of such analogs of naturally occurring amino acids is that, when incorporated into a protein, the protein exhibits a specific reactivity to antibodies induced by proteins composed entirely of the same, but entirely, naturally occurring amino acids. The terms “polypeptide,” “peptide,” and “protein” also include modifications, including but not limited to glycosylation, lipid attachment, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0059] The term "enzyme" in this document refers to a protein that has a catalytic function. In cases where a protein catalyzes a biological reaction, the terms "protein" and "enzyme" may be used interchangeably herein. When referring to enzymes in the Enzyme Classification (EC), an enzyme class is a category in which an enzyme is classified or can be classified according to the enzyme nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which can be found at http: / / www.chem.qmul.ac.uk / iubmb / enzyme / . It is intended to include other suitable enzymes that are not yet (not yet) classified in the specified category but can be so classified.

[0060] If a protein or nucleic acid sequence (such as a gene) is referred to in this document by a reference accession number, unless otherwise specified, that number is specifically used to refer to a protein or nucleic acid sequence (gene) that can be found via www.ncbi.nlm.nih.gov / (available as of October 1, 2020).

[0061] Each nucleic acid sequence encoding a polypeptide in this paper also includes variants of any conserved modifications thereof. This includes, by reference to the genetic code, every possible silent variant of the nucleic acid. The term “conserved variant” applies to both amino acids and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conserved variant refers to those nucleic acids that encode the same amino acid sequence or a variant of a conserved modified amino acid sequence due to the degeneracy of the genetic code. The term “degeneracy of the genetic code” refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where the codon specifies alanine, the codon can be changed to any of the described corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are “silent variants” and represent a variant of a conserved modification.

[0062] As used herein, the term "functional homolog" (or simply "homology") of a gene having a specific sequence (e.g., "SEQ ID NO:X") refers to a polypeptide and / or amino acid sequence containing the specific sequence, or a nucleic acid sequence containing a polypeptide and / or amino acid sequence encoding the specific sequence, provided that one or more amino acids are mutated, substituted, deleted, added, and / or inserted, and that the polypeptide has (qualitatively) the same enzymatic function for substrate transformation.

[0063] As used herein, the term "functional homolog" (or simply "homolog") of a polynucleotide and / or nucleic acid sequence having a specific sequence (e.g., "SEQ ID NO:X") refers to a polynucleotide and / or nucleic acid sequence containing said specific sequence, provided that one or more nucleic acids are mutated, substituted, deleted, added, and / or inserted, and that the polynucleotide encodes a polypeptide sequence having (qualitatively) the same enzymatic function for substrate transformation. Regarding nucleic acid sequences, the term "functional homolog" is intended to include nucleic acid sequences that differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[0064] In this document, sequence identity is defined as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. Typically, sequence identity or similarity is compared over the entire length of the sequences being compared. In this art, “identity” also means the degree of sequence correlation between amino acid or nucleic acid sequences, as determined by matching strings of such sequences.

[0065] Amino acid or nucleotide sequences are said to be homologous when they exhibit a certain level of similarity. Two sequences being homologous indicates a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "identity percentage" or "similarity percentage," which are high or low, respectively. Although controversial, "homology level" or "homology percentage" is often used interchangeably to indicate "identity percentage" or "similarity percentage." Sequence comparisons and the determination of the identity percentage between two sequences can be accomplished using mathematical algorithms. Technicians will realize that several different computer programs can be used to align two sequences and determine their homology (Kruskal et al., "An overview of sequence comparison: Time warps, string edits, and macromolecules", (1983), Society for Industrial and Applied Mathematics (SIAM), Vol. 25, No. 2, pp. 201-237; and the handbook edited by D. Sankoff and J.B. Kruskal, "Time warps, string edits and macro molecules: the theory and practice of sequence comparison", (1983), pp. 1-44, published by Addison-Wesley Publishing Company, Massachusetts USA).

[0066] The Needleman and Wunsch algorithm can be used to align two sequences to determine the percentage of identity between the two amino acid sequences. (Needleman et al., "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins" (1970), J. Mol. Biol., Vol. 48, pp. 443-453). This algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or later, see Rice et al., "EMBOSS: The European Molecular Biology Open Software Suite", (2000), Trends in Genetics, Vol. 16, (6), pp. 276-277, http: / / emboss.bioinformatics.nl / ) was used. For protein sequences, EBLOSUM62 was used as the substitution matrix. For nucleotide sequences, EDNAFULL was used. Other matrices may be specified. Optional parameters for amino acid sequence alignment are a vacancy opening penalty of 10 and a vacancy expansion penalty of 0.5. Those skilled in the art will understand that all these different parameters will produce slightly different results, but the overall percentage of identity between the two sequences does not change significantly when different algorithms are used.

[0067] Homology or identity is the percentage of identical matches between two complete sequences across the total aligned region, including any vacancies or extensions. Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions in both sequences showing the same amino acid in the alignment divided by the total length of the alignment, including vacancies. Identity, as defined herein, is available from NEEDLE and is labeled "IDENTITY" in the program's output.

[0068] Homology or identity between two aligned sequences is calculated as follows: the number of positions in the alignment showing the same amino acid in both sequences is divided by the total length of the alignment after subtracting the total number of vacancies in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "longest-identity" in the program's output.

[0069] Variants of the nucleotide or amino acid sequences disclosed herein may also be defined as nucleotide or amino acid sequences having one or more mutations, substitutions, insertions, and / or deletions compared to the nucleotide or amino acid sequences specifically disclosed herein (e.g., in the sequence listing).

[0070] Optionally, when determining the degree of amino acid similarity, those skilled in the art may also consider so-called “conservative” amino acid substitutions, which will be clear to them. Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. In one embodiment, the group of conserved amino acid substitutions is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are variants in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted at its position. Preferably, the amino acid changes are conserved. In one embodiment, the conserved substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0071] The nucleotide sequences of the present invention can also be defined by their ability to partially hybridize with specific nucleotide sequences disclosed herein, respectively, under moderate hybridization conditions or preferably under stringent hybridization conditions. Stringent hybridization conditions are defined herein as conditions that allow nucleic acid sequences of at least about 25 nucleotides, preferably about 50, 75, or 100 nucleotides, and most preferably about 200 or more nucleotides, to hybridize at about 65°C in a solution containing about 1 M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to be washed at 65°C in a solution containing about 0.1 M or less salt (preferably 0.2x SSC or any other solution with equivalent ionic strength). Preferably, hybridization is performed overnight, i.e., at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with about 90% or higher sequence identity. In this document, moderate conditions are defined as conditions that allow nucleic acid sequences of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at about 45°C in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to wash at room temperature in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent ionic strength). Preferably, hybridization is performed overnight, i.e., at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with up to 50% sequence identity. Those skilled in the art will be able to modify these hybridization conditions to specifically identify sequences with identity varying between 50% and 90%.

[0072] "Expression" refers to the transcription of genes into structural RNA (rRNA, tRNA) or messenger RNA (mRNA), which is then translated into proteins.

[0073] "Overexpression" refers to the expression of a gene (corresponding nucleic acid sequence) in recombinant cells exceeding its expression in the corresponding wild-type cells. Such overexpression can be arranged, for example, by increasing the transcription frequency of one or more nucleic acid sequences, such as by operatively linking the nucleic acid sequence to a functional promoter in the recombinant cell; and / or by increasing the copy number of a nucleic acid sequence.

[0074] The term "upregulation" and its variations refer to the process by which cells increase the amount of cellular components, such as RNA or proteins. This upregulation can be in response to or caused by gene modification.

[0075] In this article, the term "pathway" or "metabolic pathway" is understood as a series of chemical reactions that build up and break down molecules in the cell.

[0076] Nucleic acid sequences (i.e., polynucleotides) or proteins (i.e., polypeptides) can be native or heterologous to the host cell's genome.

[0077] The terms "natural," "homologous," or "endogenous" in relation to a host cell mean that the nucleic acid sequence is indeed naturally present in the host cell's genome, or that the protein is naturally produced by that cell. The terms "natural," "homologous," and "endogenous" are used interchangeably in this document.

[0078] As used herein, “heterologous” or “exogenous” can refer to a nucleic acid sequence or a protein. For example, in relation to a host cell, “heterologous” can refer to a polynucleotide that is not naturally present in the genome of the host cell in this manner, or a polypeptide or protein that is not naturally produced by the cell in this manner. A heterologous nucleic acid sequence is a nucleic acid derived from a foreign species, or, if from the same species, substantially modified relative to its natural form in terms of composition and / or genomic loci through deliberate human intervention. For example, a promoter operatively linked to a natural structural gene is derived from a species different from the species from which the structural gene is derived, or, if from the same species, one or both are substantially modified relative to their original form. A heterologous protein can be derived from a foreign species, or, if from the same species, substantially modified relative to its original form through deliberate human intervention. That is, heterologous protein expression involves the expression of a protein that is not naturally expressed in the host cell in this manner. The term “heterologous expression” refers to the expression of a heterologous nucleic acid in a host cell. The expression of heterologous proteins in eukaryotic host cell systems (such as *Escherichia coli*) is well known to those skilled in the art. Polynucleotides containing nucleic acid sequences encoding genes for proteins or enzymes with specific activities can be expressed in such eukaryotic systems. In some embodiments, transformed / transfected cells can be used as expression systems for enzymes. The expression of heterologous proteins in *E. coli* is well known. *The Cold Spring Harbor Laboratory* is a recognized work describing various methods that can be used to express proteins in *E. coli*.

[0079] As used herein, a "promoter" is a DNA sequence that directs the transcription of a (structural) gene or other (partial) nucleic acid sequence. Appropriately, the promoter is located in the 5' region of the gene, near the transcription start site of the (structural) gene. The promoter sequence can be constitutive, inducible, or repressive. In one embodiment, no (external) inducer is required.

[0080] As used herein, the term "vector" includes references to autosomal expression vectors and integration vectors for integration into chromosomes.

[0081] The term "expression vector" refers to a linear or circular DNA molecule containing a segment encoding a target polypeptide, which is controlled (i.e., operatively linked to) additional nucleic acid segments that provide for its transcription. These additional segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both.

[0082] "Plasmid" refers to autonomously replicating extrachromosomal DNA that does not integrate into the genome of a microorganism and is usually circular in nature.

[0083] In this paper, "host cell" is understood to be a cell (such as an E. coli cell) that will be transformed with one or more nucleic acid sequences encoding one or more heterologous proteins to construct a transformed cell (also known as a recombinant cell). For example, the transformed cell may contain a vector and may support the replication and / or expression of the vector.

[0084] As used herein, “transformation” refers to the insertion of a foreign polynucleotide into a host cell, regardless of the method used for insertion, such as direct uptake, transduction, f-conjugation, or electroporation. The foreign polynucleotide may be maintained as a non-integrating vector (e.g., a plasmid) or alternatively integrated into the host cell genome.

[0085] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0086] Those skilled in the art will understand that the gene or its functional homologs disclosed herein comprise a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the exemplarily listed sequences, or having one or more mutated, substituted, inserted, and / or deleted amino acid sequences compared to the amino acid sequence it encodes.

[0087] Preferably, compared with the amino acid sequence encoded by a gene, the amino acid sequence encoded by any functional homolog of that gene has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 amino acid mutations, substitutions, insertions, and / or deletions.

[0088] Exemplary, and not limiting, the functional homolog of the BASP gene shown in SEQ ID NO.1 comprises a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of those nucleic acid sequences, or its functional homolog comprises a nucleic acid sequence having one or more mutations, substitutions, insertions, and / or deletions compared to any of those nucleic acid sequences; preferably, compared to such nucleic acid sequences, the nucleic acid sequence of any such functional homolog has no more than 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 10, or 5 nucleic acid mutations, substitutions, insertions, and / or deletions. More preferably, the functional homolog of the BASP gene comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the polypeptide shown by SEQ ID NO. 2.

[0089] Those skilled in the art will understand that other sequences include the above-described cases.

[0090] Unless otherwise specified herein, the terms and terminology used herein should be understood in accordance with the conventional knowledge and usage of those skilled in the art. Unless otherwise stated, the specific operational methods (including: preparation processes, experimental steps, detection methods, etc.) employed in this application utilize conventional techniques in the fields of biochemistry, cell biology, molecular biology, gene editing (e.g., recombinant DNA technology), zoology, and related areas. These techniques are well-described in existing literature. For details, please refer to Sam Brook et al., *Molecular Cloning: a Laboratory Manual*, 4th edition, Cold Spring Harbor Laboratory Press, 2012; Ausubel et al., *Current Protocols in Molecular Biology*, Wiley Online Press, updated irregularly; Kursad Turksen et al., *Embryonic StemCell Protocols*, 3rd edition, Springer Press, 2016; P. Nagarajan et al., *Essentials of Laboratory Animal Science: Principles and Practices*, Springer Press, 2021; and Jann Hau et al., *Handbook of Laboratory Animal Science: Essential Principles and Practices*, 4th edition, CRC Press, 2021.

[0091] The Escherichia coli Trans1-T1 competent cells in the following examples are products of Beijing TransGen Biotech, product catalog number CD501.

[0092] Carrier pBAD / HisB: Invitrogen, catalog number V430-01.

[0093] Tyrosol: Purchased from Aladdin, catalog number H306005

[0094] Sugar: Purchased from Aladdin, catalog number S112224

[0095] Rhodioloside: Purchased from Aladdin, catalog number S101157

[0096] Salicylol: Purchased from Aladdin, catalog number NBC-242871

[0097] Salicin: Purchased from Aladdin, catalog number S104922 Detailed Implementation

[0098] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0099] The liquid LB medium (pH 7.0) in the following examples contains 1 g / 100 mL NaCl, 1 g / 100 mL tryptone, 0.5 g / 100 mL yeast extract, and the remainder is water. The solid medium is obtained by adding agarose to the liquid medium.

[0100] The *E. coli* strains used in the following examples may specifically be one of *E. coli* MG1655, BW25113, or MC02. *E. coli* MG1655 (CGSC#: 6300) and BW25113 (CGSC#: 76376) are products of the Yale University Genetic Collection Center for *E. coli*. *E. coli* MC02 is deposited at the China General Microbiological Culture Collection Center (CGMCC) (accession number CGMCC No. 34378).

[0101] Table 1. List of Sequence Fragments

[0102] Serial Number Segment Name Sequence features Sequence (5'-3') SEQ ID NO.1 BASP gene Gene sequence of Bifidobacterium adolescentis (BASP) sucrose phosphatase atgaaaaacaaggtgcagctcatcacttacgccgaccgccttggcgacggcaccatcaagtcgatgaccgacattctgcgcacccgcttcgacggcgtgtacgacggcgttcacatcctgccgttcttcaccccgttcgacggcgccgacgcaggcttcgacccgatcgaccacaccaaggtcgacgaacgtctcggcagctgggacgacgtcgccgaactctccaagacccacaacatcatggtcgacgccatcgtcaaccacatgagttgggaatccaagcagttccaggacgtgctggccaagggcgaggagtccgaatactatccgatgttcctcaccatgagctccgtgttcccgaacggcgccaccgaagaggacctggccggcatctaccgtccgcgtccgggcctgccgttcacccactacaagttcgccggcaagacccgcctcgtgtgggtcagcttcaccccgcagcaggtggacatcgacaccgattccgacaagggttgggaatacctcatgtcgattttcgaccagatggccgcctctcacgtcagctacatccgcctcgacgccgtcggctatggcgccaaggaagccggcaccagctgcttcatgaccccgaagaccttcaagctgatctcccgtctgcgtgaggaaggcgtcaagcgcggtctggaaatcctcatcgaagtgcactcctactacaagaagcaggtcgaaatcgcatccaaggtggaccgcgtctacgacttcgccctgcctccgctgctgctgcacgcgctgagcaccggccacgtcgagcccgtcgcccactggaccgacatacgcccgaacaacgccgtcaccgtgctcgatacgcacgacggcatcggcgtgatcgacatcggctccgaccagctcgaccgctcgctcaagggtctcgtgccggatgaggacgtggacaacctcgtcaacaccatccacgccaacacccacggcgaatcccaggcagccactggcgccgccgcatccaatctcgacctctaccaggtcaacagcacctactattcggcgctcgggtgcaacgaccagcactacatcgccgcccgcgcggtgcagttcttcctgccgggcgtgccgcaagtctactacgtcggcgcgctcgccggcaagaacgacatggagctgctgcgtaagacgaataacggccgcgacatcaatcgccattactactccaccgcggaaatcgacgagaacctcaagcgtccggtcgtcaaggccctgaacgcgctcgccaagttccgcaacgagctcgacgcgttcgacggcacgttctcgtacaccaccgatgacgacacgtccatcagcttcacctggcgcggcgaaaccagccaggccacgctgacgttcgagccgaagcgcggtctcggtgtggacaacactacgccggtcgccatgttggaatgggaggattccgcgggagaccaccgttcggatgatctgatcgccaatccgcctgtcgtcgcctga SEQ ID NO.2 Protein sequence of Bifidobacterium adolescentis (BASP) sucrose phosphatase MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEE DLAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYD FALPPLLLHALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVY YVGALAGKNDMELLRKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISFTWRGETSQATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVA* SEQ ID NO.3 PPGPK gene Gene sequence of PPGPK (glucosamine phosphokinase) from *Actinomyces ferruginea* atggcatctcggggacgggtcgggctggggattgacatcgggggaagcgggatcaaaggcgcccctgtggacttggaccggggaacgttcgtggtggaccgggtcaagatcgctactccgcagcccgcaacccctgaggcggtggctgcggtggtggcggagatagtcaccgcgttcgccgacgatgtgccgcaggatgcaccgttgggggtgacgtttcccgcggtgatccagcacggggtggcgcgcagcgccgccaacgtggaccgctcgtggatcggcaccaacgtcgaggagctgctgtctgcggtgacggggcggcgggtgctggtggtcaacgacgctgacgccgcagcgatggcggagcaccgctacggcgctgcctcaggcgtcgacggggtggtgctgttgactactttgggtaccggtattggtacggcggtgctagtggacggggtgctgctccccaacacggagttcgggcacttggagatcgacggctacgacgctgagacccgggcctctgctagcgctaaggagcgcgagaacctctcctacaaggagtgggctgaggagcggctgcagcgctactactcggtgatcgaggatttgctgtggccggacttgatcgtggtgggcggcggggtcagccgcaaggcggacaagtttttgccgcatctccgcttgcgcgcgccgatcgtgccggcgaagttgcgcaataccgcggggatcgtgggtgcggccgtgctggccgcggagcggctggggggtgaccgggtctctgcctag SEQ ID NO.4 Protein sequence of glucose phosphate kinase PPGPK from Actinopycnodysgenetes sp. MASRGRVGLGIDIGGSGIKGAPVDLDRGTFVVDRVKIATPQPATPEAVAAVVAEIVTAFADDVPQDAPLGVTFPAVIQHGVARSAANVDRSWIGTNVEELLSAVTGRRVLVVNDADAAAMAEHRYGAASGV DGVVLLTTLGTGIGTAVLVDGVLLPNTEFGHLEIDGYDAETRASASAKERENLSYKEWAEERLQRYYSVIEDLLWPDLIVVGGGVSRKADKFLPHLRLRAPIVPAKLRNTAGIVGAAVLAAERLGGDRVSA* SEQ ID NO.5 PGI gene Gene sequence of Escherichia coli glucose-6-phosphate isomerase PGI atgaaaaacatcaatccaacgcagaccgctgcctggcaggcactacagaaacacttcgatgaaatgaaagacgttacgatcgccgatctttttgctaaagacggcgatcgtttttctaagttctccgcaaccttcgacgatcagatgctggtggattactccaaaaaccgcatcactgaagagacgctggcgaaattacaggatctggcgaaagagtgcgatctggcgggcgcgattaagtcgatgttctctggcgagaagatcaaccgcactgaaaaccgcgccgtgctgcacgtagcgctgcgtaaccgtagcaataccccgattttggttgatggcaaagacgtaatgccggaagtcaacgcggtgctggagaagatgaaaaccttctcagaagcgattatttccggtgagtggaaaggttataccggcaaagcaatcactgacgtagtgaacatcgggatcggcggttctgacctcggcccatacatggtgaccgaagctctgcgtccgtacaaaaaccacctgaacatgcactttgtttctaacgtcgatgggactcacatcgcggaagtgctgaaaaaagtaaacccggaaaccacgctgttcttggtagcatctaaaaccttcaccactcaggaaactatgaccaacgcccatagcgcgcgtgactggttcctgaaagcggcaggtgatgaaaaacacgttgcaaaacactttgcggcgctttccaccaatgccaaagccgttggcgagtttggtattgatactgccaacatgttcgagttctgggactgggttggcggccgttactctttgtggtcagcgattggcctgtcgattgttctctccatcggctttgataacttcgttgaactgctttccggcgcacacgcgatggacaagcatttctccaccacgcctgccgagaaaaacctgcctgtactgctggcgctgattggcatctggtacaacaatttctttggtgcggaaactgaagcgattctgccgtatgaccagtatatgcaccgtttcgcggcgtacttccagcagggcaatatggagtccaacggtaagtatgttgaccgtaacggtaacgttgtggattaccagactggcccgattatctggggtgaaccaggcactaacggtcagcacgcgttctaccagctgatccaccagggaaccaaaatggtaccgtgcgatttcatcgctccggctatcacccataacccgctctctgatcatcaccagaaactgctgtctaacttcttcgcccagaccgaagcgctggcgtttggtaaatcccgcgaagtggttgagcaggaatatcgtgatcagggtaaagatccggcaacgcttgactacgtggtgccgttcaaagtattcgaaggtaaccgcccgaccaactccatcctgctgcgtgaaatcactccgttcagcctgggtgcgttgattgcgctgtatgagcacaaaatctttactcagggcgtgatcctgaacatcttcaccttcgaccagtggggcgtggaactgggtaaacagctggcgaaccgtattctgccagagctgaaagatgataaagaaatcagcagccacgatagctcgaccaatggtctgattaaccgctataaagcgtggcgcggttaa SEQ ID NO.6 Protein sequence of Escherichia coli glucose-6-phosphate isomerase PGI * SEQ ID NO.7 TKPGM gene Gene sequence of TKPGM, a glucose-phosphate mutase from Thermococcus Kodakini atgggaaaactattcggtacatttggggtacgaggtattgcgaacgaggagattaccccagagttcgcattgaagattggtatggcatttggtactctgttgaagcgcgagggtcgtgagcgtccgctggtcgtggtaggtcgtgatacgcgtgtttcgggcgagatgctgaaagacgcactaatcagcggcctgttgagcaccggttgtgacgttatcgacgtgggcatcgcaccgaccccagcaattcaatgggctaccaatcacttcaatgcggacggcggggccgttatcactgcgagccacaacccgcctgaatataacgggattaagctgctggaaccgaacggcatgggtttgaagaaagaacgtgaggctatcgtggaagagttatttttcagcgaagatttccatcgtgcgaagtggaatgaaattggtgaattgcgtaaagaggacattatcaaaccgtacattgaggcgatcaagaaccgcgttgacgtcgaggccatcaagaaacgccgtccgtttgttgtggttgataccagcaatggtgcgggttccctgaccctgccgtatctgctgcgtgagctgggctgcaaagttgttagcgttaatgcacacccggacggccattttccggcgcgtaatccggaaccgaacgaggagaacctgaagggttttatggaaattgtgaaagccctgggtgcggacttcggcgttgcccaggatggtgatgcagaccgtgctgtgtttatcgacgaaaacggtcgctttatccaaggtgataaaacctttgcgctcgtggctgacgcggttttgcgggagaacggcggaggcttgttggtgaccaccatcgccacctctaacctgcttgatgatattgccaagcgcaacggcgcgaaggtgatgcgtaccaaagtgggcgacctgattgtcgcgagagcgctgttagagaataacggaacaatcggtggcgaagagaacggtggtgtgatcttcccggatttcgtgcttggtcgcgatggcgctatgaccaccgctaagatcgtcgagatcttcgctaagtctggtaagaagttcagcgaactgattgatgaactcccaaaatactaccagtttaaaactaaacgccatgttgagggcgatcgtaaagcgatcgttgctaaggtggcggaactggcagaaaaaaaaggttataaaatcgacacgacggatggcaccaaaatcatcttcgacgacggttgggttctggtgcgtgcgtccggcacggaaccgattattcgtattttctcagaagcgaagtccgaagagaaggcacgcgagtacctggagctgggcatcaaactgcttgaagaagcgctgaaaggttaa SEQ ID NO.8 Protein sequence of TKPGM, a glucose phosphate mutase from Thermococcus Kodakini MGKLFGTFGVRGIANEEITPEFALKIGMAFGTLLKREGRERPLVVVGRDTRVSGEMLKDALISGLLSTGCDVIDVGIAPTPAIQWATNHFNADGGAVITASHNPPEYNGIKLLEPNGMGLKKEREAIVEELFFSEDFHRAKWNEIGELRKEDIIKPYIEAIKNRVDVEAIKKRRPFVVVDTSNGAGSLTLPYLLRELGCKVVSVNAHPDGHFPARNPEPNEENLKGFMEIVKALGADFGVAQDGDADRAVFIDENGRFIQGDKTFALVADAVLRENGGGLLVTTIATSNLLDDIAKRNGAKVMRTKVGDLIVARALLENNGTIGGEENGGVIFPDFVLGRDGAMTTAKIVEIFAKSGKKFSELIDELPKYYQFKTKRHVEGDRKAIVAKVAELAEKKGYKIDTTDGTKIIFDDGWVLVRASGTEPIIRIFSEAKSEEKAREYLELGIKLLEEALKG* SEQ ID NO.9 GALU gene Gene sequence of UTP-glucose-1-phosphate uridylyltransferase GALU from Escherichia coli () atggctgccattaatacgaaagtcaaaaaagccgttatccccgttgcgggattaggaaccaggatgttgccggcgacgaaagccatcccgaaagagatgctgccacttgtcgataagccattaattcaatacgtcgtgaatgaatgtattgcggctggcattactgaaattgtgctggttacacactcatctaaaaactctattgaaaaccactttgataccagttttgaactggaagcaatgctggaaaaacgtgtaaaacgtcaactgcttgatgaagtgcagtctatttgtccaccgcacgtgactattatgcaagttcgtcagggtctggcgaaaggcctgggacacgcggtattgtgtgctcacccggtagtgggtgatgaaccggtagctgttattttgcctgatgttattctggatgaatatgaatccgatttgtcacaggataacctggcagagatgatccgccgctttgatgaaacgggtcatagccagatcatggttgaaccggttgctgatgtgaccgcatatggcgttgtggattgcaaaggcgttgaattagcgccgggtgaaagcgtaccgatggttggtgtggtagaaaaaccgaaagcggatgttgcgccgtctaatctcgctattgtgggtcgttacgtacttagcgcggatatttggccgttgctggcaaaaacccctccgggagctggtgatgaaattcagctcaccgacgcaattgatatgctgatcgaaaaagaaacggtggaagcctatcatatgaaagggaagagccatgactgcggtaataaattaggttacatgcaggccttcgttgaatacggtattcgtcataacacccttggcacggaatttaaagcctggcttgaagaagagatgggcattaagaagtaa SEQ ID NO.10 Protein sequence of Escherichia coli () UTP-glucose-1-phosphate uridylyltransferase GALU MAAINTKVKKAVIPVAGLGTRMLPATKAIPKEMLPLVDKPLIQYVVNECIAAGITEIVLVTHSSKNSIENHFDTSFELEAMLEKRVKRQLLDEVQSICPPHVTIMQVRQGLAKGLGHAVLCAHPVVGDEPVAVILPDVILDEYESDLSQDN LAEMIRRFDETGHSQIMVEPVADVTAYGVVDCKGVELAPGESVPMVGVVEKPKADVAPSNLAIVGRYVLSADIWPLLAKTPPGAGDEIQLTDAIDMLIEKETVEAYHMKGKSHDCGNKLGYMQAFVEYGIRHNTLGTEFKAWLEEEMGIKK* SEQ ID NO. 11 BSYJIC gene Gene sequence of Bacillus subtilis glycosyltransferase BSYJIC atgaaaaagtatcacatatcaatgattaatattccggcgtacggccatgttaatccgaccctggcgctggtggaaaagctgtgcgaaaagggtcaccgtgttacctatgcgactacggaagagttcgcaccggccgtccagcaggctggcggcgaggcgttgatctaccacaccagcttgaatatcgacccgaagcagatccgtgagatgatggagaagaacgatgcgccactgtcactgttaaaagagtccttgtcgatcctgccgcaactggaggaattatataaggacgaccagccggatctgatcatttacgattttgttgcacttgcgggtaagctattcgccgagaagctgaacgtgccggtgatcaaattatgcagctcgtacgcgcagaatgaatctttccagctgggaaacgaagacatgttgaagaagatcagagaagcggaggctgaatttaaagcgtacctggagcaggaaaaactgccggccgtgagctttgaacagctggcagtgccggaggcgctgaacattgtttttatgccgaagagcttccaaattcaacacgaaacctttgacgaccgcttttgctttgtgggtccgagcctgggtgagcgtaaagagaaggagtccctgttgattgataaagacgaccgtccgctgatgctgatctccctgggcacggccttcaacgcatggcccgagttctataaaatgtgtattaaagctttccgtgatagcagctggcaggtcattatgtctgttggtaaaaccattgatccggaatccctcgaggacatcccggcgaactttaccatccgccagagcgtgccgcaactcgaggttttggagaaggcggatctgttcataagccatggtggtatgaacagcaccatggaagctatgaatgctggcgttcctctggttgttatcccgcaaatgtacgaacaggagctgaccgcgaatcgcgtcgatgagctgggtctgggcgtatatctgccaaaagaggaagtgacggtgagctccttgcaagaggcggttcaagcggtcagctctgatcaagaattgttgtctcgtgtgaaaaacatgcagaaagacgttaaagaagcgggtggcgcagagcgcgcagctgccgaaattgaagctttcatgaagaagagcgcagtgccgcaataa SEQ ID NO. 12 Protein sequence of Bacillus subtilis glycosyltransferase BSYJIC MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDD QPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDR FCFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ* SEQ ID NO. 13 PPK2 gene Gene sequence of polyphosphokinase PPK2 from *Radiata spp.* atggacatagataattatagggtaaaacccggaaagcgcgtgaagctgagcgattgggcaaccaacgacgacgctggcctgtccaaggaggagggccaggcgcagaccgcgaagctggccggtgaactggcggagtggcaagaacgtctctatgcggagggcaagcaaagcctgttgcttatcctccaagcccgtgacgccgcaggcaaggacggtgcagttaagaaggttatcggcgcgtttaacccggcgggcgttcagattaccagctttaaacaaccgtctgctgaggaattgtctcacgatttcctgtggcgtatccaccagaaagcaccggcgaaaggttatgtgggtgtgttcaatcgtagccagtatgaagatgtactggtgacccgtgtttacgacatgattgatgataagaccgcgaaaagacgtttggaacatattcgtcattttgaagagctgttgactgataatgcaacgcgtattgttaaggtgtatctgcacatctcgccggaagagcagaaagaacgcctgcaggcgcgcctggataaccctggtaaacattggaaattcaacccaggtgatctgaaagaccgcagtaattgggataaattcaacgacgtttacgaggacgccctgacgacctccaccgacgacgctccgtggtacgtggtgccggcggatcgtaaatggtaccgcgatttggttctgagccacatcctgttaggtgctctgaaggacatgaacccgcaatttccggcgattgactacgatccgagcaaagtcgtcatccactaa SEQ ID NO. 14 Protein sequence of polyphosphate kinase PPK2 of Deinococcus radiodurans () MDIDNYRVKPGKRVKLSDWATNDDAGLSKEEGQAQTAKLAGELAEWQERLYAEGKQSLLLILQARDAAGKDGAVKKVIGAFNPAGVQITSFKQPSAEELSHDFLWRIHQKAPAKGYVGVFNRSQYEDVLVTRV YDMIDDKTAKRRLEHIRHFEELLTDNATRIVKVYLHISPEEQKERLQARLDNPGKHWKFNPGDLKDRSNWDKFNDVYEDALTTSTDDAPWYVVPADRKWYRDLVLSHILLGALKDMNPQFPAIDYDPSKVVIH* SEQ ID NO. 15 IPPASE gene Gene sequence of methanococcus japonicus (IPPASE) pyrophosphatase atgcgttacgtcgtaggtcacaagaatccggacacagatagcatcgcgtccgccatcgtgctggcttatttcttggactgctatccggctcgtctcggtgacatcaacccggagacggagttcgtactgcgtaaattcggggtaatggagccagagctgatcgaatccgcaaaaggtaaagaaatcatcctggttgatcacagcgaaaagagccagtctttcgacgatctggaagaaggcaaactgattgctatcatcgaccaccacaaggttggcctgaccaccacggagcctatcctgtactatgctaagccagtgggttctaccgcaactgtaatcgcagaactgtacttcaaagacgccatcgatctgattggcggtaagaaaaaagaactgaaaccggacctggccggtctgctcctgtctgcgatcatcagcgacacagtgctgttcaaatccccaactaccaccgatctggacaaggaaatggcgaaaaaacttgcggagatcgctggtatcagcaacatcgaagagtttggtatggaaattctgaaagcgaaatctgtagtgggtaaactgaaacctgaagaaattattaacatggattttaaaaacttcgatttcaacggcaaaaaagttggcattggccaggtagaagtgatcgatgtgtccgaagtggaatctaaaaaagaagacatctataaacttctggaagaaaaactgaagaatgagggttatgatctgattgtgttcctgatcactgacatcatgaaagaaggttccgaagctctggttgttggtaacaaagaaatgtttgaaaaagcatttaatgtaaaagtggaaggtaacagcgtcttccttgaaggcgtgatgtctcgcaaaaagcaagtcgttcctccgctggagcgtgcgtacaacggctaa SEQ ID NO.16 Protein sequence of Pyrophosphatase IPPASE from Methanococcus jannaschii () MRYVVGHKNPDTDSIASAIVLAYFLDCYPARLGDINPETEFVLRKFGVMEPELIESAKGKEIILVDHSEKSQSFDDLEEGKLIAIIDHHKVGLTTTEPILYYAKPVGSTATVIAELYFKDAIDLIGGKKKELKPDLAGLLLSAIISDTVLFKSPTTTDLDKEMAKKLAEIAGISNIEEFGMEILKAKSVVGKLKPEEIINMDFKNFDFNGKKVGIGQVEVIDVSEVESKKEDIYKLLEEKLKNEGYDLIVFLITDIMKEGSEALVVGNKEMFEKAFNVKVEGNSVFLEGVMSRKKQVVPPLERAYNG* SEQ ID NO. 17 NDK gene Gene sequence of nucleoside diphosphate kinase NDK of Escherichia coli () atggctattgaacgtactttttccatcatcaaaccgaacgcggtagcaaaaaacgtcattggtaatatctttgcgcgctttgaagctgcagggttcaaaattgttggcaccaaaatgctgcacctgaccgttgaacaggcacgtggcttttatgctgaacacgatggaaaaccgttctttgatggtctggttgaattcatgacctctggcccgatcgtggtttccgtgctggaaggtgaaaacgccgttcagcgtcaccgcgatctgctgggcgcgaccaatccggcaaacgcactggctggtactctgcgcgctgattacgctgacagcctgaccgaaaacggtacccacggttctgattccgtcgaatctgccgctcgcgaaatcgcttatttctttggcgaaggcgaagtgtgcccgcgcacccgttaa SEQ ID NO. 18 Protein sequence of nucleoside diphosphate kinase NDK of Escherichia coli () MAIERTFSIIKPNAVAKNVIGNIFARFEAAGFKIVGTKMLHLTVEQARGFYAEHDGKPFFDGLVEFMTSGPIVVSVLEGENAVQRHRDLLGATNPANALAGTLRADYADSLTENGTHGSDSVESAAREIAYFFGEGEVCPRTR* SEQ ID NO. 19 RBS RBS sequence caggaggaattaacc Table 2 Primer Sequences

[0103] Primer or sequence name Nucleotide sequence (5' to 3') pBAD-R GGTTAATTCCTCCTGTTAGCCCAAAAAACG pBAD-F TGCCTGGCGGCAGTAGCGCGGTGGTCCCAC araF CTGACGCTTTTTATCGCAACTCTCT B1-F GCTAACAGGAGGAATTAACCATGAAAAACAAGGTGCAGCTCATCACTTACG B1-R CGCGCTACTGCCGCCAGGCATTAACCTTTCAGCGCTTCTTCAAGCAGTTT B2-F GCTAACAGGAGGAATTAACCATGGCTGCCATTAATACGAAAGTCAAAAAAG B2-R CGCGCTACTGCCGCCAGGCATTATTGCGGCACTGCGCCTCTTCTTCATGAA B3-F GCTAACAGGAGGAATTAACCATGGACATAGATAATTATAGGGTAAAACCCG B3-R CGCGCTACTGCCGCCAGGCATTAACGGGTGCGCGGGCACACTTCGCCTTC B4-R CGCGCTACTGCCGCCAGGCATCAGGCGACGACAGGCGGATTGGCGATCAG Example 1: Construction of plasmids and recombinant bacteria

[0104] (1) Construction and preparation of pBAD-BASP-TFPPGK-PGI-TKPGM plasmid

[0105] The pBAD fragment of the plasmid backbone was prepared by PCR amplification using pBAD / His-B plasmid as a template and primers pBAD-F / pBAD-R.

[0106] The BASP-TFPPGK-PGI-TKPGM fragment was synthesized by Qingke Biotechnology and contains the fragments of the BASP gene, RBS, TFPPGK gene, RBS, PGI gene, RBS, and TKPGM gene in sequence. Using this fragment as a template, PCR amplification was performed using primers B1-F / B1-R.

[0107] All the above PCRs were performed using high-fidelity TransStart FastPfu DNA polymerase (Beijing TransGen Biotech Co., Ltd., product catalog AP221), and the target fragments were recovered by agarose gel electrophoresis.

[0108] The BASP-TFPPGK-PGI-TKPGM fragment and the pBAD fragment were ligated using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to selpxMeral hundred kilobases. Nat. methods. 2009; 6(5):343-345) to obtain the recombinant expression vector pBAD-BASP-TFPPGK-PGI-TKPGM. E. coli DH5α competent cells (purchased from Beijing TransGen Biotech Co., Ltd., product catalog: CD201) were transformed using the CaCl2 method. The cells were evenly spread on LB agar plates containing streptomycin and incubated overnight at 37°C. Clones were selected, and those capable of amplifying the target fragment were identified using primers ara-F / B1-R and sequenced. Positive clones were selected, and plasmids were extracted. The positive plasmid was named pBAD-BASP-TFPPGK-PGI-TKPGM.

[0109] (2) Construction and preparation of plasmids pBAD-GalU-BsYjiC, pBAD-PPK2-IPPase-NDK, and pBAD-BASP

[0110] The plasmids pBAD-GalU-BsYjiC, pBAD-PPK2-IPPase-NDK, and pBAD-BASP were constructed and prepared using the same methods and primers as pBAD-BASP-TFPPGK-PGI-TKPGM in step (1). The only differences were the template fragment used for PC amplification, the primers used for the amplification fragment, and the identification primers. Specific information is shown in Table 3.

[0111] Table 3. Plasmid Construction Information

[0112] Serial Number plasmid Excerpt Fragment description Fragment amplification primers Identification primers 1 pBAD-BASP-TFPPGK-PGI-TKPGM BASP-TFPPGK-PGI-TKPGM BASP gene, RBS, TFPPGK gene, RBS, PGI gene, RBS, TKPGM gene B1-F / B1-R ara-F / B1-R 2 pBAD-GalU-BsYjiC GalU- BsYjiC GALU gene, RBS, BSYJIC gene B2-F / B2-R ara-F / B2-R 3 pBAD-PPK2-IPPase-NDK PPK2-IPPase-NDK PPK2 gene, RBS, IPPASE gene, RBS, NDK gene B3-F / B3-R ara-F / B3-R 4 pBAD-BASP BASP BASP gene B1-F / B4-R ara-F / B4-R

[0113] (3) Construction of recombinant strain I, recombinant strain II, recombinant strain III and recombinant strain IV

[0114] The expression vector pBAD-BASP-TFPPGK-PGI-TKPGM constructed in Example 1 (1) was transformed into Escherichia coli MC02 by chemical transformation. Positive clones were screened on LB plates containing ampicillin (50 μg / mL) to obtain the corresponding recombinant engineered strain, which was named recombinant strain I.

[0115] In Example 1 (2), the expression vector pBAD-GalU-BsYjiC was transformed into Escherichia coli MC02 by chemical transformation. Positive clones were screened on LB plates containing ampicillin (50 μg / mL) to obtain the corresponding recombinant engineered strain, which was named recombinant strain Ⅱ.

[0116] In Example 1 (2), the expression vector pBAD-PPK2-IPPase-NDK constructed was transformed into Escherichia coli MC02 by chemical transformation. Positive clones were screened on LB plates containing ampicillin (50 μg / mL) to obtain the corresponding recombinant engineered strain, which was named recombinant strain III.

[0117] In Example 1 (2), the expression vector pBAD-BASP was transformed into Escherichia coli MC02 by chemical transformation. Positive clones were screened on LB plates containing ampicillin (50 μg / mL) to obtain the corresponding recombinant engineered strain, which was named recombinant strain IV. Example 2 Protein expression and preparation of crude enzyme solution

[0118] The test strains were recombinant strain I, recombinant strain II, recombinant strain III, and recombinant strain IV.

[0119] 1. Take a single clone of the test strain and inoculate it into 2YT liquid medium containing 50 μg / mL ampicillin. Incubate at 37℃ and 220 rpm with shaking until OD600nm=0.6-0.8.

[0120] 2. After completing step 1, add arabinose (0.2g / 100mL) to the culture system and incubate at 25 ℃ and 220 rpm for 12 h with shaking.

[0121] 3. After completing step 2, take samples and determine the cell concentration of each recombinant bacterium. Then, take the entire culture system, centrifuge at 4 ℃ and 4500 rpm for 10 min, and collect the cell pellet.

[0122] 4. After completing step 3, dilute the collected cell pellet to 160 OD using phosphate buffer (pH=7.0) according to the cell concentration of each recombinant bacterial culture system.

[0123] 5. After completing step 4, use an ultrasonic disruptor to disrupt all the diluted recombinant bacterial solutions for 20 minutes according to the program of 30W power, 15℃ alarm temperature, 2 seconds off, 3 seconds on, until the cells are completely disrupted and crude enzyme solution is obtained.

[0124] The crude enzyme solution obtained from recombinant strain I is named crude enzyme solution I.

[0125] The crude enzyme solution obtained from recombinant strain II is named crude enzyme solution II.

[0126] The crude enzyme solution obtained from recombinant bacteria III is named crude enzyme solution III.

[0127] The crude enzyme solution obtained from recombinant bacteria IV is named crude enzyme solution IV. Example 3: Preparation of Rhodioloside using Crude Enzyme Solution

[0128] The crude enzyme solutions used in the experiments were crude enzyme solution I, crude enzyme solution II, crude enzyme solution III, and crude enzyme solution IV, prepared in step 5 of Example 2. The substrate was sucrose, and the glycosyl acceptor was tyrosol. Tyrosol and UDPG reacted with glycosyltransferases to generate rhodioloside.

[0129] Three experimental groups were set up. Experimental group 1-1 was supplemented with crude enzyme solution I, crude enzyme solution II, and crude enzyme solution III; experimental group 1-2 was supplemented with crude enzyme solution I and crude enzyme solution II; and experimental group 1-3 was supplemented with crude enzyme solution II, crude enzyme solution III, and crude enzyme solution IV. All other components were the same for all three experimental groups. The initial system was prepared by mixing the experimental crude enzyme solutions, sucrose, tyrosol, and phosphate buffer (pH=7.0). The initial system had an OD600nm value of 20. The initial sucrose concentration was 500 mM, and the tyrosol concentration was 50 mM. The initial system was reacted at 45 ℃ for 24 h (pH was monitored in real time during the reaction, and 2 M NaOH aqueous solution was used to control the pH at 7.0-7.5). The reaction was terminated after completion. The concentrations of sucrose, fructose, and rhodioloside in the system were measured.

[0130] Three replicate experiments were conducted, with three parallel experiments set up for each experimental group in each replicate.

[0131] The methods for determining sucrose, fructose, and rhodioloside concentrations are as follows (HPLC): The stop solution was centrifuged at 12000 rpm for 10 min, the supernatant was collected, diluted with sterile water to obtain a diluted solution, and then filtered through a 0.45 μm pore size filter membrane. The filtrate was used as the sample loading solution. The results are shown in Table 4.

[0132] The results showed that the rhodioloside yield in experimental group 1-1 was significantly higher than that in experimental groups 1-2 and 1-3, while the residual fructose content was significantly lower. This indicates that the reaction system provided by this invention, containing the complete enzyme composition (recombinant bacterial lysate), can achieve highly efficient catalysis of sucrose substrate to synthesize rhodioloside. Example 4: Preparation of salicin using crude enzyme solution

[0133] The crude enzyme solutions used in the experiments were crude enzyme solution I, crude enzyme solution II, crude enzyme solution III, and crude enzyme solution IV, prepared in step 5 of Example 2. The substrate was sucrose, and the glycosyl acceptor was salicylol. Salicylol and UDPG reacted with glycosyltransferase to form salicin.

[0134] Three experimental groups were set up. Experimental group 2-1 was supplemented with crude enzyme solution I, crude enzyme solution II, and crude enzyme solution III; experimental group 2-2 was supplemented with crude enzyme solution I and crude enzyme solution II; and experimental group 2-3 was supplemented with crude enzyme solution II, crude enzyme solution III, and crude enzyme solution IV. All other components were the same for the three experimental groups. The initial system was prepared by mixing the experimental crude enzyme solution, sucrose, salicylol, and phosphate buffer (pH=7.0). The OD600nm value of the initial system was 20. The initial system had a sucrose concentration of 500 mM and a tyrosol concentration of 50 mM. The initial system was reacted at 45 ℃ for 24 h (the pH was monitored in real time during the reaction, and the pH was controlled at 7.0-7.5 using 2 M NaOH aqueous solution). The reaction was terminated after completion. The concentrations of sucrose, fructose, and salicylol in the system were measured.

[0135] Three replicate experiments were conducted, with three parallel experiments set up for each experimental group in each replicate.

[0136] The methods for determining sucrose, fructose, and salicin concentrations are as follows (HPLC): The stop solution was centrifuged at 12000 rpm for 10 min, the supernatant was collected, diluted with sterile water to obtain a diluted solution, and then filtered through a 0.45 μm pore size filter membrane. The filtrate was used as the sample loading solution. The results are shown in Table 4.

[0137] The results showed that the salicin yield in experimental group 2-1 was significantly higher than that in experimental groups 2-2 and 2-3, while the residual fructose content was significantly lower. This indicates that the reaction system provided by this invention, containing the complete enzyme composition (recombinant bacterial lysate), can achieve highly efficient catalysis of salicin synthesis from sucrose substrates.

[0138] Table 4 Experimental Results

[0139]

Claims

1. An enzyme composition for producing UDP-glucose and its derivatives from sucrose, comprising enzyme composition I, enzyme composition II, and enzyme composition III, wherein, Enzyme composition I comprises a protein or polypeptide having the following functions: A) sucrose phosphatase, B) glucose phosphokinase, C) glucose-6-phosphate isomerase, D) glucose phosphomutase; enzyme composition II comprises a protein or polypeptide having the following functions: E) UTP glucose-1-phosphate uridine transferase, F) glycosyltransferase; and enzyme composition III comprises a protein or polypeptide having the following functions: G) polyphosphokinase, H) pyrophosphatase, I) nucleoside diphosphate kinase.

2. The enzyme composition according to claim 1, wherein the enzyme composition comprises one of the following characteristics: A) sucrose phosphatase is derived from *Bifidobacterium adolescentis*, B) glucose phosphokinase is derived from *Thermobifida fusca*, C) glucose-6-phosphate isomerase is derived from *Escherichia coli*, D) glucose phosphomutase is derived from *Thermococcus kodakarensis*, E) UTP glucose-1-phosphate uridine transferase is derived from *Escherichia coli*, F) glycosyltransferase is derived from *Bacillus subtilis*, G) polyphosphokinase is derived from *Deinococcus radiodurans*, H) pyrophosphatase is derived from *Methanococcus jannaschii*, and I) nucleoside diphosphate kinase is derived from *Escherichia coli*.

3. The enzyme composition according to claims 1 to 2, wherein the enzyme composition comprises one of the following characteristics: A) the amino acid sequence of sucrose phosphatase comprises the amino acid sequence shown in SEQ ID NO:2; B) the amino acid sequence of glucose phosphokinase comprises the amino acid sequence shown in SEQ ID NO:4; C) the amino acid sequence of glucose-6-phosphate isomerase comprises the amino acid sequence shown in SEQ ID NO:6; D) the amino acid sequence of glucose phosphomutase comprises the amino acid sequence shown in SEQ ID NO:8; E) the amino acid sequence of UTP glucose-1-phosphate uridine transferase comprises the amino acid sequence shown in SEQ ID NO:10; F) the amino acid sequence of glycosyltransferase comprises the amino acid sequence shown in SEQ ID NO:12; G) the amino acid sequence of polyphosphokinase comprises the amino acid sequence shown in SEQ ID NO:14; H) the amino acid sequence of pyrophosphatase comprises the amino acid sequence shown in SEQ ID NO:16; I) the amino acid sequence of nucleoside diphosphate kinase comprises the amino acid sequence shown in SEQ ID NO:

18.

4. A method for synthesizing rhodioloside, comprising: Using sucrose and tyrosol as substrates, water, an enzyme composition as described in any one of claims 1 to 3, or at least one enzyme in the enzyme composition are added to the mixture for reaction. The supernatant collected by centrifugation after the reaction is completed is the rhodioloside solution.

5. A method for synthesizing salicin, comprising: Using sucrose and salicylol as substrates, water, an enzyme composition as described in any one of claims 1 to 3, or at least one enzyme in the enzyme composition are added to react. The supernatant collected by centrifugation after the reaction is completed is the salicylol solution.

6. A group of recombinant engineered bacteria for the production of UDP-glucose and its derivatives from sucrose, including: I) Recombinant Escherichia coli I, containing an introduced or enhanced gene encoding A) sucrose phosphatase, B) glucose phosphokinase, C) glucose-6-phosphate isomerase, D) glucose phosphomutase; II) Recombinant Escherichia coli II, containing an introduced or enhanced gene encoding E) UTP glucose-1-phosphate uridine transferase, F) glycosyltransferase; III) Recombinant Escherichia coli III, containing introduced or enhanced expression of genes encoding G) polyphosphokinase, H) pyrophosphatase and I) nucleoside diphosphate kinase.

7. The group of recombinant engineered bacteria according to claim 6, wherein the recombinant engineered bacteria comprises one of the following characteristics: A) sucrose phosphatase is derived from Bifidobacterium adolescentis, B) glucose phosphokinase is derived from Thermobifida fusca, C) glucose-6-phosphate isomerase is derived from Escherichia coli, D) glucose phosphomutase is derived from Thermococcus kodakarensis, E) UTP glucose-1-phosphate uridine transferase is derived from Escherichia coli, F) glycosyltransferase is derived from Bacillus subtilis, G) polyphosphokinase is derived from Deinococcus radiodurans, H) pyrophosphatase is derived from Methanococcus jannaschii, and I) nucleoside diphosphate kinase is derived from Escherichia coli.

8. The group of recombinant engineered bacteria according to claims 6 to 7, wherein the recombinant engineered bacteria comprises one of the following characteristics: A) the amino acid sequence of sucrose phosphatase comprises the amino acid sequence shown in SEQ ID NO:2; B) the amino acid sequence of glucose phosphokinase comprises the amino acid sequence shown in SEQ ID NO:4; C) the amino acid sequence of glucose-6-phosphate isomerase comprises the amino acid sequence shown in SEQ ID NO:6; D) the amino acid sequence of glucose phosphomutase comprises the amino acid sequence shown in SEQ ID NO:8; E) the amino acid sequence of UTP glucose-1-phosphate uridine transferase comprises the amino acid sequence shown in SEQ ID NO:10; F) the amino acid sequence of glycosyltransferase comprises the amino acid sequence shown in SEQ ID NO:12; G) the amino acid sequence of polyphosphokinase comprises the amino acid sequence shown in SEQ ID NO:14; H) the amino acid sequence of pyrophosphatase comprises the amino acid sequence shown in SEQ ID NO:16; I) the amino acid sequence of nucleoside diphosphate kinase comprises the amino acid sequence shown in SEQ ID NO:

18.

9. A method for synthesizing rhodioloside, comprising: Using sucrose and tyrosol as substrates, water and recombinant engineered bacterial cells or cell fragments as described in any one of claims 6 to 8 are added to the reaction. The supernatant collected by centrifugation after the reaction is completed is the rhodioloside solution.

10. A method for synthesizing salicin, comprising: Using sucrose and salicylol as substrates, water and recombinant engineered bacterial cells or cell fragments as described in any one of claims 6 to 8 are added to the reaction. The supernatant collected by centrifugation after the reaction is completed is the salicylol solution.

11. The application of the enzyme composition or recombinant engineered bacteria according to any one of claims 1 to 10 in the production of UDP-glucose, UDP-glucose derivatives, rhodioloside, and salicin.

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