# Patent

## Title
Method for producing stevioside compounds by microorganism

## Abstract

The present invention provides a method for producing stevioside compounds by microorganism, comprising carrying out heterologous biosynthesis of stevioside compounds from geranyl geranyl pyrophosphate synthase (GGPPS), Copalyl pyrophosphate synthase (CDPS), Kaurene synthase (KS), dual-function kaurene synthase (CPS/KS), kaurene oxidase (KO), a cytochrome P450 redox protein (CPR), kaurenoic acid-13[alpha]-hydroxylase, UGT85C2 glycosyltransferase and UGTB1/IBGT glycosyltransferase (optionally comprising UGT74G1 glycosyltransferase and/or UGT76G1 glycosyltransferase).

## Background
SEQUENCE LISTING INFORMATION
A computer readable textfile, entitled “1 JV3897.txt (Sequence Listing.txt)” created on or about Aug. 26, 2016, with a file size of about 186 KB, contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
This Application claims priority to and is a national phase of PCT/CN2013/084618, filed 29 Sep. 2013, entitled “METHOD FOR PRODUCING STEVIOSIDE COMPOUNDS BY MICROORGANISM,” which claims priority to Chinese Patent Application No. 201210378341.3, filed 29 Sep. 2012, which are incorporated herein by reference.
TECHNICAL FIELD
The present invention belongs to the field of synthetic biology. Specifically, the present invention relates to a method for production of steviol glycosides by microorganisms.
BACKGROUND ART
Rebaudioside A (Reb A) is a new natural sweetener extracted from Stevia rebaudiana with intense sweetness, low calorific content and good stability, etc. Compared to the other main components of steviol glycosides, rebaudioside A (RebA) has the highest sweetness. The sweetness of rebaudioside A is at least 450 folds stronger than sucrose, but its calorific content is 300 folds less than sucrose. Besides, rebaudioside A has intense sweetness, white color, pure sweet taste and no peculiar smell. Therefore, it is the best nature alternative for sucrose and the chemically synthetic sweeteners, and it is called as “the third-generation sugar source” in the world.
FIG. 1 shows the structures of a portion of steviol glycosides extracted from Stevia rebaudiana, which has different side chain modifications due to their different R1 and R2 (see Table 1). Stevia sugar will have purer sweet taste if it contains more rebaudioside A and thus it will be accepted by more consumers. Therefore, the content of rebaudioside A in the product must be enhanced during production of stevia sugar.







TABLE 1







Steviol glycosides extracted from Stevia rebaudiana










No.
Compound
R1
R2













1
Steviol
H
H


2
Steviolmonoside
H
β-Glc


3
Steviolbioside
H
β-Glc-β-Glc(2→1)


4
Rubusoside
β-Glc
β-Glc


5
Stevioside
β-Glc
β-Glc-β-Glc(2→1)


6
Rebaudioside A
β-Glc
β-Glc-β-Glc(2→1)





β-Glc (3→1)


7
Rebaudioside B
H
β-Glc-β-Glc(2→1)





β-Glc (3→1)


8
Rebaudioside C
β-Glc
β-Glc-α-Rha(2→1)





β-Glc(3→1)


9
Rebaudioside D
β-Glc-β-Glc(2→1)
β-Glc-β-Glc(2→1)





β-Glc (3→1)


10
Rebaudioside E
β-Glc-β-Glc(2→1)
β-Glc-β-Glc(2→1)


11
Rebaudioside F
β-Glc
β-Glc-α-Xly(2→1)





β-Glc(3→1)


12
Dulcoside A
β-Glc
β-Glc-α-Rha(2→1)









Currently, steviol glycosides have been widely used in food, beverage, medicine and cosmetic industries. The recent studies show that steviol glycosides can be used to prevent hypertension, diabetes and heart disease, etc. Therefore, there is a rapid need for steviol glycosides in the recent years.
Commercially available rebaudioside A currently is mainly extracted from the leaves of Stevia rebaudiana. The preparation process mainly includes the steps of drying and grinding the leaves of Stevia rebaudiana, extracting in a liquid phase, removing impurities, treating by resin, drying by spray and refining, etc. Generally, the leaves of Stevia rebaudiana may contain up to 4% to 20% of stevia sugar calculated by dried weight. However, there are many problems, including a great quantity of lands required for planting Stevia rebaudiana, different quantity of Stevia rebaudiana used for production of stevia sugar, low conversion efficiency of the raw material and low purity of the extracted product. Therefore, it is necessary to develop a production method for safely producing rebaudioside A in a large scale, with the raw material being readily obtained and the extraction method being simple.
As the development of techniques in synthetic biology during the last ten years, it is possible to produce various heterologous compounds by microorganisms. This synthesis has advantages of low cost, small production area and easy control of product quantity. However, synthesis of rebaudioside A by heterologous organisms is not reported to date. The key technical difficulty is that the glycosyltransferase used for converting steviolmonoside to steviolbioside was not known. Therefore, there is an urgent need to overcome the existing technical difficulty to achieve microbial synthesis of rebaudioside A.
CONTENTS OF INVENTION
The present invention aims to provide a method for producing steviol glycosides by microorganism.
In the first aspect of the present invention, an isolated polypeptide is provided, which is a glycosyltransferase obtained from the non-Stevia rebaudiana source and is used to catalyze transfer of an additional glucose to the C-2′ of the O-glucosyl residue of steviol glycosides.
Preferably, the amino acid sequence of the polypeptide has a sequence identity no higher than 95%, preferably no higher than 80%, preferably, no higher than 70%, preferably no higher than 60%, preferably no higher than 50%, preferably no higher than 40%, preferably no higher than 30%, to the amino acid sequence of the enzyme having the same function and from Stevia rebaudiana. 
In a preferred embodiment, the glycosyltransferase from the non-Stevia rebaudiana source is from Starmerella bombicola or Ipomoea batatas. 
In another preferred embodiment, the glycosyltransferase from Starmerella bombicola has an amino acid sequence as set forth in SEQ ID NO: 41 (called as “UGTB1”) or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more amino acid residues in SEQ ID NO: 41.
In another preferred embodiment, the glycosyltransferase from Ipomoea batatas has an amino acid sequence as set forth in SEQ ID NO: 51 (called as “IBGT”) or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more amino acid residues in SEQ ID NO: 51.
In another aspect of the present invention, an isolated nucleotide sequence is provided, which encodes the glycosyltransferase from the non-Stevia rebaudiana source, and is used to catalyze transfer of an additional glycosyl unit to the C-2′ of the O-glucosyl residue of steviol glycosides.
In a preferred embodiment, the nucleotide sequence has (1) a sequence shown in SEQ ID NO: 42, or a sequence having 70% or more, preferably 80% or more, preferably 90% or more, more preferably 95% or more, homology to SEQ ID NO: 42; (2) a sequence shown in SEQ ID NO: 52, or a sequence having 70% or more, preferably 80% or more, preferably 90% or more, more preferably 95% or more, homology to SEQ ID NO: 52.
In another preferred embodiment, the nucleotide sequence is (1) the sequence as set forth in SEQ ID NO: 42 or (2) the sequence as set forth in SEQ ID NO: 52.
In another aspect of the invention, use of the glycosyltransferase from the non-Stevia rebaudiana source, which could catalyze the transfer of an additional glucosyl unit to the C-2′ of the O-glucosyl residue of steviol glycosides, for recombination expression in a host cell to prepare a steviol glycoside is provided.
In a preferred embodiment, the catalytic substrate of the glycosyltransferase includes, but is not limited to, steviol-13-O-glucoside (also called as steviolmonoside), rubusoside, Stevioside, and rebaudioside A. Preferably, steviolmonoside is catalyzed to produce steviolbioside.
In another aspect of the present invention, a method for synthesizing steviol glycosides is provided, which includes the step of recombinantly expressing the glycosyltransferase from the non-Stevia rebaudiana source in a host cell, which is capable of catalyzing transfer of an additional glucose to the C-2′ of the O-glucose residue of the steviol glycosides.
In a preferred embodiment, the host cell further comprises one or more of:
(a) Geranylgeranyl diphosphate synthase;
(b) An enzyme selected from (I) ent-copalyl diphosphate synthase and ent-kaurene synthase and (II) bifunctional ent-kaurene synthase;
(c) Ent-kaurene oxidase;
(d) Cytochrome P450 redox protein;
(e) Kaurenoic acid-13α-hydroxylase;
(f) UGT85C2 glycosyltransferase;
(h) UGT74G1 glycosyltransferase; and
(i) UGT76G1 glycosyltransferase.
In another preferred embodiment, the host cell further contains gene expression cassette(s) of the following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and isopentenyl-diphosphate delta-isomerase.
In another preferred embodiment, the host cell is a cell from prokaryotic microorganisms and eukaryotic microorganisms.
In another preferred embodiment, the prokaryotic microorganism is Escherichia coli, Bacillus subtilis, Acetobacteria, Corynebacterium or Brevibacterium. Preferably, the E. coli is selected from the group consisting of BL21, BLR, DH10B, HMS, C43, JM109, DH5a or Noveblue. The eukaryotic microorganism is yeast, mold, or basidiomycete. Yeast may be Pichia pastoris, Saccharomyces cerevisiae, or Kluyveromyces lactis. Preferably, Pichia pastoris may be selected from GS115, MC100-3, SMD1163, SMD1165, SMD1168 or KM71. Preferably, Saccharomyces cerevisiae may be selected from W303, CEN.PK2, S288c, FY834 or S1949. Preferably, Kluyveromyces lactis may be GG799.
In another aspect of the present invention, a method for synthesizing a steviol glycoside, comprising recombinantly, preferably heterologously, expressing in a cell the following enzymes:
(a) Geranylgeranyl diphosphate synthase (GGPPS);
(b) An enzyme selected from (I) or (II) as follows: (I) ent-copalyl diphosphate synthase (CDPS) and ent-kaurene synthase (KS) and (II) bifunctional ent-kaurene synthase (CPS/KS);
(c) Ent-kaurene oxidase (KO);
(d) Cytochrome P450 redox protein (CPR);
(e) Kaurenoic acid-13α-hydroxylase;
(f) UGT85C2 glycosyltransferase; and
(g) UGTB1/IBGT glycosyltransferase;
and culturing the cell to produce the steviol glycoside.
In a preferred embodiment, the method further contains recombinantly expressing:
(h) UGT74G1 glycosyltransferase; and/or
(i) UGT76G1 glycosyltransferase.
In another preferred embodiment, the geranylgeranyl diphosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase and UGTB1/IBGT glycosyltransferase are recombinantly expressed to synthesize steviolbioside.
In another preferred embodiment, the geranylgeranyl diphosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGTB1/IBGT glycosyltransferase and UGT74G1 glycosyltransferase are recombinantly expressed to synthesize stevioside.
In another preferred embodiment, the geranylgeranyl diphosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGTB1/IBGT glycosyltransferase, UGT74G1 glycosyltransferase and UGT76G1 glycosyltransferase are recombinantly expressed to synthesize rebaudioside A.
In another preferred embodiment, in item (b), (II) bifunctional ent-kaurene synthase is used.
In another preferred embodiment, the geranylgeranyl diphosphate synthase used in the method can be obtained from Taxus canadensis or Stevia rebaudiana, preferably from Taxus Canadensis; 
The ent-copalyl diphosphate synthase can be obtained from Stevia rebaudiana or Bradyrhizobium japonicum, preferably from Stevia rebaudiana; 
The ent-kaurene synthase can be obtained from Stevia rebaudiana or Bradyrhizobium japonicum, preferably from Stevia rebaudiana; 
The bifunctional ent-kaurene synthase can be obtained from Physcomitrella patens or Gibberella fujikuroi, preferably from Physcomitrella patens; 
The ent-kaurene oxidase can be obtained from Stevia rebaudiana, Gibberella fujikuroi, Arabidopsis thaliana, or Bradyrhizobium japonicum, preferably from Stevia rebaudiana. 
The kaurenoic acid-13α-hydroxylase can be obtained from Stevia rebaudiana or Arabidopsis thaliana, preferably from Stevia rebaudiana; 
The UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase and UGT76G1 glycosyltransferase can be obtained from Stevia rebaudiana; 
The UGTB1 glycosyltransferase can be obtained from Starmerella bombicola. The IBGT glycosyltransferase can be obtained from Ipomoea batatas. The cytochrome P450 redox protein can be obtained from Artemisia annua, Phaeosphaeria sp. L487, Gibberella fujikuroi, Stevia rebaudiana, or Arabidopsis thaliana, preferably from Phaeosphaeria sp.
In another preferred embodiment, in item (b), (II) bifunctional ent-kaurene synthase is used; the geranylgeranyl diphosphate synthase is obtained from Taxus canadensis, the bifunctional ent-kaurene synthase is obtained from Physcomitrella patens; the ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase and UGT76G1 glycosyltransferase are from Stevia rebaudiana; the UGTB1 glycosyltransferase is from Starmerella bombicola; the cytochrome P450 redox protein is from Phaeosphaeria; and the IGBT glycosyltransferase is from Ipomoea batatas. 
In another preferred embodiment, the geranylgeranyl diphosphate synthase from Taxus canadensis has its transit-peptide sequence removed from its N terminus in relative to the wild type sequence. Preferably, the 98 amino acid residues at the N terminus are truncated;
the cytochrome P450 redox protein from Artemisia annua has its transmembrane domain removed from its N terminus in relative to the wild type sequence. Preferably, the 66 amino acid residues at the N terminus are truncated.
In another preferred embodiment, the geranylgeranyl diphosphate synthase has an amino acid sequence as shown in SEQ ID NO: 1 or 45, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 1 or 45;
the ent-copalyl diphosphate synthase has an amino acid sequence as shown in SEQ ID NO: 3 or 25, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 3 or 25;
the ent-kaurene synthase has an amino acid sequence as shown in SEQ ID NO: 5 or 27, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 5 or 27;
the bifunctional ent-kaurene synthase has an amino acid sequence as shown in SEQ ID NO: 21 or 23, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 21 or 23;
the ent-kaurene oxidase has an amino acid sequence as shown in SEQ ID NO: 7, 31, 37 or 29, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 7, 31, 37 or 29;
the kaurenoic acid-13α-hydroxylase has an amino acid sequence as shown in SEQ ID NO: 9, 43, 47 or 49, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 9, 43, 47 or 49;
the UGT85C2 glycosyltransferase has an amino acid sequence as shown in SEQ ID NO: 11, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 11;
the UGT74G1 glycosyltransferase has an amino acid sequence as shown in SEQ ID NO: 13, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 13;
the UGT76G1 glycosyltransferase has an amino acid sequence as shown in SEQ ID NO: 15, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 15;
the UGTB1 glycosyltransferase has an amino acid sequence as shown in SEQ ID NO: 41, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 41;
the IBGT glycosyltransferase has an amino acid sequence as shown in SEQ ID NO: 51, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 51; or
the cytochrome P450 redox protein has an amino acid sequence as shown in SEQ ID NO: 17, 19, 33, 35 or 39, or is a derivative protein having the same function and produced by substitution, deletion or addition of one or more (such as 1-30, preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues on SEQ ID NO: 17, 19, 33, 35 or 39.
In another preferred embodiment, the coding gene of the geranylgeranyl diphosphate synthase has a nucleotide sequence as shown in SEQ ID NO: 2 or 46, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 2 or 46;
the coding gene of the ent-copalyl diphosphate synthase has a nucleotide sequence as shown in SEQ ID NO: 4 or 26, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 4 or 26;
the coding gene of the ent-kaurene synthase has a nucleotide sequence as shown in SEQ ID NO: 6 or 28, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 6 or 28;
the coding gene of the bifunctional ent-kaurene synthase has a nucleotide sequence as shown in SEQ ID NO: 22 or 24, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 22 or 24;
the coding gene of the ent-kaurene oxidase has a nucleotide sequence as shown in SEQ ID NO: 8, 32, 38 or 30, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 8, 32, 38 or 30;
the coding gene of the kaurenoic acid-13α-hydroxylase has a nucleotide sequence as shown in SEQ ID NO: 10, 44, 48 or 50, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 10, 44, 48 or 50;
the coding gene of the UGT85C2 glycosyltransferase has a nucleotide sequence as shown in SEQ ID NO: 12, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 12;
the coding gene of the UGT74G1 glycosyltransferase has a nucleotide sequence as shown in SEQ ID NO: 14, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 14;
the coding gene of the UGT76G1 glycosyltransferase has a nucleotide sequence as shown in SEQ ID NO: 16, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 16;
the coding gene of the UGTB1 glycosyltransferase has a nucleotide sequence as shown in SEQ ID NO: 42, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 42;
the coding gene of the IBGT glycosyltransferase has a nucleotide sequence as shown in SEQ ID NO: 52, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 52; or
the coding gene of the cytochrome P450 redox protein has a nucleotide sequence as shown in SEQ ID NO: 18, 20, 34, 36 or 40, or a nucleotide sequence encoding a protein having the same function and having 70% or more, 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, identity to SEQ ID NO: 18, 20, 34, 36 or 40.
In another preferred embodiment, the cell is selected from but is not limited to the cell from prokaryotic microorganisms and eukaryotic microorganisms.
In another preferred embodiment, the prokaryotic microorganism is E. coli, Bacillus subtilis, Acetobacteria, Corynebacterium and Brevibacterium. Preferably, the E. coli is selected from the group consisting of BL21, BLR, DH10B, HMS, C43, JM109, DH5α or Noveblue.
In another preferred embodiment, the eukaryotic microorganism is selected from, but is not limited to, yeast, mold, or basidiomycete. Yeast may be, but is not limited to, Pichia pastoris, Saccharomyces cerevisiae, or Kluyveromyces lactis. Preferably, Pichia pastoris may be selected from GS115, MC100-3, SMD1163, SMD1165, SMD1168 or KM71. Preferably, Saccharomyces cerevisiae may be selected from W303, CEN.PK2, S288c, FY834 or S1949. Preferably, Kluyveromyces lactis may be GG799.
In another preferred embodiment, the cell is a gram-negative strain and it uses pET, pBAD and pQE expression vectors, such as pET28a and pET21c, to recombinantly express each enzyme. Alternatively, when the cell is a yeast cell, pPIC expression vector, such as pPIC3.5, or pSY expression vector, such as pSY01, is used to recombinantly express each enzyme.
In another preferred embodiment, the method comprises the step of inserting the coding genes of (a)-(g) and optionally (h)-(i) into the recombinant expression vectors to construct gene expression cassette(s) for recombinantly expressing the enzymes.
In another aspect of the present invention, an expression construct, such as an expression vector, for synthesizing a steviol glycoside is provided, which comprises the gene expression cassette(s) of the following enzymes:
(a) Geranylgeranyl diphosphate synthase (GGPPS);
(b) An enzyme selected from (I) ent-copalyl diphosphate synthase (CDPS) and ent-kaurene synthase (KS) and (II) bifunctional ent-kaurene synthase (CPS/KS);
(c) Ent-kaurene oxidase (KO);
(d) Cytochrome P450 redox protein (CPR);
(e) Kaurenoic acid-13α-hydroxylase;
(f) UGT85C2 glycosyltransferase; and
(g) UGTB1/IBGT glycosyltransferase.
In another preferred embodiment, the expression construct for synthesizing a steviol glycoside may further comprise the gene expression cassette(s) of the following enzymes:
(h) UGT74G1 glycosyltransferase; and/or
(i) UGT76G1 glycosyltransferase.
In another aspect of the present invention, an expression construct for strengthening the precursor pathway of a steviol glycoside is provided, which comprises the gene expression cassette(s) of the following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) and isopentenyl-diphosphate delta-isomerase (IDI).
In another aspect of the present invention, a host cell for synthesizing a steviol glycoside is provided, which comprises the expression constructs for synthesizing the steviol glycoside.
In another preferred embodiment, the host cell for synthesizing a steviol glycoside is a non-generative material or a non-propagative material.
In another preferred embodiment, the cell used for synthesizing a steviol glycoside is selected from, but is not limited to, a cell from prokaryotic microorganisms and eukaryotic microorganisms.
In another preferred embodiment, the host cell for synthesizing a steviol glycoside may further comprise the expression construct for strengthening the precursor pathway of a steviol glycoside.
In another aspect of the present invention, a method for preparing ent-kaurene is provided, which comprises recombinantly, preferably heterologously, expressing in a cell from prokaryotic microorganisms and eukaryotic microorganisms (a) geranylgeranyl diphosphate synthase (GGPPS) and (b) bifunctional ent-kaurene synthase (CPS/KS).
In another preferred embodiment, the geranylgeranyl diphosphate synthase can be obtained from Taxus canadensis or Stevia rebaudiana, preferably from Taxus Canadensis; or the bifunctional ent-kaurene synthase can be obtained from Physcomitrella patens or Gibberella fujikuroi. 
In another aspect of the present invention, an expression construct for preparing ent-kaurene is provided, which comprises the gene expression cassette(s) of the following enzymes: (a) Geranylgeranyl diphosphate synthase; and (b) bifunctional ent-kaurene synthase.
In another aspect of the present invention, a host cell for preparing ent-kaurene is provided, which comprises the expression construct for preparing ent-kaurene, and/or the expression construct for strengthening the precursor pathway of a steviol glycoside.
In another preferred embodiment, the host cell for preparing ent-kaurene is a gram negative DE3 lysogenic strain or a yeast cell.
In another aspect of the present invention, use of UGTB1/IBGT glycosyltransferase is provided for converting steviolmonoside into steviolbioside, preferably adding a glycosyl group at the C-2′ of the C13 glucose of the steviolmonoside.
In another aspect of the present invention, use of bifunctional ent-kaurene synthase is provided for converting geranylgeranyl diphosphate to ent-kaurene.
In another aspect of the present invention, a combination of enzymes for preparing a steviol glycoside is provided, which comprises:
(a) Geranylgeranyl diphosphate synthase (GGPPS);
(b) An enzyme selected from (I) ent-copalyl diphosphate synthase (CDPS) and ent-kaurene synthase (KS) and (II) bifunctional ent-kaurene synthase (CPS/KS);
(c) Ent-kaurene oxidase (KO);
(d) Cytochrome P450 redox protein (CPR);
(e) Kaurenoic acid-13α-hydroxylase;
(f) UGT85C2 glycosyltransferase; and
(g) UGTB1/IBGT glycosyltransferase.
In another preferred embodiment, the combination of enzymes may further comprise:
(h) UGT74G1 glycosyltransferase;
(i) UGT76G1 glycosyltransferase.
In another aspect of the present invention, a kit for preparing a steviol glycoside is provided, which comprises the expression construct(s) for synthesizing the steviol glycoside. Preferably, the kit may further comprise the expression construct for strengthening the precursor pathway of the steviol glycoside. Alternatively, the kit may comprise the host cell for synthesizing the steviol glycoside.
In another aspect of the present invention, a combination for preparing ent-kaurene, such as a kit comprising enzymes, is provided, which comprises (a) geranylgeranyl diphosphate synthase (GGPPS) and (b) bifunctional ent-kaurene synthase (CPS/KS).
In another preferred embodiment, the combination for preparing ent-kaurene may further comprise 1-deoxy-D-xylulose-5-phosphate synthase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and isopentenyl-diphosphate delta-isomerase.
In another aspect of the present invention, a kit for preparing ent-kaurene is provided, which comprises the expression construct(s) for preparing ent-kaurene. Preferably, the kit may further comprise the expression construct for strengthening the precursor pathway of a steviol glycoside. Alternatively, the kit may comprise the host cell for synthesizing ent-kaurene.
The other aspects of the present invention will be apparent to the skilled artisan based on the above aspects of the present invention.



DESCRIPTION OF FIGURES
FIG. 1 shows the structure of steviol glycosides.
FIG. 2 shows the biosynthesis procedure of rebaudioside A.
FIG. 3A shows the structure of plasmid pET28a-ggpps with the insertion site being NcoI/HindIII.
FIG. 3B shows the plasmid pET21c-Inserted gene, wherein the inserted genes include cdps, cps/ks, ks, ko, kah, ugt85c2, ugtb1, ugt74g1 and ugt76g1, each of which was inserted between NdeI/HindIII.
FIG. 3C shows the structure of the plasmid pET21d-cpr, with insertion site being NcoI/HindIII.
FIG. 3D shows the structure of the plasmid pJF47 (pET21d-dxs-ispD-ispF-idi).
FIG. 3E shows the structure of the plasmid pZQ110 (pET28a-ggpps-cps/ks-ko-kah-ugt85c2-ugtb1-ugt74g1-ugt76g1-cpr).
FIG. 3F shows the restriction map of the plasmid pZQ110.
FIG. 3G shows the plasmid pPIC3.5K-Inserted gene, wherein the inserted genes include cdps, cps/ks, ks, ko, kah, ugt85c2, ugtb1, ugt74g1 and ugt76g1, each of which was inserted between BglII/NotII.
FIG. 3H shows the structure of the plasmid pZQ210 (pSY1-ggpps-cps/ks-ko-kah-ugt85c2-ugtb1-ugt74g1-ugt76g1-cpr).
FIG. 3I shows the PCR map of the plasmid pZQ210.
FIG. 4A shows the SDS-PAGE map for expression of the adps gene and ugt85c2 gene.
FIG. 4B shows the SDS-PAGE map for expression of the ko gene and the ks gene.
FIG. 4C shows the SDS-PAGE map for expression of the kah gene.
FIG. 4D shows SDS-PAGE map for expression of the ugt74g1 gene.
FIG. 4E shows SDS-PAGE map for expression of the ugt76g1 gene.
FIG. 5A shows the GC-MS map of ent-kaurene from the fermentation broth of Example 6.
FIG. 5B shows the yield of ent-kaurene from the fermentation broth of Example 6.
FIG. 6A shows the HPLC map of the fermentation broth of Example 7.
FIG. 6B shows the HPLC-MS map of kaurenoic acid obtained from the fermentation broth of Example 7.
FIG. 6C shows the HPLC-MS map of steviol obtained from the fermentation broth of Example 7.
FIG. 6D shows the HPLC-MS map of steviolmonoside obtained from the fermentation broth of Example 7.
FIG. 6E shows the HPLC-MS map of rebaudioside A obtained from the fermentation broth of Example 7.
FIG. 6F shows the yield of rebaudioside A obtained from the fermentation broth of Example 7.
FIG. 7 shows the yield of ent-kaurene from the fermentation broth of Example 9.
FIG. 8 shows the yield of rebaudioside A obtained from the fermentation broth of Example 10.
FIG. 9 shows the HPLC-MS maps of steviolmonoside and steviolbioside obtained from the fermentation broth of Example 11.
9a shows the HPLC-MS maps of steviolmonoside and steviolbioside obtained from fermentation broth of the engineering strain containing the pZQ107 expression vector;
9b shows the HPLC-MS maps of steviolmonoside and steviolbioside obtained from fermentation broth of the engineering strain containing the pZQ108 expression vector;
9c shows the HPLC-MS maps of steviolmonoside and steviolbioside obtained from fermentation broth of the engineering strain containing the pZQ105 expression vector;
9d shows the HPLC-MS maps of steviolmonoside and steviolbioside obtained from fermentation broth of the engineering strain containing the pZQ109 expression vector.
FIG. 10 shows the yields of steviolmonoside and steviolbioside obtained after transfecting the cells with expression plasmids from different sources, as shown in Example 11.



SPECIFIC MODE FOR CARRYING OUT THE INVENTION
After thoughtful investigation by the present inventors, the present invention firstly reveals a key enzymes used for heterologous biosynthesis of steviol glycosides, thereby realizing heterologous biosynthesis of a steviol glycoside.
Terms
As used herein, the “steviol glycosides” refers to a compound selected from steviol, steviolmonoside, steviolbioside, Stevioside, rebaudioside A or rebaudioside B.
As used herein, the “gene expression cassette” refers to a gene expression system comprising all essential elements required for expressing a target polypeptide, i.e., the enzyme in the subject invention. It generally comprises a promoter, a gene sequence encoding the polypeptide and a terminator. Optionally, it can further comprise a sequence encoding a signal peptide, etc. All the sequences are operably linked.
As used herein, the “operably linked” refers to the functional spatial arrangement of two or more nucleic acid regions or nucleotide sequences. For example, the promoter region is arranged at a special position in relative to the nucleic acid of the target gene to allow direction of the transcription of the nucleic acid by the promoter region. As such, the promoter region is “operably linked” to the nucleic acid of the target gene.
As used herein, the “expression construct” refers to a recombinant DNA molecule, which comprises a desired nucleic acid coding sequence. The construct may comprise one or more gene expression cassette(s). And the “construct” generally is contained within an expression vector.
As used herein, the “heterologous” refers to the relationship between two or more nucleic acids or proteins which are from different sources, or the relationship between the proteins (or nucleic acids) from different sources and the host cells. For example, if the combination of a nucleic acid and a host cell does not exist naturally, the nucleic acid is heterologous to the host cell. A specific sequence is “heterologous” to a cell or an organism into which it is inserted.
Proteins in the Synthetic Pathway and their Expression Systems
The prevent invention relates to the geranylgeranyl diphosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, bifunctional ent-kaurene synthase (optionally, ent-copalyl diphosphate synthase and ent-kaurene synthase), ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase, UGT76G1 glycosyltransferase, UGTB1 glycosyltransferase and cytochrome P450 redox protein involved in the synthesis of the steviol glycoside. Co-expression of the above proteins could synthesize the steviol glycoside. Preferably, enzymes that strengthen the precursor pathway at the upstream of the synthetic pathway are also involved, which may be any enzyme that converts pyruvic acid (PYR) and glyceraldehyde 3-phosphate (G3P) precursors in the central metabolic pathway to the common precursors for synthesis of terpenoids, including isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Preferably, the enzymes for strengthening the precursor pathway include 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) and isopentenyl-diphosphate delta-isomerase (IDI).
The inventors have also investigated the efficiency of enzymes from different sources for use in synthesis of an intermediate, ent-kaurene, and a final product, a steviol glycoside. Enzymes with excellent results were obtained. Therefore, as a preferred embodiment of the present invention, the geranylgeranyl diphosphate synthase is obtained from Taxus canadensis or Stevia rebaudiana, preferably from Taxus Canadensis; the ent-copalyl diphosphate synthase is obtained from Stevia rebaudiana or Bradyrhizobium japonicum, preferably from Stevia rebaudiana; the ent-kaurene synthase is obtained from Stevia rebaudiana or Bradyrhizobium japonicum, preferably from Stevia rebaudiana; the bifunctional ent-kaurene synthase is obtained from Physcomitrella patens or Gibberella fujikuroi, preferably from Physcomitrella patens; the ent-kaurene oxidase is obtained from Stevia rebaudiana, Gibberella fujikuroi, Arabidopsis thaliana, or Bradyrhizobium japonicum, preferably from Stevia rebaudiana; the kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase and UGT76G1 glycosyltransferase are from Stevia rebaudiana; the UGTB1 glycosyltransferase is obtained from Starmerella bombicola; and the IGBT glycosyltransferase is from Ipomoea batatas; or the cytochrome P450 redox protein is obtained from Artemisia annua, Phaeosphaeria sp., Gibberella fujikuroi, Stevia rebaudiana, or Arabidopsis thaliana, preferably from Phaeosphaeria sp. As a more preferred embodiment of the present invention, the bifunctional ent-kaurene synthase is used; and the geranylgeranyl diphosphate synthase is obtained from Taxus canadensis, the bifunctional ent-kaurene synthase is obtained from Physcomitrella patens; the ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase and UGT76G1 glycosyltransferase are from Stevia rebaudiana; the UGTB1 glycosyltransferase is obtained from Starmerella bombicola; and the cytochrome P450 redox protein is obtained from Phaeosphaeria sp.
As a more preferred embodiment of the present invention, the geranylgeranyl diphosphate synthase from Taxus canadensis has its transit-peptide sequence removed from its N terminus in relative to the wild type sequence. Preferably, the 98 amino acid residues at the N terminus are truncated. The cytochrome P450 redox protein from Artemisia annua has its transmembrane domain removed from its N terminus in relative to the wild type sequence. Preferably, the 66 amino acid residues at the N terminus are truncated.
In the present invention, the above enzymes or proteins may be naturally occurring enzymes or proteins. For example, they may be isolated or purified from animals, plants or microorganisms. The enzymes or proteins may also be artificially prepared. For example, the recombinant enzymes or proteins may be produced by the conventional genetic engineering recombinant technique. Preferably, recombinant enzymes or proteins are used in the present invention.
Any suitable enzyme or protein can be used in the present invention. The enzyme or protein includes the full-length enzyme or protein or their biologically active fragments (or called as “active fragment”). The active fragments of the bifunctional CPS/KS enzyme having CDPS and KS activities include YDTAWXA (SEQ ID NO: 61), DXDD (SEQ ID NO: 62), and DDXXD (SEQ ID NO: 63), or YDTAWXA (SEQ ID NO: 64), DXDD (SEQ ID NO: 65), and DEXXE (SEQ ID NO: 66). The active fragments of the UGTB1 glycosyltransferase include GHVGP (SEQ ID NO: 67) and NGGYGG (SEQ ID NO: 68). The amino acids of the enzyme or protein formed by substitution, deletion or addition of one or more amino acid residue are also encompassed by the present invention. The biologically active fragment of an enzyme or a protein refers to a polypeptide which retains the whole or partial function of the full-length enzyme or protein. Generally, the biologically active fragment retains at least 50% activity of the full-length enzyme or protein. More preferably, the active fragment can retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% activity of the full-length enzyme or protein. The enzyme or protein or biologically active fragment thereof may include some conservative substitutions. The activity of the sequence having said substitutions will not be affected or the sequence retains a partial activity. It is a common knowledge in the art to suitably replace an amino acid residue, which could readily be practiced with the biological activity of the resultant molecule being not changed. The skilled artisan will realize, based on these techniques, that generally changing one single amino acid in the non-essential region of a polypeptide will basically not change the biological activity of the polypeptide. See Watson et al., Molecular Biology of The Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co. P224.
The present invention can also use the modified or improved enzyme or protein. For example, the enzyme or protein being modified or improved to promote its half life, efficacy, metabolism and/or potency of protein can be used. The modified or improved enzyme or protein may be a conjugate, or may comprise substituted or artificial amino acid(s). The modified or improved enzyme or protein may be significantly different from the naturally occurring enzyme or protein, but can still have the same or basically identical function to the wild type enzyme or protein and will not produce the other un-favored effect. In other words, any variants that do not affect the biological activity of the enzyme or protein may be used in the present invention.
The present invention also encompasses isolated nucleic acid that encodes the biologically active fragment of the enzyme or protein, or its complementary strand. As a preferred embodiment of the present invention, the coding sequence of each enzyme or protein may be subjected to codon optimization to enhance expression efficiency. The DNA sequence encoding the biologically active fragment of the enzyme or protein may be obtained by artificial synthesis of the whole sequence or may be obtained by PCR amplification. After obtaining the DNA sequence that encodes the biologically active fragment of the enzyme or protein, the DNA sequence is linked into a suitable expression construct, such as an expression vector. The construct is then transferred into a suitable host cell. The transformed host cell is cultivated to produce the desired protein.
The present invention also includes the expression construct comprising a nucleic acid that encodes the biologically active fragment of the enzyme or protein. The expression construct may comprise one or more gene expression cassette(s) encoding the enzyme or protein, and additionally, expression regulatory sequences operably linked to the nucleic acid molecule to facilitate the expression of the protein. Techniques for designing the expression regulatory sequences are known in the art. In the expression regulatory sequence, an inducible promoter or a constitutive promoter can be used as needed. The inducible promoter can be used for more controllable protein expression and production of compound, which is beneficial for the industrial application.
As a preferred embodiment of the present invention, an expression construct is provided, which comprises the gene expression cassette(s) of the following enzymes: the gene expression cassette(s) of the following enzymes: geranylgeranyl diphosphate synthase (GGPPS); an enzyme selected from (I) ent-copalyl diphosphate synthase (CDPS) and ent-kaurene synthase (KS) and (II) bifunctional ent-kaurene synthase (CPS/KS); ent-kaurene oxidase (KO); cytochrome P450 redox protein (CPR); kaurenoic acid-13α-hydroxylase; UGT85C2 glycosyltransferase; and UGTB1/IBGT glycosyltransferase. More preferably, the expression construct may further comprise the gene expression cassette(s) of the following enzymes: UGT74G1 glycosyltransferase; and/or UGT76G1 glycosyltransferase.
Technique for establishing an expression construct was well known in the art. Therefore, after determining the desired enzyme or protein, the skilled artisan can readily construct their expression construct. The gene sequence encoding enzyme or protein may be inserted into different expression constructs, such as expression vectors, or may be inserted into the same expression construct, as long as the enzyme or protein can be effectively expressed after transfecting into cells.
Additionally, the recombinant cell comprising a nucleic acid sequence encoding the biologically active fragment of the enzyme or protein is also encompassed in the present invention. The “host cell” may include prokaryotic cell and eukaryotic cell. Common prokaryotic host cell include E. coli and Bacillus subtilis, etc. Common eukaryotic host cell includes yeast, insect cell and mammal cell. As a preferred embodiment of the present invention, the cell is selected from, but is not limited to, gram negative DE3 lysogenic strain and yeast cell. More preferably, the gram negative DE3 lysogenic strain is, but is not limited to, E. coli and Bacillus subtilis. More preferably, the E. coli is selected from the group consisting of BL21(DE3), BLR(DE3), DH10B(DE3), HMS(DE3), C43(DE3), JM109(DE3), DH5a(DE3) or Noveblue(DE3). More preferably, the yeast may be (but is not limited to) Pichia pastoris, Saccharomyces cerevisiae, or Kluyveromyces lactis. Preferably, Pichia pastoris may be selected from GS115, MC100-3, SMD1163, SMD1165, SMD1168 or KM71. Preferably, Saccharomyces cerevisiae may be selected from W303, CEN.PK2, S288c, FY834 or S1949. Preferably, Kluyveromyces lactis may be GG799.
Suitable expression vector for used in bacterial cell or fungal cell were known in the art. Therefore, the skilled artisan can readily select a suitable expression vector for use as a backbone vector for cloning the coding gene. For example, when the cell is a bacterial cell, pET series expression vectors, such as pET28a, can be used to recombinantly express each enzyme. And when the cell is a yeast cell, the pPICC series expression vectors, such as pPICC3.5, or the pSY series expression vectors, such as pSY01, could be used.
Conventional techniques known in the art can be used to transform a host cell with a recombinant DNA. When the host is a prokaryotic organism, such as E. coli, the competent cells that can adsorb DNA can be harvested after exponential growth by treated by, such as CaCl2 or MgCl2. The steps as used are well known in the art. If required, transformation can be carried out by electroporation. When the host is a eukaryotic cell, the following DNA Transformation methods could be used: calcium phosphate co-precipitation method and conventional mechanical methods, such as microinjection, electroporation and liposomal packaging.
The resultant transformant could be cultured by a conventional method for expressing the enzyme or protein encoded by the gene of the present invention. Based on the used host cell, the culture medium used can be selected from various conventional culture mediums. Host cells are cultured under conditions suitable for their growth.
Methods for Synthesizing Steviol Glycosides
Disclosed is a method for heterologous synthesis of a steviol glycoside by Microorganism. Enzymes involved in the biosynthesis of a steviol glycoside, which are from different sources, are artificially combined together by techniques of synthetic biology to obtain the steviol glycoside, including rebaudioside A, in a host cell.
The biosynthesis process for synthesizing a steviol glycoside, as disclosed in the present invention, is shown in FIG. 2. Pyruvic acid (PYR) and glyceraldehyde 3-phosphate (G3P) in the central metabolic pathway are used as precursors to obtain the common precursors for synthesis of terpenoids: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Ent-kaurene is obtained sequentially through geranylgeranyl diphosphate synthase (GGPPS, encoded by ggpps), ent-copalyl diphosphate synthase (CDPS, encoded by cdps), and ent-kaurene synthase (KS, encoded by ks). Preferably, CDPS and KS are replaced by the bifunctional ent-kaurene synthase (CPS/KS, encoded by cps/ks). Thereafter, kaurene is catalyzed to kaurenoic acid by cytochrome P450 redox protein and ent-kaurene oxidase (KO, encoded by ko), and kaurenoic acid is oxidized by kaurenoic acid hydroxylase (KAH, encoded by kah) to obtain the core structure of diterpene, steviol. Steviol is converted to steviolmonoside via UGT85C2 glycosyltransferase and steviolmonoside is further glycosylated to produce steviolbioside. Steviolbioside is converted to stevioside by UGT74G1 glycosyltransferase and stevioside is converted to rebaudioside A via the UGT76G1 glycosyltransferase.
Heterologous biosynthesis of steviol glycosides was firstly achieved by the present inventors by using the above enzymes. UGTB1/IBGT glycosyltransferases, which could incorporate a glycosyl group to the C-2′ site at the C-13 glucose of steviolmonoside, are specifically used to convert steviolmonoside to steviolbioside. Thus, the technical problem of the prior art that steviolmonoside cannot be converted to steviolbioside is solved. The glycosyltransferases required for converting steviolmonoside to steviolbioside in one glycosylation step in the biosynthesis of a steviol glycoside were not known in the prior art. After thoughtful investigation by screening a lot of glycosyltransferase genes, the present inventors finally found that UGTB1 or IBGT glycosyltransferase could further glycosylate the C-2′ site in the C-13 glucose of the substrate.
Suitable UGTB1/IBGT glycosyltransferases can function as uridine-5′-diphosphoglucosyl: steviol-13-O-glucosyltransferase (also called as steviol-13-monoglucoside 1,2-glucosylase) to transfer a glucose moiety to the C-2′ of the 13-O-glucose of the receptor molecule, the steviol-13-gluco side.
Generally, suitable UGTB1/IBGT glycosyltransferases can function as uridine-5′-diphosphoglucosyl: rubusoside transferase to transfer a glucose moiety to the C-2′ of the 13-O-glucose of the receptor molecule, rubusoside.
Suitable UGTB1/IBGT glycosyltransferases can further catalyze the reaction utilizing stevioside substrate other than steviol-13-O-glucoside and rubusoside. For example, they can utilize stevioside as a substrate to transfer a glucose moiety to the C-2′ of the 19-O-glucose residue to produce rebaudioside E. They can also use rebaudioside A as a substrate to transfer a glucose moiety to the C-2′ of the 19-O-glucose residue to produce rebaudioside D. However, UGTB1/IBGT glycosyltransferases generally will not transfer a glucose moiety to a steviol compounds having 1,3-bound glucose at the C-13 position, that is, will not transfer a glucose moiety to steviol 1,3-diglycoside and 1,3-stevioside.
Suitable UGTB1/IBGT glycosyltransferases can transfer a glucose moiety except for uridine diphosphoglucose. For example, suitable UGTB1/IBGT glycosyltransferases can function as uridine 5′-diphosphoric acid D-xylosyl: steviol-13-O-glucoside transferase to transfer the xylose moiety to C-2′ of the 13-O-glucose of the receptor molecule, steviol-13-O-glucoside. In another example, suitable UGTB1/IBGT glycosyltransferases can function as uridine 5′-diphosphoric acid L-rhamnosyl: steviol-13-O-glucoside transferase to transfer the rhamnose moiety to C-2′ of the 13-O-glucose of the receptor molecule, steviol-13-O-glucoside.
The common precursors IPP and DMAPP for synthesis of terpenoids can be obtained via known techniques in the art. As a preferred embodiment of the present invention, to obtain IPP and DMAPP, the inventors simplify the technical solutions known in the prior art which utilize genes such as dxr, ispE, ispG and ispH, etc. Specifically, in the present invention, pyruvic acid (PYR) and glyceraldehyde 3-phosphate (G3P) precursors in the central metabolic pathway are used as precursors, and the common precursors for synthesis of terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are obtained through the 1-deoxyxylose-5-phosphoric acid pathway, which orderly includes catalytic synthesis by 1-deoxy-D-xylulose-5-phosphate synthase (DXS, encoded by the dxs gene), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS, encoded by the ispD gene), and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS, encoded by the ispF gene). With the isopentenyl-diphosphate delta-isomerase (encoded by the idi gene), IPP and DMAPP can be converted to each other.
Method for Synthesizing Ent-Kaurene
The steviol glycoside is a reaction product, the backbone of which is a diterpene compound, ent-kaurene. Ent-kaurene is a major intermediate product. Enhanced production efficiency of ent-kaurene will facilitate highly effective synthesis of the down-stream steviol glycoside.
To improve the efficiency of synthesizing kaurene, the inventors finally find, after repeated experiments, that the efficiency of synthesizing kaurene could be significantly improved by replacing ent-copalyl diphosphate synthase (CDPS) and ent-kaurene synthase (KS) by the bifunctional ent-kaurene synthase (CPS/KS), which is a bifunctional enzyme having the CDPS and KS activities.
The Effective Results of the Present Invention
By utilizing the methods of the present invention, the yield of the crucial intermediate product, ent-kaurene, can reach 1 g/L or more, and the yield of rebaudioside A can reach 10 mg/L or more. The methods of the present invention can be used to replace the plant extraction method to obtain steviol glycosides, especially rebaudioside A which is of great market value. Thus, the subject invention has a wide application prospect and development potential.
As compared to the traditional extraction from plant, which is time-consuming, sensitive to environment and deleterious to the natural resources, the subject application has advantages of low cost, small production area and easy control of product quantity.
The subject invention is further illustrated by making reference to the specific Examples. It should be understood that these examples are provided for illustrate the invention but not for limit the scope of the invention. The experimental method in the examples, in which the specific conditions are not indicated, is practiced generally according to Joseph Sambrook, Molecular Cloning: A Laboratory Manual, the 3th edition, Science Press, 2002, or according to the conditions recommended by the manufacturer. Unless otherwise indicated, the percentage and part are calculated by weight.
Example 1
Obtaining the Proteins Used in the Heterologous Biosynthesis of a Steviol Glycoside
Gene sources and synthesis of the geranylgeranyl diphosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, bifunctional ent-kaurene synthase, ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGTB1 glycosyltransferase, UGT74G1 glycosyltransferase, UGT76G1 glycosyltransferase, and cytochrome P450 redox protein involved in the synthesis of a steviol glycoside, rebaudioside A.
Geranylgeranyl diphosphate synthase (GGPPS) from Taxus canadensis and Stevia rebaudiana; ent-copalyl diphosphate synthase (CDPS) from Stevia rebaudiana and Bradyrhizobium japonicum; ent-kaurene synthase (KS) from Stevia rebaudiana and Bradyrhizobium japonicum; bifunctional ent-kaurene synthase (CPS/KS) from Physcomitrella patens and Gibberella fujikuroi; ent-kaurene oxidase (KO) from Stevia rebaudiana, Gibberella fujikuroi, Arabidopsis thaliana and Bradyrhizobium japonicum; kaurenoic acid hydroxylase (KAH), UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase and UGT76G1 glycosyltransferase from Stevia rebaudiana; UGTB1 glycosyltransferase from Starmerella bombicola; IBGT glycosyltransferase from Ipomoea batatas; and cytochrome P450 redox protein from Artemisia annua, Phaeosphaeria sp. L487, Stevia rebaudiana, Arabidopsis thaliana and Gibberella fujikuroi were selected from NCBI. Table 2 shows the information of the selected enzymes.







TABLE 2







Genes Involved in the Heterologous Pathway Construction









Name of Gene
Genbank No.
Source





GGPP synthase
AAD16018
Taxus canadensis


GGPP synthase
ABD92926.2
Stevia rebaudiana


ent-copalyl diphosphate synthase
AAB87091
Stevia rebaudiana


ent-copalyl diphosphate synthase
BAC47414
Bradyrhizobium




japonicum


ent-kaurene synthase
AAD34294
Stevia rebaudiana


ent-kaurene synthase
BAC47415
Bradyrhizobium




japonicum


bifunctional ent-kaurene synthase
BAF61135
Physcomitrella patens


bifunctional ent-kaurene synthase
Q9UVY5.1
Gibberella fujikuroi


bifunctional ent-kaurene synthase
CAH18005.1
Fusarium proliferatum


bifunctional ent-kaurene synthase
BAA22426
Phaeosphaeria




sp. L487


bifunctional ent-kaurene synthase
CAP07655
Sphaceloma




manihoticola


ent-kaurene oxidase
AAQ63464
Stevia rebaudiana


ent-kaurene oxidase
O94142.1
Gibberella fujikuroi


ent-kaurene oxidase
AF047719
Arabidopsis thaliana


ent-kaurene oxidase
NP_768785
Bradyrhizobium




japonicum


kaurenoic acid-13α-hydroxylase
ABD60225
Stevia rebaudiana


kaurenoic acid-13α-hydroxylase
AEH65419
Stevia rebaudiana


kaurenoic acid-13α-hydroxylase
AED93376.1
Arabidopsis thaliana


kaurenoic acid-13α-hydroxylase
AED93377.1
Arabidopsis thaliana


UGT85C2 glycosyltransferase
AAR06916
Stevia rebaudiana


UGT74G1 glycosyltransferase
AAR06920
Stevia rebaudiana


UGT76G1 glycosyltransferase
AAR06912
Stevia rebaudiana


UGTB1 glycosyltransferase
ADT71703
Starmerella bombicola


cytochrome P450 redox protein
ABM88789
Artemisia annua


cytochrome P450 redox protein
BAG85333
Phaeosphaeria




sp. L487


cytochrome P450 redox protein
CAE09055.1
Gibberella fujikuroi


cytochrome P450 redox protein
ABB88839
Stevia rebaudiana


cytochrome P450 redox protein
X66016
Arabidopsis thaliana


IBGT glycosyltransferase
ABL74480.1
Ipomoea batatas









The coding sequences of the selected enzymes were optimized by conventional optimization method, such as Optimizer (genomes.urv.es/OPTIMIZER). These enzymes were synthesized as follows.
The amino acid sequence of the geranylgeranyl diphosphate synthase (GGPPS) from Taxus Canadensis is shown in SEQ ID NO: 1. The 98 amino acid residues (transit-peptide sequence) at the N terminus of the wild type GGPP synthase were removed and a methionine was added, so as to obtain the modified recombinant GGPP synthase. Then codon optimization was carried out and the optimized DNA sequence is shown in SEQ ID NO: 2.
The amino acid sequence of ent-copalyl diphosphate synthase (CDPS) from Stevia rebaudiana is shown in SEQ ID NO: 3, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 4.
The amino acid sequence of ent-kaurene synthase (KS) from Stevia rebaudiana is shown in SEQ ID NO: 5, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 6.
The amino acid sequence of ent-kaurene oxidase (KO) from Stevia rebaudiana is shown in SEQ ID NO: 7, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 8.
The amino acid sequence of kaurenoic acid-13α-hydroxylase (KAH) from Stevia rebaudiana is shown in SEQ ID NO: 9, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 10.
The amino acid sequence of UGT85C2 glycosyltransferase from Stevia rebaudiana is shown in SEQ ID NO: 11, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 12.
The amino acid sequence of UGT74G1 glycosyltransferase from Stevia rebaudiana is shown in SEQ ID NO: 13, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 14.
The amino acid sequence of UGT76G1 glycosyltransferase from Stevia rebaudiana is shown in SEQ ID NO: 15, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 16.
The amino acid sequence of cytochrome P450 redox protein (CPR) from Artemisia annua is shown in SEQ ID NO: 17, with the 66 amino acid residues at the N terminus of wild type CPR protein being truncated and the N terminus of the CPR protein (AAU10466) from C. tropicalis being added to obtain the modified CPR protein of the subject invention. The DNA sequence obtained after codon optimization is shown in SEQ ID NO: 18.
The amino acid sequence of cytochrome P450 redox protein from Phaeosphaeria is shown in SEQ ID NO: 19, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 20.
The amino acid sequence of bifunctional ent-kaurene synthase (CPS/KS) from Physcomitrella patens is shown in SEQ ID NO: 21, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 22.
The amino acid sequence of bifunctional ent-kaurene synthase (CPS/KS) from Gibberella fujikuroi is shown in SEQ ID NO: 23, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 24.
The amino acid sequence of ent-copalyl diphosphate synthase from Bradyrhizobium japonicum is shown in SEQ ID NO: 25, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 26.
The amino acid sequence of ent-kaurene synthase from Bradyrhizobium japonicum is shown in SEQ ID NO: 27, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 28.
The amino acid sequence of ent-kaurene oxidase from Bradyrhizobium japonicum is shown in SEQ ID NO: 29, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 30.
The amino acid sequence of ent-kaurene oxidase from Gibberella fujikuroi is shown in SEQ ID NO: 31, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 32.
The amino acid sequence of cytochrome P450 redox protein from Gibberella fujikuroi is shown in SEQ ID NO: 33, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 34.
The amino acid sequence of cytochrome P450 redox protein from Stevia rebaudiana is shown in SEQ ID NO: 35, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 36.
The amino acid sequence of ent-kaurene oxidase from Arabidopsis thaliana is shown in SEQ ID NO: 37, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 38.
The amino acid sequence of cytochrome P450 redox protein from Arabidopsis thaliana is shown in SEQ ID NO: 39, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 40.
The amino acid sequence of UGTB1 glycosyltransferase from Starmerella bombicola is shown in SEQ ID NO: 41, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 42.
The amino acid sequence of kaurenoic acid-13α-hydroxylase (KAH) from Stevia rebaudiana is shown in SEQ ID NO: 43, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 44.
The amino acid sequence of the geranylgeranyl diphosphate synthase (GGPPS) from Stevia rebaudiana is shown in SEQ ID NO: 45, the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 46.
The amino acid sequence of kaurenoic acid-13α-hydroxylase (KAH) from Arabidopsis thaliana (Genbank accession No. AED93376.1) is shown in SEQ ID NO: 47, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 48.
The amino acid sequence of kaurenoic acid-13α-hydroxylase (KAH) from Arabidopsis thaliana (Genbank accession No. AED93377.1) is shown in SEQ ID NO: 49, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 50.
The amino acid sequence of glycosyltransferase from Ipomoea batatas (Genbank accession No. ABL74480.1) is shown in SEQ ID NO: 51, and the DNA sequence obtained after codon optimization is shown in SEQ ID NO: 52.
Optimized DNA sequences of other proteins can be obtained by the same method.
Furthermore, the inventors analyzed the sequence homology of the proteins from different source, which were identified to have the same function. Table 3 shows the results.







TABLE 3







Homology Analysis of Amino Acids from Different Sources











Homology


Protein
Source
(NCBI blastP)





GGPPS
Stevia rebaudiana vs. Taxus Canadensis
68%


CDPS
Stevia rebaudiana vs. Physcomitrella patens
50%


CDPS
Stevia rebaudiana vs. Gibberella fujikuroi
58%


CDPS
Stevia rebaudiana vs. Bradyrhizobium
57%



japonicum



KS
Stevia rebaudiana vs. Bradyrhizobium
43%



japonicum



KO
Stevia rebaudiana vs. Bradyrhizobium
55%



japonicum



KO
Stevia rebaudiana vs. Gibberella fujikuroi
25%


KO
Stevia rebaudiana vs. Arabidopsis thaliana
61%


CPR
Stevia rebaudiana vs. Phaeosphaeria sp.
34%



L487



CPR
Stevia rebaudiana vs. Gibberella fujikuroi
75%


CPR
Stevia rebaudiana vs. Arabidopsis thaliana
66%


KS
Stevia rebaudiana vs. Physcomitrella patens
36%


KS
Stevia rebaudiana vs. Gibberella fujikuroi
64%


CPS/KS
Gibberella fujikuroi vs. Physcomitrella patens
34%


KS vs.
KS: Stevia rebaudiana vs. CPSKS:
36%


CPS/KS
Physcomitrella patens



KS vs.
KS: Stevia rebaudiana vs.CPSKS: Gibberella
64%


CPS/KS
fujikuroi



UGTB1 vs.
UGTB1: Starmerella bombicola vs.
33%


UGT85C2
UGT85C2: Stevia rebaudiana



UGTB1 vs.
UGTB1: Starmerella bombicola vs.
83%


UGT74G1
UGT74G1: Stevia rebaudiana



UGTB1 vs.
UGT76G1: Starmerella bombicola vs.
45%


UGT76G1
UGT76G1: Stevia rebaudiana



IBGT vs.
IBGT: Ipomoea batatas vs. UGT85C2: Stevia
31%


UGT85C2
rebaudiana



IBGT vs.
IBGT: Ipomoea batatas vs. UGT74G1: Stevia
36%


UGT74G1
rebaudiana



IBGT vs.
IBGT: Ipomoea batatas vs. UGT76G1: Stevia
28%


UGT76G1
rebaudiana



UGTB1 vs.
UGT76G1: Starmerella bombicola vs. IBGT:
22%


IBGT
Ipomoea batatas









From Table 3, it can be found that the proteins from different sources but having the same function have low homology. For example, CDPS from Stevia rebaudiana has only 50% and 58% homology, respectively, to CPS/KS having CDPS and KS bifunctional enzymatic activity from Physcomitrella patens and Gibberella fujikuroi. KS from Stevia rebaudiana has only 36% and 64% homology, respectively, to CPS/KS having CDPS and KS bifunctional enzymatic activity from Physcomitrella patens and Gibberella fujikuroi. Furthermore, the UGTB1 glycosyltransferase from Starmerella bombicola has only 33%, 83% and 45% homology, respectively, to UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase and UGT76G1 glycosyltransferase from Stevia rebaudiana. 
CPS/KS from several fungal sources has relatively high homology, but CPS/KS from Physcomitrella patens has a relatively low homology. Nevertheless, all of them contain regions rich in aspartic acid, which are fragments having CPS and KS enzymatic activity. The active fragment of CPS starts from YDTAWXA with an N terminal sequence of DXDD; while the active fragment of KS starts from DDXXD or DEXXE at the C terminus, wherein X is any amino acid. The active fragments of UGTB1 include GHVGP, which locates at positions 16 to 20, and NGGYGG, which locates at positions 338 to 343.
Example 2
Construction of the Prokaryotic Expression Vector
The optimized genes of geranylgeranyl diphosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, bifunctional ent-kaurene synthase, ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase, UGT76G1 glycosyltransferase and cytochrome P450 redox protein were cloned into related plasmids to construct the gene expression vectors used in the rebaudioside A synthesis pathway in bacterium.
A SpeI restriction site was inserted after the terminator codon TAA of the optimized ggpps gene, then the resultant gene was cloned into plasmid pET28a (Novagen) at the NcoI/HindIII restriction site to produce pET28a-ggpps (FIG. 3A).
The SpeI site was inserted after the terminator codon TAA of the optimized cdps, cps/ks, ks, ko, kah, ugt85c2, ugtb1, ugt74g1, and ugt76g1, respectively, then the resultant genes were cloned into pET21a (Novagen), respectively, at the NdeI/BamHI site, to obtain pET21a-cdps, pET21a-cps/ks, pET21a-ks, pET21a-ko, pET21a-kah, pET21a-ugt85c2, pET21a-ugtb1, pET21a-ugt74g1, and pET21a-ugt76g1, respectively (see FIG. 3B). In FIG. 3B, the inserted genes refer to cdps, cps/ks, ks, ko, kah, ugt85c2, ugtb1, ugt74g1 and ugt76g1, which were respectively inserted at the NdeI/HindIII site of individual plasmids.
The SpeI restriction site was inserted after the terminator codon TAA of the optimized cpr gene, then the resultant gene was cloned into plasmid pET21d (Novagen) at the NcoI/HindIII restriction site to produce pET21d-cpr (FIG. 3C).
As an example, the plasmid pZQ110 was constructed as follows. The heterologous genes were assembled in series by a method similar to the method provided by the BioBrick Assembly Kit of New England Biolab. Specifically, the plasmid pET28a-ggpps (Taxus Canadensis) was digested by SpeI/HindIII and the plasmid pET21a-cps/ks (Physcomitrella patens) was digested by XbaI/HindIII. The pET28a-ggpps vector was recovered by PCR purification kit and the cps/ks DNA fragment was recovered by gel. The cps/ks DNA fragment was ligated to the pET28a-ggpps vector by T4 DNA ligase to construct plasmid pET28a-ggpps-cps/ks. The same method was performed to ligate ko (Stevia rebaudiana), kah (Stevia rebaudiana), ugt85c2 (Stevia rebaudiana), ugtb1 (Starmerella bombicola), ugt74g1 (Stevia rebaudiana), ugt76g1 (Stevia rebaudiana) and cpr (Phaeosphaeria sp. L487) in series, to obtain a gene expression vector pZQ110 for synthesizing rebaudioside A in bacterium (FIG. 3E). The restriction map of the plasmid (XbaI/HindIII double digestion) was shown in FIG. 3F, in which M1 is Marker 1 with a molecular weight of DS15000, 1 indicates the negative control product, 2-4 indicate pZQ110 products, M2 is Marker 2 with a molecular weight of DS5000. According to FIG. 3F, two bands with molecular weights of 5300/16500 were obtained after digesting the plasmid pZQ110 by XbaI/HindIII. Thus, it can confirm that the pZQ110 was correctly constructed.
Additional recombinant expression plasmids for expressing intermediates or products were also constructed by methods similar to construction of pZQ110, as shown in Table 4.







TABLE 4







Information about the expression plasmids for expressing the


genes involved in the synthesis of rebaudioside A in bacterium










Plasmids
Whole Name





Synthesis of
pZQ3
pET28a-GGPPS-CDPS-KS


ent-kaurene,

GGPPS: Taxus canadensis (inserted into


an intermediate

pET28a at the NcoI/HindIII site)


for

CDPS: Stevia rebaudiana (inserted into the


synthesizing

pET28a at the XbaI/HindIII site)


stevia sugar

KS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)



pSY32
pET28a-GGPPS-CPS/KS




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into




the pET28a at the XbaI/HindIII site)



pSY33
pET28a-GGPPS-CPS/KS




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)



pZQ101
pET28a-GGPPS-CPS/KS




GGPPS: Stevia rebaudiana (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into




the pET28a at the XbaI/HindIII site)



pZQ102
pET28a-GGPPS-CPS/KS




GGPPS: Stevia rebaudiana (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)


Synthesis of
pZQ104
pET28a-GGPPS-CDPS-KS-KO-KAH-CPR-


steviolmonoside

UGT85C2




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CDPS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)



pZQ105
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into




the pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)



pZQ106
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)


Synthesis of
pZQ107
pET28a-GGPPS-CDPS-KS-KO-KAH-CPR-


steviolbioside

UGT85C2-UGTB1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CDPS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ108
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into




the pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ109
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the X




XbaI/HindIII site)



pSY200
pET28a-GGPPS-CDPS-KS-KO-KAH-CPR-




UGT85C2-UGTB1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CDPS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




IBGT glycosyltransferase (Ipomoea batatas)




(inserted into the pET28a at the XbaI/HindIII




site)



pSY201
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into




the pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




IBGT glycosyltransferase (Ipomoea batatas)




(inserted into the pET28a at the XbaI/HindIII




site)



pSY202
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




IBGT glycosyltransferase (Ipomoea batatas)




(inserted into the pET28a at the XbaI/HindIII




site)


Ligation of the
pZQ9
pET28a-GGPPS-CDPS-KS-KO-KAH-CPR-


genes involved

UGT85C2--UGTB1-UGT74G1-UGT76G1


in the synthesis

GGPPS: Taxus canadensis (inserted into the


of stevia sugar

pET28a at the NcoI/HindIII site)


for

CDPS: Stevia rebaudiana (inserted into the


synthesizing

pET28a at the XbaI/HindIII site)


rebaudioside A

KS: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT74G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT76G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ110
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into




the pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT74G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT76G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ120
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT74G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT76G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ111
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPPS: Taxus canadensis (inserted into the 




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into 




the pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the 




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44) 




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT74G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT76G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ121
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT74G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT76G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ112
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Physcomitrella patens (inserted into




the pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Arabidopsis thaliana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT74G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT76G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)



pZQ122
pET28a-GGPPS-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPPS: Taxus canadensis (inserted into the




pET28a at the NcoI/HindIII site)




CPS/KS: Gibberella fujikuroi (inserted into the




pET28a at the XbaI/HindIII site)




KO: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




KAH: Arabidopsis thaliana (SEQ ID NO: 44)




(inserted into the pET28a at the XbaI/HindIII




site)




CPR: Stevia rebaudiana (inserted into the




pET28a at the XbaI/HindIII site)




UGT85C2: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT74G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGT76G1: Stevia rebaudiana (inserted into




the pET28a at the XbaI/HindIII site)




UGTB1 glycosyltransferase (Starmerella




bombicola) (inserted into the pET28a at the




XbaI/HindIII site)









Example 3
Construction of the Fungal Expression Vectors
The optimized genes for the geranylgeranyl diphosphate synthase, ent-copalyl diphosphate synthase, ent-kaurene synthase, bifunctional ent-kaurene synthase, ent-kaurene oxidase, kaurenoic acid-13α-hydroxylase, UGT85C2 glycosyltransferase, UGTB1 glycosyltransferase, UGT74G1 glycosyltransferase, UGT76G1 glycosyltransferase, and cytochrome P450 redox protein, obtained in Example 1, were cloned into corresponding plasmids to construct gene expression plasmids for synthesizing rebaudioside A in fungus.
Firstly, the initial pAO815 vector (Invitrogen) was modified by introducing BamHI and XhoI restriction sites after pAO815 terminator by site-directed mutagenesis PCR. The modified pAO815 was named pSY01. The BamHI site in the pET28a-ggpps gene was removed by site-directed mutagenesis PCR and the BglII site in the pET21a-ks gene was removed by site-directed mutagenesis PCR.
The ggpps gene was amplified by PCR by using the pET28a-ggpps as template and introduce the EcoRI restriction sites at its two ends (introduction four A bases before ATG). The PCR fragment and the pSY01 vector were cleaved by EcoRI. The pSY01 vector and the ggpps fragment were recovered by a purification kit. Then the ggpps fragment was ligated to the pSY01 vector by T4 DNA ligase to construct the pSY01-ggpps plasmid.
The cdps gene (Taxus canadensis) was amplified by PCR by using the pES21a-cdps as template and introduce the BglII and NotI restriction sites its two ends (introduction 4 A bases before ATG). The PCR fragment was digested by BglII and NotI, and the pPIC3.5K (Invitrogen) was digested by BamHI and Nod. The pPIC3.5K and the cdps fragment were recovered by a purification kit and then the cdps fragment was ligated to the pPIC3.5K vector by T4 DNA ligase to construct pPIC3.5K-cdps. The same method was used to construct cps/ks, ks, ko, kah, ugt85c2, ugt74g1, ugt76g1 and cpr into pPIC3.5K, respectively, at the site indicated as “inserted gene” in the Figures.
The 5′ AOX-cps/ks-TT was amplified by PCR by using the pPIC3.5K-cps/ks as template and introduce the B gill and XhoI restriction sites at its two ends. The PCR fragment was digested by BglII and XhoI, and the pSY01-ggpps vector was digested by BamHI and XhoI. The pSY01-ggpps vector and the 5′AOX-cps/ks-TT fragment were recovered by a purification kit and then the 5′AOX-cps/ks-TT fragment was ligated to the pSY01-ggpps vector by T4 DNA ligase to construct a plasmid. The same method was used to ligate cps/ks (Physcomitrella patens), ko (Stevia rebaudiana), kah (Stevia rebaudiana), ugt85c2 (Stevia rebaudiana), ugtb1 (Starmerella bombicola), ugt74g1 (Stevia rebaudiana), ugt76g1 (Stevia rebaudiana) and cpr (Phaeosphaeria sp. L487) in series to finally obtain the expression plasmid pSY210. See FIG. 3G. FIG. 3I shows the PCR verification map of the plasmid pSY210, in which M1 is Marker with a molecular weight of DS2000, 1 indicates the positive control product, 2 and 3 indicate the pSY210 products. According to FIG. 3I, a band of about 1383 bp was obtained after verifying the pSY210 plasmid by PCR by using primers specific to the full-length coding gene of UGT74G1 glycosyltransferase. Thus, the pSY210 was correctly constructed.
Additional recombinant expression plasmids for expressing intermediates or products were also constructed by methods similar to construction of pZQ210, as shown in Table 5.







TABLE 5







Information about the expression plasmids for expressing the


genes involved in the synthesis of rebaudioside A in fungus










Names of




Plasmids
Alias





Synthesis
pSY16
pSY01-GGPP-CDPS-KS


of Kaurene

GGPP: Taxus Canadensis (inserted into the




pSY01 at the EcoRI site)




CDPS: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




KS: Stevia rebaudiana (inserted into the pSY01




at the BamHI/XhoI site)



pZQ132
pSY01-GGPP-CPS/KS




GGPP: Taxus Canadensis (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Physcomitrella patens (inserted into




the pSY01 at the BamHI/XhoI site)



pZQ133
pSY01-GGPP-CPS/KS




GGPP: Taxus Canadensis (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Gibberella fujikuroi (inserted into the




pSY01 at the BamHI/XhoI site)



pZQ201
pSY01-GGPP-CPS/KS




GGPP: Stevia rebaudiana (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Physcomitrella patens (inserted into




the pSY01 at the BamHI/XhoI site)



pZQ202
pSY01-GGPP-CPS/KS




GGPP: Stevia rebaudiana (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Gibberella fujikuroi (inserted into the




pSY01 at the BamHI/XhoI site)


Ligation of
pSY22
pSY01-GGPP-CDPS-KS-KO-KAH-CPR-


the genes

UGT85C2-UGTB1-UGT74G1-UGT76G1


involved in

GGPP: Taxus Canadensis (inserted into the


the synthesis

pSY01 at the EcoRI site)


of stevia sugar

CDPS: Stevia rebaudiana (inserted into the


for

pSY01 at the BamHI/XhoI site)


synthesizing

KS: Stevia rebaudiana (inserted into the pSY01


rebaudioside A

at the BamHI/XhoI site)




KO: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




KAH: Stevia rebaudiana (SEQ ID NO: 10 or




SEQ ID NO: 44) (inserted into the pSY01




at the BamHI/XhoI site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pSY01 at the BamHI/XhoI site)




UGT85C2: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT74G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT76G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGTB1 glycosyltransferase (inserted into the




pSY01 at the BamHI/XhoI site)



pZQ210
pSY01-GGPP-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPP: Taxus Canadensis (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Physcomitrella patens (inserted into




the pSY01 at the BamHI/XhoI site)




KO: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




KAH: Stevia rebaudiana (SEQ ID NO: 10)




(inserted into the pSY01 at the BamHI/XhoI




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pSY01 at the BamHI/XhoI site)




UGT85C2: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT74G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT76G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGTB1 glycosyltransferase (inserted into the




pSY01 at the BamHI/XhoI site)



pZQ220
pSY01-GGPP-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPP: Taxus Canadensis (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Gibberella fujikuroi (inserted into the




pSY01 at the BamHI/XhoI site)




KO: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pSY01 at the BamHI/XhoI




site)




CPR: Phaeosphaeria sp. L487 (inserted into




the pSY01 at the BamHI/XhoI site)




UGT85C2: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT74G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT76G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGTB1 glycosyltransferase (inserted into the




pSY01 at the BamHI/XhoI site)



pZQ211
pSY01-GGPP-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPP: Taxus Canadensis (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Physcomitrella patens (inserted into




the pSY01 at the BamHI/XhoI site)




KO: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




KAH: Stevia rebaudiana (SEQ ID NO: 10)




(inserted into the pSY01 at the BamHI/XhoI




site)




CPR: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT85C2: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT74G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT76G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGTB1 glycosyltransferase (inserted into the




pSY01 at the BamHI/XhoI site)



pZQ221
pSY01-GGPP-CPS/KS-KO-KAH-CPR-




UGT85C2-UGTB1-UGT74G1-UGT76G1




GGPP: Taxus Canadensis (inserted into the




pSY01 at the EcoRI site)




CPS/KS: Gibberella fujikuroi (inserted into the




pSY01 at the BamHI/XhoI site)




KO: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




KAH: Stevia rebaudiana (SEQ ID NO: 44)




(inserted into the pSY01 at the BamHI/XhoI




site)




CPR: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT85C2: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT74G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGT76G1: Stevia rebaudiana (inserted into the




pSY01 at the BamHI/XhoI site)




UGTB1 glycosyltransferase (inserted into the




pSY01 at the BamHI/XhoI site)









Example 4
Expression Profiles of Each Gene in E. coli 
The plasmids pET21a-cdps, pET21a-ks, pET21a-ko, pET21a-kah, pET21a-ugt85c2, pET21a-ugt74g1 and pET21a-ugt76g1 obtained in Example 2 were transformed into host cell BL21(DE3).
Each clone was respectively picked up to 2 ml LB culture medium (100 mg/L ampicillin), cultured at 37° C. overnight, and then inoculated, in 1% (v/v) inoculation amount, to 2 ml fresh LB culture medium supplemented with the same antibiotic. The culture was cultivated at 37° C. until OD600 reached 0.3-0.4. IPTG was added to a final concentration of 0.1 mM, and then expression was induced for 6 h at 18° C. After that, each fermentation broth was subjected to SDS-PAGE analysis.
Results were shown in FIGS. 4A, 4B, 4C, 4D and 4E. According to FIG. 4A, the genes cdps and ugt85C2 expressed evidently. According to FIG. 4B, the genes ko and ks expressed evidently. According to FIG. 4C, the kah gene expressed evidently. According to FIG. 4D, the ugt74g1 gene had less expression. And, according to FIG. 4E, the ugt76g1 gene expressed evidently.
Example 5
Transformation of Vectors and Prokaryotic Expression
Host cells, E. coli BL21(DE3), were co-transformed by the gene expression plasmids for synthesizing rebaudioside A or intermediates obtained in Example 2 and the expression plasmid pJF47 (FIG. 3D) for strengthening the precursor pathway to produce recombinant E. coli BL21(DE3) transformed with the gene expression plasmids for synthesizing rebaudioside A or intermediates and pJF47.
The genome of E. coli MG1655 was firstly extracted and then each gene was amplified by using the primers shown in the Table below. Each gene was cloned into the plasmid pET21d at the NcoI/EcoRI site. The pET21c-ispF was digested by SpeI/EcoRI, and the pET21d-idi was digested by XbaI/EcoRI. The pET21c-ispF vector and the idi gene were recovered and ligated together to construct a plasmid pET21d-ispF-idi. The plasmid pET21c-ispD was digested by SpeI/EcoRI, and the pET21d-ispF-idi was digested by XbaI/EcoRI. The pET21c-ispD and ispF-idi gene fragment were recovered and ligated together to construct pET21d-ispD-ispF-idi. pET21d-dxs was digested by SpeI/EcoRI, and pET21d-ispD-ispF-idi was digested by XbaI/EcoRI. The pER21d-dxs and ispD-ispF-idi fragment were recovered and ligated together to construct pET21d-dxs-ispD-ispF-idi, which is designated as pJF47.













Gene
Genbank No.
Primers
Cloning Site







dxs
NP_414954.1
dxs-F: CATGCCATGGGCATGAGTTTTGATATTGCCAAATACCCG
NcoI/EcoRI




(SEQ ID NO: 53)



 




dxs-R: CGGAATTCACTAGTTTATGCCAGCCACCTT





(SEQ ID NO: 54)



 


ispD
NP_417227.1
ispD-F: CATGCCATGGGCATGGCAACCACTCATTTGGATGTT
NcoI/EcoRI




(SEQ ID NO: 55)



 




ispD-R: CGGAATTCACTAGTTTATGTATTCTCCTGATGGATGGTT





(SEQ ID NO: 56)



 


ispF
NP_417226.1
ispF-F: CATGCCATGGGCATGCGAATTGGACACGGTTTTG
NcoI/EcoRI




(SEQ ID NO: 57)



 




ispF-R: CGGAATTCACTAGTTCATTTTGTTGCCTTAATGAGTAG





(SEQ ID NO: 58)



 


idi
NP_417365.1
idi-F: CATGCCATGGGCATGCAAACGGAACACGTCATTTTA
NcoI/EcoRI




(SEQ ID NO: 59)



 




idi-R: CGGAATTCTTATTTAAGCTGGGTAAATGCAG





(SEQ ID NO: 60)









Clones were picked up to LB liquid culture medium containing ampicillin (100 mg/L) and kanamycin (50 mg/L), and cultured at 37° C. for 8 h. The thalli were collected by centrifugation and 10% (v/v) glycerin were added to prepare an inoculum. The inoculum was stored at −80° C.
The inoculum was inoculated, in 5% (v/v) inoculation amount, into 10 ml M9 culture medium (purchased from Shanghai Sangon, containing 100 mg/L ampicillin and 50 mg/L kanamycin) in a 100 ml shake flask, cultured at 37° C. After the OD reaches about 0.4, 0.05 mM IPTG was added for induction culture. The culture was cultivated at 22° C. for 5 days. The fermentation broth was collected and stored at −80° C.
Example 6
Detection of Ent-Kaurene in Some Recombinant Cells for Synthesizing Ent-Kaurene, an Intermediate for Stevia Sugar, Prepared in Example 5
The recombinant E. coli BL21(DE3) containing the gene expression plasmids for synthesizing rebaudioside A or intermediates thereof and the expression plasmid pJF47 for strengthening the precursor pathway was fermented for detection ent-kaurene.
50 μl 2M HCl were added into 1 ml fermentation broth of Example 5 and then the same volume of ethyl acetate were added. The mixture was subjected to ultrasonic treatment in an ice bath for 1 min and then vortexed at ambient temperature for 20 min. After that, the mixture was centrifuged at 12000 rpm for 1 min to separate the organic phase. The organic phase was sucked out and the residual water phase was extracted by the same volume of ethyl acetate for one time. The organic phases were pooled and extracted to obtain the product containing ent-kaurene. The ent-kaurene was directly detected by GC-MS.
The ent-kaurene was detected by GC-MS under the conditions as follows: Agilent 7890-5975 GC-MS system was used. The column was HP-5MS, the carrier gas was helium gas. The flow rate was 1 ml/min. The loading amount was 5 μl without splitflow and the loading temperature was 250° C. The warming procedure of the column included: 100° C. for 2 min, warming to 250° C. by 5° C./min, and 250° C. for 15 min. Solvent was loaded in a delay of 4.50 min. Ion scanning (m/z 272) was used for scanning. The dissociation voltage was 70 eV.
The mass spectrogram of ent-kaurene was shown in FIG. 5A. The appearance time of ent-kaurene was 11.212 min. The yield of ent-kaurene produced by the recombinant E. coli BL21(DE3) strains transformed by various vectors was shown in FIG. 5B and Table 6.







TABLE 6







The yield of ent-kaurene in a portion of fermentation broth obtained in


Examples 6 and 8










Host
Plasmids
OD600
Yield of ent-kaurene (mg/L)













BL21 (DE3)
pET28a + pJF47
24.7
0



(control, blank





plasmid)




BL21 (DE3)
pZQ3 + pJF47
31.8
189


BL21 (DE3)
pSY32 + pJF47
24.0
1105


BL21 (DE3)
pSY33 + pJF47
24.2
876


BL21 (DE3)
pZQ101 + pJF47
20.1
403


BL21 (DE3)
pZQ102 + pJF47
22.3
296









According to FIG. 6, except the blank vector pET28a, the lowest ent-kaurene yield was obtained by the cells transformed by pZQ3 (GGPP was from Taxus Canadensis and CDPS and KS were from Stevia rebaudiana), which was merely 189 mg/L. The highest ent-kaurene yield was obtained from the cells transformed by pSY32 (GGPP was from Taxus Canadensis and CDPKS were from Physcomitrella patens), which was up to 1105 mg/L.
The results show that in the E. coli expression system, the bifunctional CPS/KS module was superior in expressing synthetic ent-kaurene than the CDPS and KS modules, and the CPS/KS from Physcomitrella patens and GGPPS from Taxus Canadensis produce the preferred results.
Example 7
Detection of Product Produced by Some Recombinant Cells for Synthesizing Rebaudioside A Prepared in Example 5
One milliliter ethyl acetate was added into 1 ml fermentation broth of Example 5. The mixture was subjected to ultrasonic treatment in an ice bath for 1 min and then vortexed at ambient temperature for 20 min. After that, the mixture was centrifuged at 12000 rpm for 1 min to separate the organic phase. The organic phase was sucked out and the residual water phase was extracted by the same volume of ethyl acetate for one time. The organic phases were pooled and extracted to obtain the extraction product containing steviol glycosides, including kaurenoic acid, steviol, steviolmonoside and rebaudioside A. The organic phase was dried under vacuum. 500 μl acetonitrile were added to re-dissolve the residues and the product was detected by HPLC-MS.
Steviol glycosides, including kaurenoic acid, steviol, were detected by HPLC-MS (Aglient, LC1200/MS-QTOF6520) equipped with C18 reversed phase chromatographic column (Waters, Xterra, 2.1×50 mm). The mobile A phase is methanol+0.1% formic acid, B phase is water+0.1% formic acid. Gradient elution conditions included that the A phase increased from 30% to 100% and the B phase decreased from 70% to 0 within 0-35 min. The flow rate was 0.2 ml/min and the loading amount was 8 μl. Mass spectrum conditions: Negative ion scanning was used and the scanning range (m/z) was 100-1500.
The results were shown in FIG. 6. FIG. 6B shows the detection results of kaurenoic acid, in which the results obtained from high resolution mass spectrum show that there is the 303.2291 ion by positive ion scanning FIG. 6C shows the detection results of steviol, in which the results obtained from high resolution mass spectrum show that there is the 319.2244 ion by positive ion scanning FIG. 6D shows the detection results of steviolmonoside, in which the results obtained from high resolution mass spectrum show that there is the 481.2754 ion by positive ion scanning FIG. 6E shows the detection results of rebaudioside A, in which the results obtained from high resolution mass spectrum show that there is the 967.43 ion by positive ion scanning. The results could demonstrate that the recombinant E. coli could successfully synthesize kaurenoic acid, steviol, steviolmonoside and rebaudioside A. The yield of rebaudioside A produced by cells transformed by various vectors are shown in FIG. 6F and Table 7.









TABLE 7







Yield


Sample
OD600
of rebaudioside A (mg/L)

















BL21 (DE3) (pET28a + pJF47)
23.9
0


(Control, blank plasmid)




BL21 (DE3) (pZQ9 + pJF47)
25.6
1.8


BL21 (DE3) (pZQ110 + pJF47)
20.2
11


BL21 (DE3) (pZQ120 + pJF47)
22.3
8.4


BL21 (DE3) (pZQ111 + pJF47)
21
4.0


BL21 (DE3) (pZQ121 + pJF47)
21.2
3.0


BL21 (DE3) (pZQ112 + pJF47)
22.5
2









From the synthesis pathway of rebaudioside A, it can be found that the UGTB1 glycosyltransferases from Starmerella bombicola could successfully further glycosylated the C-2′ site at the C-13 glucose of steviolmonoside to produce steviolbioside. By comparing the yield of rebaudioside A, it can be found that proteins from different sources could influence the yield. The highest yield was produced by pZQ110, which could reach to 11 mg/L. In the pZQ110, GGPPS was from Taxus canadensis, CPS/KS was from Physcomitrella patens, KO and KAH were from Stevia rebaudiana, CPR was from Phaeosphaeria, UGT76G1, UGT74G1 and UGT85C2 were from Stevia rebaudiana, and UGTB1 glycosyltransferase was from Starmerella bombicola. 
Example 8
Transformation of Fungal Vector and Expression in Yeast
The expression plasmids obtained in Example 3 for synthesizing rebaudioside A was digested by SalI to produce a linearized vector. The linearized vector was transformed by electroporation into Pichia pastoris KM71 (purchased from Invitrogen) to produce recombinant Pichia pastoris KM71 having plasmids integrated into the genome at the His site. Clone was picked up to BMGY liquid culture medium and cultured at 30° C. for 24 hours. The cells were collected by centrifugation and 10% (v/v) glycerol was added to prepare an inoculum. The inoculum was stored at −80° C.
Clone was picked up to 50 ml BMGY in 500 ml shake flask and subjected to 28° C. overnight. The culture was cultured at 250 rpm until OD600 reached 2-5 (about 16-20 hours). The cells were collected by centrifugation and the supernate was discarded. The cells were inoculated to 10 ml BMMY in 100 ml shake flask and then cultured at 28° C. and 250 rpm. 50 μl methanol was added every 24 hours. After culturing 5 days, the fermentation broth was collected and stored at −80° C.
Example 9
Detection of Ent-Kaurene in Some Recombinant Cells for Synthesizing Ent-Kaurene, an Intermediate for Stevia Sugar, Prepared in Example 8
The product, ent-kaurene, produced by fermenting the recombinant Pichia pastoris KM71 obtained in Example 8 was detected.
50 μl 2M HCl were added into 1 ml fermentation broth of Example 8 and then the same volume of ethyl acetate were added. The mixture was subjected to ultrasonic treatment in an ice bath for 1 min and then vortexed at ambient temperature for 20 min. After that, the mixture was centrifuged at 12000 rpm for 1 min to separate the organic phase. The organic phase was sucked out and the residual water phase was extracted by the same volume of ethyl acetate for one time. The organic phases were pooled. The ent-kaurene was detected by GC-MS. The ent-kaurene was successfully detected and its yield was shown in FIG. 7 and Table 8.









TABLE 8







Yield


Sample
OD600 Value
of ent-Kaurene (mg/L)

















KM71 (pPIC3.5K) (Control,
24.7
0


blank control)




KM71 (pSY16)
124
189


KM71 (pZQ132)
143
1172


KM71 (pZQ133)
135
827


KM71 (pZQ201)
120
438


KM71 (pZQ202)
123
342









According to the data on the yield, in the Pichia pastoris expression system, the bifunctional CPS/KS combination was superior in synthesizing ent-kaurene than the CDPS/KS combination. With the most preferred expression vector pZQ132, the yield of ent-kaurene could reach 1172 mg/L, which was 5.4 folds higher than the yield obtained by the pSY16 expression vector.
Example 10
Detection of Product Produced by Some Recombinant Yeast Cells for Synthesizing Rebaudioside A Prepared in Example 8
One milliliter ethyl acetate was added into 1 ml fermentation broth of Example 8. The mixture was subjected to ultrasonic treatment in an ice bath for 1 min and then vortexed at ambient temperature for 20 min. After that, the mixture was centrifuged at 12000 rpm for 1 min to separate the organic phase. The organic phase was sucked out and the residual water phase was extracted by the same volume of ethyl acetate for one time. The organic phases were pooled and extracted to obtain the steviol glycosides, including kaurenoic acid, steviol, steviolmonoside and rebaudioside A. The organic phase was dried under vacuum. 500 μl acetonitrile were added to re-dissolve the residues and the product was detected by HPLC-MS. The results of high resolution mass spectrum obtained by positive ion scanning show that there are 303.2291, 319.2244, 481.2754 and 967.43 ions, indicating that kaurenoic acid, steviol, steviolmonoside and rebaudioside A were successfully synthesized in the recombinant Pichia pastoris. The yields of rebaudioside A of each recombinant yeast cells were shown in FIG. 8 and Table 9.









TABLE 9





Sample
OD600
Yield of rebaudioside A (mg/L)

















KM71 (pPIC3.5K) (Control,
24.7
0


blank plasmid)




KM71 (pSY22)
120
1.7


KM71 (pZQ210)
132
12


KM71 (pZQ220)
141
8.9


KM71 (pZQ211)
118
4.5


KM71 (pZQ221)
119
3.6









According to FIG. 8 and Table 9, the highest yield of rebaudioside A was obtained by pZQ210, which reached 12 mg/L. In pZQ210, GGPP was from Taxus canadensis, CPS/KS was from Physcomitrella patens, KO and KAH were from Stevia rebaudiana, CPR was from Phaeosphaeria, UGT76G1, UGT74G1 and UGT85C2 were from Stevia rebaudiana, and UGTB1 glycosyltransferase was from Starmerella bombicola. 
Example 11
Study on Function of UGTB1 or IBGT Glycosyltransferase
One milliliter ethyl acetate was added into 1 ml fermentation broth of Example 5. The mixture was subjected to ultrasonic treatment in an ice bath for 1 min and then vortexed at ambient temperature for 20 min. After that, the mixture was centrifuged at 12000 rpm for 1 min to separate the organic phase. The organic phase was sucked out and the residual water phase was extracted by the same volume of ethyl acetate for one time. The organic phases were pooled and extracted to obtain steviolmonoside and steviolbioside. The organic phase was dried under vacuum. 500 μl acetonitrile were added to re-dissolve the residues and the product was detected by HPLC-MS. The results were shown in FIG. 9. According to FIG. 9C, the fermentation broth of pZQ104, pZQ105 or pZQ106 which only contains UGT85c2 glycosyltransferase but does not contain UGTB1 glycosyltransferase or IBGT glycosyltransferase only contained steviolmonoside. No steviolbioside was detected in these broths. The pZQ107, pZQ108, pZQ109, pSY200, pSY201 and pSY202 contain both UGT85c2 glycosyltransferase and UGTB1 glycosyltransferase or IBGT glycosyltransferase. Steviolbioside was successfully detected in their fermentation broths (FIGS. 9A, 9B and 9D).
The inventors have also detected the yields of steviolmonoside and steviolbioside produced by prokaryotic cells transformed by different expression vectors. The results were shown in FIG. 10 and Table 10.










TABLE 10







Yield of
Yield of




steviolmonoside
steviolbioside


Sample
OD600
(mg/L)
(mg/L)


















BL21(DE3) (pET28a + pJF47)
23.0
0
0


(Control, blank plasmid)





BL21(DE3) (pZQ104 + pJF47)
24.9
24
0


BL21(DE3) (pZQ105 + pJF47)
20.6
150
0


BL21(DE3) (pZQ106 + pJF47)
22.1
98
0


BL21(DE3) (pZQ107 + pJF47)
21.3
0.08
33


BL21(DE3) (pZQ108 + pJF47)
21.9
1.3
206


BL21(DE3) (pZQ109 + pJF47)
22.7
0.7
134


BL21(DE3) (pSY200 + pJF47)
21.1
0.09
31


BL21(DE3) (pSY200 + pJF47)
21.3
1.2
215


BL21(DE3) (pSY200 + pJF47)
22.8
0.6
140









According to FIG. 10 and Table 10, the expression vectors pZQ104, pZQ105 and pZQ106 that do not contain UGTB1 glycosyltransferase or IBGT glycosyltransferase and the negative pET28a could only produce steviolmonoside but did not produce steviolbioside. From the biocatalytic efficiency, UGTB1 glycosyltransferase or IBGT glycosyltransferase could produce very high conversion efficiency when catalyzing steviolmonoside, which could reach up to 99%.
All literature references cited herein are hereby incorporated by reference, just as each reference was cited independently. Moreover, it should be understood that the skilled in the art can change or modify the present invention based on the above disclosure and the equivalent arrangements are also included in the scope of the claims attached in the application.

## Claims

1. A method for synthesizing a steviol glycoside with an additional glucose unit, the method comprising:
providing an isolated recombinant host cell transformed with an expression construct encoding a glycosyltransferase polypeptide comprising the amino acid sequence of SEQ ID NO: 41 or 51, and wherein said host cell comprises a steviol glycoside with an O-glucose residue;
allowing the glycosyltransferase to catalyze transfer of glucose to the O-glucose residue of the steviol glycoside to produce a steviol glycoside with an additional glucose unit; and
isolating the steviol glycoside with the additional glucose unit.

2. The method according to claim 1, wherein catalytic substrates of the glycosyltransferase include steviolmonoside, rubusoside, stevioside, or rebaudioside A.

3. The method according to claim 2, wherein the glycosyltransferase catalyzes production of steviolbioside from steviolmonoside.

4. The method according to claim 1, wherein the host cell further comprises one or more of the following enzymes:
Geranylgeranyl diphosphate synthase;
Bifunctional ent-kaurene synthase, or a combination of ent-copalyl diphosphate synthase and ent-kaurene synthase;
Ent-kaurene oxidase;
Cytochrome P450 redox protein;
Kaurenoic acid-13α-hydroxylase;
UGT85C2 glycosyltransferase;
UGT74G1 glycosyltransferase; or
UGT76G1 glycosyltransferase.

5. The method according to claim 1, wherein the host cell further comprises one or more genes encoding: 1-deoxy-D-xylulose-5-phosphate synthase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, or isopentenyl-diphosphate delta-isomerase.

6. The method according to claim 1, wherein the host cell is a prokaryotic cell.

7. The method according to claim 6, wherein the prokaryotic cell is Escherichia coli, Bacillus subtilis, Acetobacteria, Corynebacterium or Brevibacterium.

8. The method according to claim 7, wherein the E. coli is BL21, BLR, DH10B, HMS, C43, JM109, DH5α, or Noveblue.

9. The method according to claim 1, wherein the host cell is a eukaryotic cell.

10. The method according to claim 9, wherein the eukaryotic cell is yeast, mold, or basidiomycete.

11. The method according to claim 10, wherein the yeast is Pichia pastoris, Saccharomyces cerevisiae, or Kluyveromyces lactis.

12. The method according to claim 11, wherein the Pichia pastoris is GS115, MC100-3, SMD1163, SMD1165, SMD1168, or KM71.

13. The method according to claim 11, wherein the Saccharomyces cerevisiae is W303, CEN.PK2, S288c, FY834, or S1949.

14. The method according to claim 11, wherein the Kluyveromyces lactis is GG799.