CN105985938B - Glycosyltransferase mutant protein and its application - Google Patents

Abstract

The invention discloses a glycosyltransferase mutein and application thereof. Specifically, the invention utilizes homology alignment and site-directed mutagenesis to find a novel mutant protein of glycosyltransferase, wherein the mutant protein is a non-natural protein, has glycosyltransferase catalytic activity, and contains the following core amino acids related to the enzymatic activity: amino acid 142 is A; amino acid 144 is H or F; and amino acid 339 is G; wherein the amino acid position numbering is based on the sequence set forth in SEQ ID NO. 13. Experiments prove that after modification of key sites in glycosyltransferase, the enzyme which is inactive or has poor activity originally can be changed or new enzyme activity can be endowed. Furthermore, the present inventors have found that the glycosyltransferase of the present invention has a significant increase in its expression level after terminal truncation.

Description

Glycosyltransferase mutant protein and its application

Technical Field

The present invention relates to the fields of biotechnology and plant biology, and in particular to a glycosyltransferase mutant protein and application thereof.

Background Art

Ginsenoside is a general term for saponins isolated from ginseng and its congener plants (such as Panax notoginseng, American ginseng, etc.). It belongs to the triterpenoid saponin and is the main active ingredient in ginseng. At present, at least 100 saponins have been isolated from ginseng. From a structural point of view, ginsenosides are bioactive small molecules formed by glycosylation of sapogenins. There are only a limited number of sapogenins in ginsenosides, mainly dammarane-type protopanaxadiol and protopanaxatriol, as well as oleanolic acid. After glycosylation, sapogenins can increase water solubility and produce different physiological activities. Protopanaxatriol has a hydroxyl group at C3, C6 and C20. The protopanaxatriol-type saponins discovered so far are all glycosylated on the hydroxyl groups of C6 and (or C20). Protopanaxatriol-type saponins with glycosyl groups at C3 have not been reported. The glycosyl group can be glucose, rhamnose, xylose, and arabinose.

Some protopanaxatriol saponins have been shown to have a wide range of physiological functions and medicinal values, including anti-allergic and anti-inflammatory, immune regulation, anti-fatigue, and heart protection. Ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) belongs to the protopanaxatriol saponin, and its content in ginseng is also very low, and it is also a rare ginsenoside. The structure of ginsenoside F1 is very similar to CK, and there is also a glucose group attached to the C-20 hydroxyl group of the sapogenin. Ginsenoside F1 also has unique medicinal value. It has anti-aging and antioxidant functions. Ginsenoside Rh1 (6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol) belongs to the protopanaxatriol saponin, and its content in ginseng is also very low, and it is also a rare ginsenoside. The structure of ginsenoside Rh1 is very similar to that of F1, but its glycosylation site is the hydroxyl group at the C6 position. Ginsenoside Rh1 also has special physiological functions and can resist allergies and inflammation. Ginsenoside Rg1 (6-O-β-(D-glucopyranosyl)-20-O-β-(D-glucopyranosyl)-protopanaxadiol) belongs to the protopanaxatriol type saponin, which has anti-fatigue and heart protection functions.

The degree of glycosylation modification of rare ginsenosides is generally low (containing only 1-2 sugar groups), while the degree of glycosylation modification of rich saponins is higher (sugar chains composed of multiple sugar groups). The traditional production method of rare ginsenoside monomers is to use the total saponins or rich saponins of ginseng or Panax notoginseng as raw materials, relying on chemical, enzymatic and microbial fermentation hydrolysis methods. Since wild ginseng resources have been basically exhausted, ginsenoside resources currently come from the artificial cultivation of ginseng or Panax notoginseng, and the growth cycle of artificial cultivation is long (generally more than 5-7 years), and is subject to geographical restrictions. It is also often affected by pests and diseases and requires the application of large amounts of pesticides. Therefore, the artificial cultivation of ginseng or Panax notoginseng has serious continuous cropping obstacles (ginseng or Panax notoginseng planting areas need to be fallow for more than 5-15 years to overcome the continuous cropping obstacles), so the yield, quality and safety of ginsenosides are facing challenges.

The development of synthetic biology has provided new opportunities for the heterologous synthesis of natural products from plants. Using yeast as the chassis, through the assembly and optimization of metabolic pathways, it has been realized to use cheap monosaccharides to ferment and synthesize artemisinic acid or dihydroartemisinic acid, and then produce artemisinin through a one-step chemical transformation method, which shows the great potential of synthetic biology in the drug synthesis of natural products. Rare ginsenoside monomers are heterologously synthesized by synthetic biology methods using yeast chassis cells. The raw materials are cheap monosaccharides, and the preparation process is a safe and controllable fermentation process, avoiding any external pollution (for example, pesticides used when the raw material plants are artificially planted). Therefore, the preparation of rare ginsenoside monomers by synthetic biology technology not only has cost advantages, but also can ensure the quality and safety of the finished product. Use synthetic biology technology to prepare sufficient amounts of various high-purity rare ginsenoside monomers for activity determination and clinical trials, and promote the development of innovative drugs for rare ginsenosides.

The use of synthetic biology methods to artificially synthesize these triol-type ginsenosides with medicinal activity requires not only the construction of a metabolic pathway for the synthesis of protopanaxatriol, but also the identification of glycosyltransferases that catalyze the glycosylation of ginsenosides. The function of glycosyltransferases is to transfer the glycosyl on the glycosyl donor (nucleoside diphosphate sugar, such as UDP-glucose) to different glycosyl acceptors. Depending on the amino acid sequence, there are currently 94 families of glycosyltransferases. More than a hundred different glycosyltransferases have been found in the currently sequenced plant genomes. The glycosyl acceptors of these glycosyltransferases include sugars, lipids, proteins, nucleic acids, antibiotics and other small molecules.

Therefore, more research and development of glycosyltransferases is needed in this field to obtain rare ginsenosides in an economical and efficient manner through more high-yield glycosyltransferases.

Summary of the invention

The invention provides a mutant protein of glycosyltransferase, and the enzyme activity thereof can be improved or the expression amount thereof can be increased by adopting the mutant protein.

In a first aspect of the present invention, a mutant protein of a glycosyltransferase is provided, wherein the mutant protein is a non-natural protein, and the mutant protein has glycosyltransferase catalytic activity, and the mutant protein contains the following core amino acids related to the enzyme catalytic activity:

The 142nd amino acid is A;

Amino acid at position 144 is H or F; and

The 339th amino acid is G;

Wherein, the amino acid position numbering is based on the sequence shown in SEQ ID NO.:13.

In another preferred embodiment, except for amino acids 142, 144, and 339, the remaining amino acids of the mutant protein are identical or substantially identical to the sequence shown in SEQ ID NO.:13.

In another preferred embodiment, the basic identity means that there are at most 50 (preferably 1-20, more preferably 1-10) amino acid differences, wherein the differences include amino acid substitutions, deletions or additions, and the mutant protein still has glycosyltransferase activity.

In another preferred embodiment, the homology with the sequence shown in SEQ ID NO.: 13 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98%.

In another preferred embodiment, the mutant protein is shown in SEQ ID NO.: 13 or 16.

In another preferred embodiment, the mutant protein is not the sequence shown in SEQ ID NO.: 1, 2 or 4.

In another preferred embodiment, the glycosyltransferase catalytic activity includes catalyzing one or more of the following reactions:

(a) transferring a glycosyl group from a glycosyl donor to the hydroxyl group at the C-20 position of a tetracyclic triterpenoid compound;

(b) Transferring the glycosyl group from the glycosyl donor to the hydroxyl group at the C-6 position of the tetracyclic triterpenoid compound.

In another preferred embodiment, the mutant protein is as shown in SEQ ID NO.: 6, 11-12, and the glycosyltransferase catalytic activity includes transferring the glycosyl from the glycosyl donor to the C-20 position and the C-6 hydroxyl group of the tetracyclic triterpenoid compound.

In another preferred embodiment, the mutant protein is as shown in SEQ ID NO.: 6, 8, 11-12, 16, and the glycosyltransferase activity includes transferring the glycosyl from the glycosyl donor to the C-6 hydroxyl group of the tetracyclic triterpenoid compound.

In another preferred embodiment, the mutant protein is as shown in SEQ ID NO.: 6-7, 9-13, and the glycosyltransferase activity includes transferring the glycosyl from the glycosyl donor to the C-20 hydroxyl group of the tetracyclic triterpenoid compound.

In another preferred embodiment, the glycosyltransferase catalytic activity includes catalyzing one or more of the following reactions:

(A)

Wherein, the compound of formula (I) is protopanaxatriol, the compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), and the mutant protein includes SEQ ID NO.: 6-7, 9-13;

(B)

Wherein, the compound of formula (I) is protopanaxatriol, and the compound of formula (III) is ginsenoside Rh1 (6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol); or

The compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), and the compound of formula (IV) is ginsenoside Rg1 (6-O-β-(D-glucopyranosyl)-20-O-β-(D-glucopyranosyl)-protopanaxatriol);

The mutant proteins include SEQ ID NO.: 6, 8, 11-12, 16;

(C)

Wherein, the compound of formula (I) is protopanaxatriol, the compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), the compound of formula (IV) is ginsenoside Rg1 (6-O-β-(D-glucopyranosyl)-20-O-β-(D-glucopyranosyl)-protopanaxatriol), and the mutant protein includes SEQ ID NO.: 6, 11-12;

(D)

Wherein, the compound of formula (I) is protopanaxatriol, the compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), the compound of formula (III) is ginsenoside Rh1 (6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), and the mutant protein includes SEQ ID NO.: 11-12.

In another preferred embodiment, the glycosyl donor includes a nucleoside diphosphate sugar. Preferably, the glycosyl donor is selected from the following group: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose, TDP-galactose, CDP-galactose, GDP-galactose, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-rhamnose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, or other nucleoside diphosphate hexoses or nucleoside diphosphate pentoses, or a combination thereof.

In another preferred embodiment, the glycosyl donor includes uridine diphosphate (UDP) sugar, preferably, the glycosyl donor is selected from the following group: UDP-glucose, UDP-galacturonic acid, UDP-galactose, UDP-arabinose, UDP-rhamnose, or other uridine diphosphate hexose or uridine diphosphate pentose, or a combination thereof.

In another preferred embodiment, the pH of the reaction system is: pH 4.0-10.0, preferably pH 6.0-8.5.

In another preferred embodiment, the temperature of the reaction system is 10°C-105°C, preferably 25°C-35°C.

In another preferred embodiment, the mutant protein is selected from:

(a1) the sequence shown in SEQ ID NO.: 13; or

(b1) A mutant protein having the glycosyltransferase catalytic activity after one or more (preferably 1-50, preferably 1-20, more preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and/or deletions of the sequence shown in SEQ ID NO.: 13.

In another preferred embodiment, the substitution is 1-50 conservative amino acid substitutions, preferably 1-20, more preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In another preferred embodiment, the deletion is a deletion of 1-5 amino acids at the C-terminus, preferably a deletion of 2-4 amino acids.

In another preferred embodiment, the mutant protein sequence is shown in SEQ ID NO.: 6-8.

In another preferred embodiment, when 1-5 amino acids are missing from the C-terminus of the mutant protein, its expression level is increased by at least 50%, more preferably 70%-100%, compared with the protein shown in SEQ ID NO.: 13.

In another preferred embodiment, the addition is the addition of a tag sequence, a signal sequence or a secretion signal sequence.

In another preferred embodiment, the mutant protein further contains one or more of the following amino acids:

The 10th amino acid is V;

The 13th amino acid is F;

Amino acid at position 82 is C; and

The 186th amino acid is L.

The second aspect of the present invention provides a polynucleotide, wherein the polynucleotide encodes the mutant protein described in the first aspect of the present invention.

In another preferred embodiment, the polynucleotide sequences are as shown in SEQ ID NOs.: 19 and 21, encoding the sequences shown in SEQ ID NOs.: 13 and 16, respectively.

The third aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the second aspect of the present invention.

In another preferred embodiment, the vector includes an expression vector, a shuttle vector, and an integration vector.

The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

In another preferred embodiment, the host cell is a ginseng cell.

In a fifth aspect, the present invention provides a method for modifying glycosyltransferase, comprising the steps of:

(i) performing homology comparison between the glycosyltransferase and the sequence shown in SEQ ID NO.: 13;

(ii) screening out the sequence in step (i) having at least 80% homology to the sequence shown in SEQ ID NO.: 13; preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98%;

(iii) modifying the sequence selected in step (ii), wherein the modified glycosyltransferase contains the following core amino acids:

The 142nd amino acid is A;

Amino acid at position 144 is H or F; and

The 339th amino acid is G;

Wherein, the amino acid position numbering is based on the sequence shown in SEQ ID NO.: 13,

And at least one of the core amino acids is artificially modified.

In another preferred embodiment, the modified glycosyltransferase further contains the following core amino acids:

The 10th amino acid is V;

The 13th amino acid is F;

The amino acid at position 82 is C; and/or

The 186th amino acid is L;

In another preferred embodiment, the C-terminus of the modified glycosyltransferase lacks 1-5 amino acids.

In a sixth aspect, the present invention provides the use of the mutant protein described in the first aspect of the present invention, wherein the mutant protein is used to catalyze one or more of the following reactions, or is used to prepare a catalytic preparation for catalyzing one or more of the following reactions:

(a) transferring a glycosyl group from a glycosyl donor to the hydroxyl group at the C-20 position of a tetracyclic triterpenoid compound;

(b) Transferring the glycosyl group from the glycosyl donor to the hydroxyl group at the C-6 position of the tetracyclic triterpenoid compound.

In another preferred embodiment, the mutant protein is used to catalyze one or more of the following reactions or is used to prepare a catalytic preparation for catalyzing one or more of the following reactions:

The seventh aspect of the present invention provides a method for performing a glycosylation catalytic reaction, comprising the steps of: performing a glycosylation reaction in the presence of the mutant protein described in the first aspect of the present invention.

In another preferred embodiment, the glycosylation reaction comprises transferring a glycosyl donor to the following site of the tetracyclic triterpenoid compound in the presence of the mutant protein according to the first aspect of the present invention:

C-20 and/or C-6, thereby forming a glycosylated tetracyclic triterpenoid compound.

In another preferred embodiment, the substrates of the sugar-catalyzed reaction include dammarane-type tetracyclic triterpenoid compounds, lanolin-type tetracyclic triterpenoid compounds, gansuane-type tetracyclic triterpenoid compounds, cycloartanane (cycloartinane)-type tetracyclic triterpenoid compounds, cucurbitane-type tetracyclic triterpenoid compounds, or meline-type tetracyclic triterpenoid compounds.

In an eighth aspect, the present invention provides a method for increasing the expression level of glycosyltransferase, comprising the steps of:

(i) performing homology comparison between the glycosyltransferase and the sequence shown in SEQ ID NO.: 13;

(ii) screening out the sequence in step (i) having at least 80% homology to the sequence shown in SEQ ID NO.: 13; preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98%;

(iii) deleting 1 to 5 amino acids at the C-terminus of the sequence selected in step (ii).

The ninth aspect of the present invention provides the use of the host cell described in the fourth aspect of the present invention for preparing glycosyltransferase, or as a catalytic cell, or for producing glycosylated tetracyclic triterpenoid compounds.

The tenth aspect of the present invention provides a method for producing transgenic plants, comprising the steps of: regenerating the host cell described in the fourth aspect of the present invention into a plant, wherein the host cell is a plant cell.

In the eleventh aspect of the present invention, a mutant protein of a glycosyltransferase is provided, wherein the mutant protein is a non-natural protein, and the mutant protein has glycosyltransferase catalytic activity, and the mutant protein contains the following core amino acids related to the enzyme catalytic activity:

The amino acid at position 82 is C; and/or

The 144th amino acid is F

Wherein, the amino acid position numbering is based on the sequence shown in SEQ ID NO.: 11, and the glycosyltransferase catalytic activity includes transferring the glycosyl from the glycosyl donor to the C-20 position and the C-6 hydroxyl group of the tetracyclic triterpene compound.

In another preferred embodiment, the mutant protein described in the eleventh aspect of the present invention has a sequence homology of at least 90%, preferably at least 95%, and more preferably at least 98% with the sequence shown in SEQ ID NO.: 11.

In another preferred embodiment, except for amino acid 82 and/or amino acid 144, the remaining amino acids of the mutant protein described in the eleventh aspect of the present invention are identical or substantially identical to the sequence shown in SEQ ID NO.: 11.

In another preferred embodiment, the basic identity is that there are at most 50 (preferably 1-20, more preferably 1-10) amino acids that are different, wherein the difference includes amino acid substitution, deletion or addition, and the mutant protein has glycosyltransferase activity of transferring the glycosyl from the glycosyl donor to the C-20 hydroxyl of the tetracyclic triterpene compound.

In a twelfth aspect of the present invention, a mutant protein of a glycosyltransferase is provided, wherein the mutant protein is a non-natural protein, and the mutant protein has glycosyltransferase catalytic activity, and the mutant protein contains the following core amino acids related to the enzyme catalytic activity:

The 142nd amino acid is A;

The amino acid at position 186 is L; and

The 338th amino acid is G

Wherein, the amino acid position numbering is based on the sequence shown in SEQ ID NO.: 16, and the glycosyltransferase catalytic activity includes transferring the glycosyl from the glycosyl donor to the C-6 hydroxyl group of the tetracyclic triterpene compound.

In another preferred embodiment, the mutant protein described in the twelfth aspect of the present invention has a sequence homology of at least 90% with that of SEQ ID NO.: 16, preferably at least 95%, and more preferably at least 98%.

In another preferred embodiment, except for amino acids 142, 186, and 338, the remaining amino acids of the mutant protein described in the twelfth aspect of the present invention are identical or substantially identical to the sequence shown in SEQ ID NO.:16.

In another preferred embodiment, the basic identity is that there are at most 50 (preferably 1-20, more preferably 1-10) amino acids that are different, wherein the difference includes amino acid substitution, deletion or addition, and the mutant protein has glycosyltransferase activity that transfers the glycosyl from the glycosyl donor to the C-6 hydroxyl of the tetracyclic triterpene compound.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Amino acid sequence alignment of five glycosyltransferases.

Figure 2. HPLC images of five glycosyltransferases catalyzed in vitro. 1, UGTPg1 catalyzes PPT; 2, UGTPg100 catalyzes PPT; 3, UGTPg101 catalyzes PPT; 4, UGTPg102 catalyzes PPT; 5, UGTPg103 catalyzes PPT; 6, pET28a empty vector as control; 7, UGTPg101 catalyzes F1; 8, UGTPg101 catalyzes Rh1.

Figure 3. Key amino acid sites that determine the glycosylation of C20-OH catalyzed by glycosyltransferases UGTPg1 and UGTPg102. The HPLC graphs are as follows: 1, PPT catalyzed by UGTPg1; 2, PPT catalyzed by UGTPg102; 3, PPT catalyzed by UGTPg1-L38F; 4, PPT catalyzed by UGTPg1-A40S; 5, PPT catalyzed by UGTPg1-F85L; 6, PPT catalyzed by UGTPg1-T134I; 7, PPT catalyzed by UGTPg1-H144Y; 8, PPT catalyzed by UGTPg1-Q388H; 9, PPT catalyzed by UGTPg102-Y144H.

Figure 4. Key amino acid sites that determine the glycosylation of C6-OH catalyzed by glycosyltransferases UGTPg100 and UGTPg103. The HPLC graphs are as follows: 1, PPT catalyzed by UGTPg100; 2, PPT catalyzed by UGTPg103; 3, PPT catalyzed by UGTPg100-A142T; 4, PPT catalyzed by UGTPg100-L186S; 5, PPT catalyzed by UGTPg100-W205R; 6, PPT catalyzed by UGTPg100-G338R; 7, PPT catalyzed by UGTPg103-T142A; 8, PPT catalyzed by UGTPg103-T142A/S186L; 9, PPT catalyzed by UGTPg103-T142A/S186L/G338R.

Figure 5. Key amino acid sites that change the substrate regiospecificity of UGTPg1. The HPLC graphs are as follows: 1, UGTPg1 catalyzed PPT; 2, UGTPg100 catalyzed PPT; 3, UGTPg1-A10V catalyzed PPT; 4, UGTPg1-I13F catalyzed PPT; 5, UGTPg1-H82C catalyzed PPT; 6, UGTPg1-H144F catalyzed PPT.

Figure 6. Truncated C-terminal amino acids of UGTPg1, UGTPg100, and UGTPg101 enhance their soluble expression in E. coli. A, Western blot of cell lysate supernatant after wild-type UGTs and C-terminally truncated UGTs were expressed in E. coli. C2, C4, and C5 indicate C-terminally truncated 2, 4, and 5 amino acids, respectively. B, TLC of PPT catalyzed by wild-type UGTs and C-terminally truncated UGTs.

DETAILED DESCRIPTION

Through extensive and in-depth research, the inventors determined the key amino acid sites of glycosyltransferase catalyzing the glycosylation of C-6 or C-20 hydroxyl groups by homology comparison, site-directed mutagenesis, etc. The present invention found that after the key sites in the glycosyltransferase were modified, the originally inactive or poorly active enzymes could be changed, or new enzyme activities could be conferred. In addition, the inventors also found that after the terminal truncation of the glycosyltransferase of the present invention, its expression level had a significant increase. On this basis, the present invention was completed.

Mutant protein and its encoding nucleic acid of the present invention

As used herein, the terms "mutant protein", "mutant protein of the present invention", and "glycosyltransferase of the present invention" all refer to non-naturally occurring glycosyltransferase mutant proteins, and the mutant protein is a protein shown in SEQ ID NO.: 13 (or SEQ ID NO.: 16), or a protein artificially modified based on the protein shown in SEQ ID NO.: 13 (or SEQ ID NO.: 16), wherein the mutant protein contains core amino acids related to enzyme catalytic activity, and at least one of the core amino acids is artificially modified; and the mutant protein of the present invention has glycosyltransferase activity that catalyzes one or more of the following reactions:

(a) transferring a glycosyl group from a glycosyl donor to the hydroxyl group at the C-20 position of a tetracyclic triterpenoid compound;

(b) Transferring the glycosyl group from the glycosyl donor to the hydroxyl group at the C-6 position of the tetracyclic triterpenoid compound.

Unless otherwise specified, the ginsenosides and sapogenins mentioned in this article are ginsenosides and sapogenins with S configuration at the C20 position.

The term "core amino acid" refers to a sequence based on SEQ ID NO.: 13 (or SEQ ID NO.: 16) and having at least 80%, such as 84%, 85%, 90%, 92%, 95%, 98% homology to SEQ ID NO.: 13 (or SEQ ID NO.: 16), wherein the corresponding positions are specific amino acids described herein, such as based on the sequence shown in SEQ ID NO.: 13, the core amino acids are:

The 142nd amino acid is A;

Amino acid at position 144 is H or F; and

The amino acid at position 339 is G, and a mutant protein containing the core amino acid has a catalytic activity of transferring a glycosyl group from a glycosyl donor to a hydroxyl group at position C-20 of a tetracyclic triterpenoid compound.

For example, based on the sequence shown in SEQ ID NO.: 16, the core amino acids are:

The 142nd amino acid is A;

The amino acid at position 186 is L; and

The amino acid at position 338 is G, and a mutant protein containing the core amino acid has a catalytic activity of transferring a glycosyl group from a glycosyl donor to a hydroxyl group at the C-6 position of a tetracyclic triterpenoid compound.

It should be understood that the amino acid numbering in the mutant protein of the present invention is based on SEQ ID NO.: 13 (or SEQ ID NO.: 16). When the homology between a specific mutant protein and the sequence shown in SEQ ID NO.: 13 (or SEQ ID NO.: 16) reaches 80% or more, the amino acid numbering of the mutant protein may be misplaced relative to the amino acid numbering of SEQ ID NO.: 13 (or SEQ ID NO.: 16), such as misplacement of 1-5 positions to the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misplacement is within a reasonable range, and mutant proteins with a homology of 80% (such as 90%, 95%, 98%) and the same or similar glycosyltransferase activity should not be excluded from the scope of the mutant protein of the present invention due to the misplacement of amino acid numbering.

Mutants of the present invention are synthetic proteins or recombinant proteins, i.e., they can be the product of chemical synthesis, or they can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the mutagen of the present invention can be glycosylated, or it can be non-glycosylated. The mutagen of the present invention may also include or not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of the mutant protein. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity of the mutant protein.

Mutant protein fragments, derivatives or analogs of the present invention can be (i) substituted mutant proteins with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) mutant proteins with substituent groups in one or more amino acid residues, or (iii) mutant proteins formed by fusion of mature mutant proteins with another compound (such as a compound that prolongs the half-life of the mutant protein, such as polyethylene glycol), or (iv) mutant proteins formed by fusion of additional amino acid sequences to the mutant protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the mutant protein, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the well-known scope of those skilled in the art. In the present invention, the conservatively substituted amino acids are preferably produced by amino acid substitution according to Table I.

Table I

Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

The active mutant protein of the present invention has glycosyltransferase activity and can catalyze one or more of the following reactions:

(A)

Wherein, the compound of formula (I) is protopanaxatriol, the compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), and the mutant protein includes SEQ ID NO.: 6-7, 9-13;

(B)

Wherein, the compound of formula (I) is protopanaxatriol, and the compound of formula (III) is ginsenoside Rh1 (6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol); or

The compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), and the compound of formula (IV) is ginsenoside Rg1 (6-O-β-(D-glucopyranosyl)-20-O-β-(D-glucopyranosyl)-protopanaxatriol);

The mutant proteins include SEQ ID NO.: 6, 8, 11-12, 16;

(C)

Wherein, the compound of formula (I) is protopanaxatriol, the compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), the compound of formula (IV) is ginsenoside Rg1 (6-O-β-(D-glucopyranosyl)-20-O-β-(D-glucopyranosyl)-protopanaxatriol), and the mutant protein includes SEQ ID NO.: 6, 11-12;

(D)

Wherein, the compound of formula (I) is protopanaxatriol, the compound of formula (II) is ginsenoside F1 (20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), the compound of formula (III) is ginsenoside Rh1 (6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol), and the mutant protein includes SEQ ID NO.: 11-12.

Generally, in addition to the above core amino acids, the mutant protein of the present invention may also have one or more of the following amino acids:

The 10th amino acid is V;

The 13th amino acid is F;

Amino acid at position 82 is C; and

The 186th amino acid is L.

Preferably, the mutant protein is as shown in SEQ ID NO.: 9-13, 16, and the mutant protein is not the sequence shown in SEQ ID NO.: 1-3.

The mutant proteins of the present invention also include mutant proteins obtained by deleting (truncating) the C-terminus of the mutant protein having glycosyltransferase activity, such as the sequences shown in SEQ ID NO.: 6-8.

It should be understood that the mutant protein of the present invention generally has a higher homology (identity) than the sequence shown in SEQ ID NO.: 13. Preferably, the homology of the mutant protein to the sequence shown in SEQ ID NO.: 13 is at least 80%, more preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98%.

In addition, the mutant protein of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization forms of mutant proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those mutant proteins produced by glycosylation modification during the synthesis and processing of mutant proteins or in further processing steps. This modification can be accomplished by exposing the mutant protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are mutant proteins that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

The term "polynucleotide encoding a mutant protein" may include a polynucleotide encoding a mutant protein of the present invention, or may include a polynucleotide further including additional coding and/or non-coding sequences.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the mutant protein encoded by it.

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%.

The mutant proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

The method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

Wild-type glycosyltransferase

In the present invention, a series of genes with C20-OH and C6-OH glycosylation activities were screened out from glycosyltransferases for research. Among them:

The UGTPg101 gene has the nucleotide sequence of SEQ ID NO:17 in the sequence list. The nucleotides 1-1428 from the 5' end of SEQ ID NO:17 are the open reading frame of UGTPg1, the nucleotides 1-3 from the 5' end of SEQ ID NO:17 are the start codon ATG of the UGTPg1 gene, and the nucleotides 1426-1428 from the 5' end of SEQ ID NO:17 are the stop codon TAA of the UGTPg1 gene. The glycosyltransferase gene UGTPg1 encodes a protein UGTPg1 containing 475 amino acids, having the amino acid residue sequence of SEQ ID NO:1. The theoretical molecular weight of the protein predicted by the software is 53.4 kDa, and the isoelectric point pI is 5.01. The amino acids 344 to 387 from the amino terminal of SEQ ID NO:1 are the conserved PSPG motifs of the glycosyltransferase modified by plant small molecule glycosylation.

The UGTPg1 gene has the nucleotide sequence of SEQ ID NO: 18 in the sequence table. The nucleotides 1-1428 from the 5' end of SEQ ID NO: 18 are the open reading frame (ORF) of UGTPg1, the nucleotides 1-3 from the 5' end of SEQ ID NO: 18 are the start codon ATG of the UGTPg1 gene, and the nucleotides 1426-1428 from the 5' end of SEQ ID NO: 18 are the stop codon TAA of the UGTPg1 gene. The glycosyltransferase gene UGTPg1 encodes a protein UGTPg1 containing 475 amino acids, having the amino acid residue sequence of SEQ ID NO:, and the theoretical molecular weight of the protein predicted by the software is 53.4 kDa, and the isoelectric point pI is 5.14. The amino acid from position 344 to position 387 of the amino terminal of SEQ ID NO: 2 is a conserved PSPG (Plant Secondary Product Glycosyltransferase) motif of a glycosyltransferase that modifies plant small molecule glycosylation.

The UGTPg102 gene has the nucleotide sequence of SEQ ID NO:19 in the sequence list. The nucleotides 1-1428 from the 5' end of SEQ ID NO:19 are the open reading frame of UGTPg1, the nucleotides 1-3 from the 5' end of SEQ ID NO:19 are the start codon ATG of the UGTPg1 gene, and the nucleotides 1426-1428 from the 5' end of SEQ ID NO:19 are the stop codon TAA of the UGTPg1 gene. The glycosyltransferase gene UGTPg1 encodes a protein UGTPg1 containing 475 amino acids, having the amino acid residue sequence of SEQ ID NO:3. The theoretical molecular weight of the protein predicted by the software is 53.6 kDa, and the isoelectric point pI is 5.08. The amino acids 344 to 387 from the amino terminal of SEQ ID NO:3 are the conserved PSPG motifs of the glycosyltransferase modified by plant small molecule glycosylation.

The UGTPg100 gene has the nucleotide sequence of SEQ ID NO:20 in the sequence list. The nucleotides 1-1419 from the 5' end of SEQ ID NO:20 are the open reading frame of UGTPg1, the nucleotides 1-3 from the 5' end of SEQ ID NO:20 are the start codon ATG of the UGTPg1 gene, and the nucleotides 1417-1419 from the 5' end of SEQ ID NO:20 are the stop codon TAA of the UGTPg1 gene. The glycosyltransferase gene UGTPg1 encodes a protein UGTPg1 containing 472 amino acids, having the amino acid residue sequence of SEQ ID NO:4. The theoretical molecular weight of the protein predicted by the software is 53.1 kDa, and the isoelectric point pI is 5.08. The amino acids 343 to 386 from the amino terminal of SEQ ID NO.:4 are the conserved PSPG motifs of glycosyltransferases for plant small molecule glycosylation modification.

The UGTPg103 gene has the nucleotide sequence of SEQ ID NO:21 in the sequence list. The nucleotides 1-1419 from the 5' end of SEQ ID NO:21 are the open reading frame of UGTPg1, the nucleotides 1-3 from the 5' end of SEQ ID NO:21 are the start codon ATG of the UGTPg1 gene, and the nucleotides 1417-1419 from the 5' end of SEQ ID NO:21 are the stop codon TAA of the UGTPg1 gene. The glycosyltransferase gene UGTPg1 encodes a protein UGTPg1 containing 472 amino acids, having the amino acid residue sequence of SEQ ID NO:5. The theoretical molecular weight of the protein predicted by the software is 53.2 kDa, and the isoelectric point pI is 5.23. The amino acids 343 to 386 from the amino terminal of SEQ ID NO.:5 are the conserved PSPG motifs of glycosyltransferases for plant small molecule glycosylation modification.

The sequence information of the wild protein, mutant protein of the present invention and its derivative protein involved above is shown in Table 2:

Table 2

Expression vector

The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

The polynucleotide sequences of the present invention can be used to express or produce recombinant mutant proteins by conventional recombinant DNA techniques. Generally, the following steps are involved:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a mutant protein of the present invention, or a recombinant expression vector containing the polynucleotide;

(2) Host cells cultured in a suitable culture medium;

(3) Isolation and purification of proteins from culture medium or cells

In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the mutant protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; λ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

Beneficial effects of the present invention:

After extensive screening and modification, the present invention discovered the catalytic active site of glycosyltransferase. After modifying the relevant site, the glycosyltransferase that originally had no catalytic activity can be transformed into a new protein with activity, and the substrate specificity of highly homologous proteins can be artificially changed after modifying the key sites. In addition, after further deleting several amino acids at the C-terminus, the expression level of the mutant protein can be effectively increased.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

Example 1. Expression and catalytic function identification of glycosyltransferases UGTPg1, UGTPg101, UGTPg102, UGTPg100 and UGTPg103 in Escherichia coli BL21 (DE3)

Two primers with nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 23 were synthesized respectively. Two restriction sites, BamHI and XhoI, were set at both ends of the synthesized primers SEQ ID NO: 22 and SEQ ID NO: 23, and PCR was performed using pMDT-UGTPg1, pMDT-UGTPg101, pMDT-UGTPg102, pMDT-UGTPg100 and pMDT-UGTPg103 as templates.

PCR amplification program: 94°C for 2 min; 94°C for 15 s, 58°C for 30 s, 68°C for 1.5 min, for a total of 35 cycles; 68°C for 10 min, then reduced to 10°C. The PCR products were separated and recovered by agarose gel electrophoresis, then double-digested with BamHI and XhoI, and ligated into the pET28a vector (Novagen) that was also double-digested with BamHI and XhoI using T4 DNA ligase from NEB.

The obtained recombinant plasmids were named pET28a-UGTPg1, pET28a-UGTPg101, pET28a-UGTPg102, pET28a-UGTPg100 and pET28a-UGTPg103. These recombinant plasmids were transformed into Escherichia coli BL21 (DE3) (Novagen) to construct recombinant bacteria pET28a-UGTPg1-BL21, pET28a-UGTPg101-BL21, pET28a-UGTPg102-BL21, pET28a-UGTPg100-BL21 and pET28a-UGTPg103-BL21.

The five recombinant strains were picked from the plate and inoculated into LB test tubes containing 50 μg/mL kanamycin, shaken and cultured at 37°C 200rpm overnight, and inoculated into 50mL LB medium at a ratio of 1%, shaken and cultured at 37°C 200rpm until OD600 was 0.6-0.8, cooled in an ice-water bath, and IPTG was added at a final concentration of 0.1mM, and induced at 110rpm at 16°C for 18h. 12000g, centrifuged for 3min to collect the cells, added 10mL PBS buffer (pH8.0) per gram of wet weight of the cells, and then the cells were lysed after resuspending, and centrifuged at 12000g for 20min to obtain the supernatant as a crude enzyme solution. At the same time, the BL21 strain pET28a-BL21 containing the pET28a empty vector was induced to prepare a crude enzyme solution as a control for subsequent examples.

The reaction system for synthesizing rare ginsenosides from ginsenoside aglycones catalyzed by glycosyltransferase is as follows: 200 μL of reaction solution contains PBS buffer (pH 8.0), 5 mM UDP-glucose, 0.5 mM protopanaxatriol (PPT), 1% Tween-20 and 150 μL crude enzyme solution. The reaction is carried out in a 40°C water bath for 4 h, and an equal volume of n-butanol is added to terminate the reaction and extract. The n-butanol phase is concentrated in vacuo, and the product is dissolved in methanol. The HPLC results are shown in Figure 2.

The results showed that UGTPg1 catalyzed PPT to produce F1, i.e., adding a sugar group to C20-OH; UGTPg101 catalyzed the glycosylation of C20-OH of PPT to produce F1, and then glycosylated F1 at C6-OH to produce a small amount of Rg1; UGTPg100 catalyzed PPT to produce Rh1, i.e., adding a molecule of glucose to C6-OH of PPT; while no enzyme activity was detected for UGTPg102 and UGTPg103.

Example 2. Key amino acid sites determining glycosyltransferase UGTPg1 and UGTPg102 catalyzing C20-OH glycosylation

Protein homology modeling of UGTPg1 was performed by SWISS-MODEL, and the three-dimensional structure was analyzed by PyMOL software (DeLanoScientific). H15 and D117 are the conserved catalytic active sites of UGTPg1. Six different amino acid sites between UGTPg1 and UGTPg102 were found near these two sites, namely L38, A40, F85, T134, H144 and Q388 of UGTPg1, which correspond to F38, S40, L85, I134, Y144 and H388 of UGTPg102, respectively. These six amino acids of UGTPg1 were mutated to the corresponding amino acids of UGTPg102, and expressed in Escherichia coli BL21 (DE3), and the enzyme activity was measured.

Synthesize two primers with nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25 in the sequence table. Use plasmid pET28a-UGTPg1 as template and use Stratagen's QuickChange II site-directed mutagenesis kit to perform site-directed mutagenesis at the L38 site of UGTPg1. The PCR amplification program is: 95℃30s; 95℃30s, 55℃1min, 68℃7min, 16 cycles in total; reduce to 10℃. The PCR product is digested with DpnI, reacted in a water bath at 37℃ for 2h, and 10μL is taken to transform Escherichia coli TOP 10 competent cells.

Single clones were picked and plasmids were extracted. The mutation sites were verified by sequencing. The obtained plasmid was named pET28a-UGTPg1-L38F. The plasmid pET28a-UGTPg1-L38F was transformed into Escherichia coli BL21 (DE3) to construct the recombinant strain pET28a-UGTPg1-L38F-BL21. The steps of inducing expression were the same as those in Example 1. Similarly, corresponding recombinant plasmids pET28a-UGTPg1-A40S, pET28a-UGTPg1-F85L, pET28a-UGTPg1-T143I, pET28a-UGTPg1-H144Y and pET28a-UGTPg1-Q388H after UGTPg1 mutation were constructed, and recombinant strains pET28a-UGTPg1-A40S-BL21, pET28a-UGTPg1-F85L-BL21, pET28a-UGTPg1-T143I-BL21, pET28a-UGTPg1-H144Y-BL21 and pET28a-UGTPg1-Q388H-BL21 were constructed. The method of site-directed mutagenesis is the same as above (the sequences of the primers for point mutations are SEQ ID NO: 26-SEQ ID NO: 35, respectively), and the methods of inducing expression and enzyme activity determination are the same as those in Example 1.

The results showed that UGTPg1-L38F, UGTPg1-A40S, UGTPg1-F85L, UGTPg1-T143I and UGTPg1-Q388H still had the enzyme activity to catalyze the glycosylation of C20-OH, that is, catalyze PPT to generate F1, while UGTPg1-H144Y had no enzyme activity detected (Figure 3). The results showed that H144 is very important for the catalytic function of UGTPg1.

Synthesize two primers having the nucleotide sequences of SEQ ID NO:36 and SEQ ID NO:37 in the sequence table, respectively. Using plasmid pET28a-UGTPg102 as a template, the Y144 site of UGTPg102 was subjected to site-directed mutagenesis using Stratagen's QuickChange II site-directed mutagenesis kit to construct the recombinant plasmid pET28a-UGTPg102-Y144H and recombinant strain pET28a-UGTPg102-Y144H-BL21 after the mutation of UGTPg102. The method of site-directed mutagenesis is the same as above, and the methods of induced expression and enzyme activity determination are the same as in Example 2. UGTPg102-Y144H has acquired the function of catalyzing C20-OH glycosylation, that is, it can catalyze PPT to generate F1 (Figure 3).

Conclusion: The identity of the amino acid sequences of UGTPg1 and UGTPg102 is as high as 95.4%, and UGTPg1 has C20-OH glycosyltransferase activity, while UGTPg102 has no enzyme activity. This example proves that the replacement of key amino acids leads to the loss of catalytic function of UGTPg102.

Example 3. Key amino acid sites determining glycosyltransferases UGTPg100 and UGTPg103 catalyzing C6-OH glycosylation

The identity of the amino acid sequences of UGTPg100 and UGTPg103 is as high as 98.7%, that is, there are only 6 amino acid differences between the two. Protein homology modeling of UGTPg100 was performed using SWISS-MODEL, and the three-dimensional structure was analyzed using PyMOL software (DeLanoScientific). H15 and D117 are the conserved catalytic active sites of UGTPg100. Two different amino acid sites between UGTPg100 and UGTPg103 were found near these two sites, namely A142 and L186 of UGTPg100, corresponding to T142 and S186 of UGTPg102, respectively. The other two amino acid sites are located in the C-terminal and N-terminal domains, respectively, which are G338 and W205 in UGTPg100, corresponding to R338 and R205 of UGTPg103. The four amino acids of UGTPg100 were mutated to the corresponding amino acids of UGTPg103, and the enzyme activity was measured after expression in E. coli BL21 (DE3). In addition, two amino acids were not studied in detail because the structures and properties of their amino acid side chains are very similar, and mutual substitution may have little effect on enzyme activity, that is, V111 and I349 in UGTPg100 correspond to I111 and V349 in UGTPg103, respectively.

Plasmid pET28a-UGTPg100 was used as a template, and the A142, L186, W205 and G338 sites of UGTPg100 were subjected to site-directed mutagenesis using Stratagen's QuickChange II site-directed mutagenesis kit to construct the mutated recombinant plasmids pET28a-UGTPg100-A142T, pET28a-UGTPg100-L186S, pET28a-UGTPg100-W205R and pET28a-UGTPg100-G338R (the sequences of the point mutation primers are SEQ ID NO: 38-SEQ ID NO: 38, respectively). IDNO: 45), and recombinant strains pET28a-UGTPg100-A142T-BL21, pET28a-UGTPg100-L186S-BL21, pET28a-UGTPg100-W205-BL21 and pET28a-UGTPg100-G338R-BL21. The method of site-directed mutagenesis is the same as above, and the method of induced expression and enzyme activity determination is the same as Example 1.

The results are shown in FIG4 . UGTPg100-A142T, UGTPg100-L186S and UGTPg100-G338R all lost the activity of C6-OH glycosyltransferase; whereas UGTPg100-W205R still retained the enzyme activity.

Using plasmid pET28a-UGTPg103 as a template, the QuickChange II site-directed mutagenesis kit of Stratagen was used to perform site-directed mutagenesis on T142, S186 and R338 of UGTPg100 (the sequences of the point mutation primers were SEQ ID NO:46-SEQ ID NO:47, respectively), and the recombinant plasmids pET28a-UGTPg103-T142A, pET28a-UGTPg103-S186L and the recombinant strain pET28a-UGTPg103-T142A-BL21 after mutating UGTPg103 were constructed. The method of site-directed mutagenesis was the same as above, and the methods of induced expression and enzyme activity determination were the same as in Example 2. As shown in Figure 4, no enzyme activity was detected in UGTPg103-T142A.

Using plasmid pET28a-UGTPg103-T142A as template and nucleotide sequences SEQ ID NO:48 and SEQ ID NO:49 as primers, the S186 site of pET28a-UGTPg103-T142A was site-directed mutagenesis kit from Stratagen was used to construct the recombinant plasmid pET28a-UGTPg103-T142A-S186L with double mutation of UGTPg103 and the recombinant strain pET28a-UGTPg103-T142A-S186L-BL21. Similarly, the recombinant plasmid pET28a-UGTPg103-T142A-S186L-R338G and the recombinant strain pET28a-UGTPg103-T142A-S186L-R338G-BL21 with triple mutations of UGTPg103 were constructed. The site-directed mutagenesis method was the same as above, and the methods of induced expression and enzyme activity determination were the same as in Example 1.

The results are shown in Figure 4. UGTPg103-T142A-S186L also does not have the function of catalyzing C6-OH glycosylation, while UGTPg103-T142A-S186L-R338G has the function of catalyzing C6-OH glycosylation, that is, it can catalyze PPT to produce Rh1.

Conclusion: The identity of the amino acid sequences of UGTPg100 and UGTPg103 is as high as 98.7%, that is, there are only 6 amino acids different between the two. UGTPg100 has C6-OH glycosyltransferase activity, while UGTPg103 has no enzyme activity, indicating that the replacement of key amino acids has led to the loss of catalytic function of UGTPg103.

Example 4. Key amino acid positions and regions that determine the substrate regiospecificity of glycosyltransferases UGTPg1 and UGTPg100

The identity of the amino acid sequences of UGTPg1 and UGTPg100 is as high as 84.1%, and UGTPg1 has C20-OH glycosyltransferase activity, while UGTPg100 has C6-OH glycosyltransferase activity, indicating that the replacement of key amino acids has changed their substrate specificity. Protein homology modeling of UGTPg1 and UGTPg100 was performed by SWISS-MODEL, and the three-dimensional structure was analyzed by PyMOL software (DeLanoScientific), as shown in the figure. H15 and D117 are the conserved catalytic active sites of UGTPg1 and UGTPg100. Four different amino acid sites of UGTPg1 and UGTPg100 were found near these two sites, namely A10, I13, H82 and H144 of UGTPg1, corresponding to V10, F13, C82 and F144 of UGTPg100, respectively. The four amino acids of UGTPg1 were mutated into the corresponding amino acids of UGTPg100, respectively, and expressed in Escherichia coli BL21 (DE3), and the enzyme activity was measured.

The nucleotide sequences of SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 in the sequence listing were synthesized as primers. Using plasmid pET28a-UGTPg1 as a template, the A10, I13, H82 and H144 sites of UGTPg1 were subjected to site-directed mutagenesis using Stratagen's QuickChange II site-directed mutagenesis kit to construct recombinant plasmids pET28a-UGTPg1-A10V, pET28a-UGTPg1-I13F, pET28a-UGTPg1-H82C and pET28a-UGTPg1-H144F after UGTPg1 mutation, as well as recombinant strains pET28a-UGTPg1-A10V-BL21, pET28a-UGTPg1-I13F-BL21, pET28a-UGTPg1-H82C-BL21 and pET28a-UGTPg1-H144F-BL21. The site-directed mutagenesis method was the same as above, and the methods for inducing expression and enzyme activity determination were the same as in Example 2. As shown in Figure 5, the C20-OH glycosyltransferase activity of UGTPg1-A10V, UGTPg1-I13F and UGTPg1-H82C was reduced; UGTPg1-H82C and UGTPg1-H144F acquired the function of catalyzing C6-OH glycosylation, but the activity of UGTPg1-H144F in catalyzing C6-OH glycosylation was very weak.

Example 5. C-terminal truncation of glycosyltransferases UGTPg1, UGTPg101 and UGTPg100 promotes soluble expression

Glycosyltransferases UGTPg1, UGTPg101 and UGTPg100 expressed in E. coli mainly exist in the form of inclusion bodies, and the soluble expression level is low. After truncating 5 amino acids at the N-terminus, UGTPg1, UGTPg101 and UGTPg100 have no enzyme activity for the substrate PPT. After truncating 5 amino acids at the C-terminus, the enzyme activity of UGTPg1, UGTPg101 and UGTPg100 for the substrate PPT is reduced, but the soluble expression level is significantly increased. After truncating 2 and 4 amino acids at the C-terminus, it was found that the soluble expression level of UGTPg1 and UGTPg101 increased by at least 50% relative to the wild type (Figure 6A), but the enzyme activity did not change significantly (Figure 6B). Although the soluble expression level of UGTPg100 increased by at least 50% relative to the wild type after truncating 2 and 4 amino acids at the C-terminus, its enzyme activity was significantly reduced (Figure 6).

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (11)

1. A mutant protein of a glycosyltransferase, wherein the mutant protein is a non-natural protein, the mutant protein has glycosyltransferase catalytic activity, and the mutant protein is shown as SEQ ID NO. 13 or 16.

2. The mutein of claim 1, wherein the glycosyltransferase catalytic activity comprises catalyzing one or more of the following reactions:

(a) Transferring a glycosyl group from a glycosyl donor to a hydroxyl group at the C-20 position of a tetracyclic triterpene compound;

(b) The glycosyl group from the glycosyl donor is transferred to the hydroxyl group at the C-6 position of the tetracyclic triterpene compound.

3. The mutein of claim 1 or 2, wherein the glycosyltransferase catalytic activity comprises catalyzing one or more of the following reactions:

(A)

wherein the compound of formula (I) is protopanaxatriol (protopanaxatriol), the compound of formula (II) is ginsenoside F1 (20-O-beta-D-glucopyranosyl-20 (S) -protopanaxatriol), and the mutein comprises SEQ ID NO. 13;

(B)

Wherein the compound of formula (I) is protopanaxatriol (protopanaxatriol), and the compound of formula (III) is ginsenoside Rh1 (6-O-beta-D-glucopyranosyl-20 (S) -protopanaxatriol); or (b)

The compound of formula (II) is ginsenoside F1 (20-O-beta-D-glucopyranosyl-20 (S) -protopanaxatriol), and the compound of formula (IV) is ginsenoside Rg1 (6-O-beta- (D-glucopyranosyl) -20-O-beta- (D-glucopyranosyl) -protopanaxatriol);

the mutant protein comprises SEQ ID NO. 16.

4. The protein of claim 1, wherein the mutein is set forth in SEQ ID No. 16 and wherein the glycosyltransferase activity comprises the transfer of a glycosyl group from a glycosyl donor to the hydroxyl group at the C-6 position of a tetracyclic triterpene compound; and/or

The mutein is shown in SEQ ID No. 13 and the glycosyltransferase activity comprises the transfer of a glycosyl group from a glycosyl donor to the hydroxyl group at the C-20 position of a tetracyclic triterpene compound.

5. A polynucleotide encoding the mutein of any one of claims 1-4.

6. A carrier, characterized in that, the vector contains the polynucleotide of claim 5.

7. A host cell comprising the vector of claim 6, or having integrated into its genome the polynucleotide of claim 5, and which does not comprise a plant cell.

8. Use of a mutein according to claim 1, characterized in that the mutein is used for catalyzing one or more of the following reactions or is used for the preparation of a catalytic preparation for catalyzing one or more of the following reactions:

(a) Transferring a glycosyl group from a glycosyl donor to a hydroxyl group at the C-20 position of a tetracyclic triterpene compound;

(b) The glycosyl group from the glycosyl donor is transferred to the hydroxyl group at the C-6 position of the tetracyclic triterpene compound.

9. A method of performing a glycosyl catalytic reaction comprising the steps of: the glycosylation reaction is performed in the presence of the mutein of claim 1.

10. The use of a host cell according to claim 7 for the preparation of a glycosyltransferase, or as a catalytic cell, or for the production of glycosylated tetracyclic triterpenes.

11. A method of producing a transgenic plant comprising the steps of: introducing the vector of claim 6 into a plant cell and regenerating the plant cell into a plant.