Cytochrome P450 mutant protein and uses

Abstract

The present invention provides cytochrome P 450 (CYP 716 A 53 v 2) mutant proteins and uses thereof. [Solution] The mutation is performed on the wild-type CYP 716 A 53 v 2 protein at a site selected from the group consisting of F 167 V, T 451 A, I 117 S, and L 208 C, or a combination thereof. The resulting CYP 716 A 53 v 2 mutant has improved enzyme catalytic activity and can increase the production of protopanaxatriol.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and natural product drugs. Specifically, the present invention relates to a mutant protein of cytochrome P450 (CYP716A53v2) and its use.

Background Art

[0002] Ginsenoside is the main active substance in plants of the genus Panax (such as Korean ginseng, Japanese ginseng, American ginseng, etc.) of the Araliaceae family. In recent years, ginsenoside has also been discovered in the cucurbitaceous plant Trichosanthes kirilowii Maxim. At present, scientists at home and abroad have already isolated at least more than 100 kinds of ginsenosides from plants such as Korean ginseng and Trichosanthes kirilowii Maxim., but the difference in the content of these saponins in Korean ginseng is very large. Among them, some triterpenoid saponins with remarkable therapeutic effects have extremely low contents in total natural saponins (also called rare saponins), and the extraction cost is high, so the price is very expensive. At present, various saponins have already been applied to drugs and health products. For example, Shenyi Capsule, a drug with ginsenoside Rg3 monomer as the main component, can improve the qi deficiency symptoms of tumor patients and enhance the body's immune function; Redsenol Capsule, with a mixture of 16 rare ginsenosides such as ginsenoside Rh1 as the main component, can inhibit the angiogenesis of the tumor site, promote the apoptosis of cancer cells, and reduce chemotherapy resistance.

[0003] Rare ginsenosides often have unique biological activities or more remarkable therapeutic effects. Therefore, traditionally prepared rare ginsenosides are prepared from a large amount of saponins extracted from Panax japonicus Torr. or Panax ginseng C. A. Mey. through chemical hydrolysis, enzymatic hydrolysis, and microbial hydrolysis. Since the wild resources of Panax japonicus Torr. are basically depleted, the total saponin resources of Panax japonicus Torr. currently mainly come from the artificial cultivation of Panax japonicus Torr. or Panax ginseng C. A. Mey. However, its artificial cultivation has a long growth cycle (generally taking more than 5 - 7 years), is restricted by regions, and is often affected by pests and diseases, requiring a large amount of pesticides to be administered. In addition, there are serious continuous cropping obstacles in the artificial cultivation of Panax japonicus Torr. or Panax ginseng C. A. Mey. (the cultivation land of Panax japonicus Torr. or Panax ginseng C. A. Mey. cannot overcome continuous cropping obstacles unless it is fallowed for more than 5 - 15 years). Therefore, the production volume, quality, and safety of ginsenosides are all facing challenges. On the other hand, when manufacturing single-component saponins using total saponins of Panax japonicus Torr. as raw materials, a large number of components in the total saponins cannot yet be converted into the target ginsenoside monomer (such as protopanaxatriol-type saponins), which not only causes waste of resources but also raises the extraction and purification costs.

[0004] The development of synthetic biology has provided new opportunities for the heterologous synthesis of plant-derived natural products. Using yeast as a chassis, through the assembly and optimization of metabolic pathways, it has been realized to ferment and synthesize artemisinic acid or dihydroartemisinic acid using inexpensive monosaccharides, and further produce artemisinin through a one-step chemical conversion method, which demonstrates the great potential of synthetic biology in the drug synthesis of natural products. Using yeast chassis cells, heterologously synthesize rare ginsenoside monomers by synthetic biology methods. The raw material is inexpensive monosaccharides, and the manufacturing process is a fermentation process with controllable safety, avoiding any foreign contamination (for example, pesticides used in the artificial cultivation of raw material plants). Therefore, manufacturing rare ginsenoside monomers through synthetic biology technology not only has a cost advantage but also can guarantee 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 measurement and clinical experiments to promote the innovative drug development of rare ginsenosides.

[0005] To artificially synthesize protopanaxatriol-type ginsenosides with medicinal activity using synthetic biology methods, first, it is necessary to analyze and reconstruct the synthetic metabolic pathway of protopanaxatriol PPT. Since ginsenosides belong to triterpene compounds, the MVA and MEP metabolic pathways in plants provide the common precursors IPP and DMAPP of terpene compounds, laying the foundation for the synthesis of the triterpene compound precursors squalene and 2,3-epoxysqualene. In 2006, scientists from South Korea and Japan cloned and identified the synthase DS that converts epoxy squalene to dammarenediol from Angelica gigas (Han, J.Y. et al., Plant Cell Physiol, 2006. 47(12): p. 1653-62.; Tansakul, P. et al., FEBS Lett, 2006. 580(22): p. 5143-9); the South Korean researcher Han JY cloned and identified the crucial cytochrome P450, CYP716A47 and CYP716A53v2, which synthesize protopanaxadiol and protopanaxatriol from the cDNA library of Angelica gigas in 2011 (Han, J.Y. et al., Plant Cell Physiol, 2011. 52(12): p. 2062-73) and 2012 (Han, J.Y. et al., Plant Cell Physiol, 2012. 53(9): p. 1535-45), respectively. CYP716A47 can catalyze the hydroxylation of the 12th position of dammarenediol to produce protopanaxadiol PPD, and CYP716A53v2 can catalyze the hydroxylation of the 6th position of protopanaxadiol to produce protopanaxatriol PPT. In WAT21 yeast, co-expression of DS, these two cytochromes P450, and the P450 reductase ATR2-1 derived from Arabidopsis thaliana yielded a recombinant strain capable of producing protopanaxadiol and protopanaxatriol. Further research shows that the conversion of protopanaxadiol to protopanaxatriol catalyzed by CYP716A53v2 is an important rate-limiting step in the entire synthetic pathway.

[0006] Therefore, in this technical field, in order to promote the synthesis efficiency of ginsenoside cell factories, it is necessary to conduct more research and modification to obtain a more efficient cytochrome P450 protein element for cytochrome P450 CYP716A53v2.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention performs mutation and optimization on the protein coding sequence of cytochrome P450 CYP716A53v2 to obtain a new mutant sequence, and expressing this mutant sequence in cells that produce protopanaxadiol can significantly increase the production amount of protopanaxatriol.

[0009] In one aspect of the present invention, a method for improving the catalytic activity of cytochrome P450 CYP716A53v2 is provided, which includes mutating the amino acid sequence of cytochrome P450 CYP716A53v2. Corresponding to the wild-type cytochrome P450 CYP716A53v2, the mutation is selected from the sites of the following group or a combination thereof: position 167, position 451, position 117, position 208.

[0010] In a preferred example, the amino acid position numbers are based on the amino acid sequence shown in SEQ ID NO:1.

[0011] In another preferred example, the mutation at position 167 is VAL (V), the mutation at position 451 is Ala (A), the mutation at position 117 is Ser (S), and the mutation at position 208 is Cys (C).

[0012] In another aspect of the present invention, a cytochrome P450 CYP716A53v2 mutant is provided, which is: (a) a protein in which the amino acid sequence corresponds to wild-type cytochrome P450 CYP716A53v2 and one or a combination of sites selected from the following groups is mutated: position 167, position 451, position 117, position 208 (preferably, they are core amino acid mutations); (b) formed by substitution, deletion or addition of one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, e.g., 5, 3) amino acid residues from the amino acid sequence of the protein in (a), having the function of the protein in (a), and being derived from (a), and the amino acids at positions 167, 451, 117, and 208 corresponding to wild-type cytochrome P450 CYP716A53v2 are the same as the amino acids after mutation at the corresponding positions of the protein in (a); (c) having a homology of 80% or more (preferably 85% or more; more preferably 90% or more; still more preferably 95% or more, e.g., 98%, 99%) with the amino acid sequence of the protein in (a), having the function of the protein in (a), and being derived from (a), and the amino acids at positions 167, 451, 117, and 208 corresponding to wild-type cytochrome P450 CYP716A53v2 are the same as the amino acids after mutation at the corresponding positions of the protein in (a); or (d) a polypeptide formed after adding a tag sequence to the N or C terminus of the polypeptide described in any one of (a) to (c), or adding a signal peptide sequence or a secretion signal sequence to its N terminus.

[0013] In a preferred example, the cytochrome P450 CYP716A53v2 mutant has significantly higher catalytic activity than its wild type.

[0014] In another preferred example, in the cytochrome P450 CYP716A53v2 mutant, the mutation at position 167 is Val (V).

[0015] In another preferred example, in the cytochrome P450 CYP716A53v2 mutant described above, the 451st mutation is Ala (A).

[0016] In another preferred example, in the cytochrome P450 CYP716A53v2 mutant described above, the 117th mutation is Ser (S).

[0017] In another preferred example, in the cytochrome P450 CYP716A53v2 mutant described above, the 208th mutation is Cys (C).

[0018] In another preferred example, the cytochrome P450 CYP716A53v2 mutant described above comprises a protein selected from the following group: corresponding to wild-type cytochrome P450 CYP716A53v2, (1) the 117th mutation is Ser and the 451st mutation is Ala; (2) the 117th mutation is Ser, the 208th mutation is Cys, the 167th mutation is Val, and the 451st mutation is Ala; (3) the 117th mutation is Ser and the 208th mutation is Cys; (4) the 117th mutation is Ser, the 208th mutation is Cys, and the 451st mutation is Ala; (5) the 167th mutation is Val; (6) the 451st mutation is Ala; (7) the 117th mutation is Ser; (8) the 208th mutation is Cys.

[0019] In another aspect of the present invention, there is provided an isolated polynucleotide, wherein the nucleic acid encodes the cytochrome P450 CYP716A53v2 mutant according to any one of the above.

[0020] In another aspect of the present invention, there is provided a vector containing the polynucleotide described above. In a preferred example, the vector includes an expression vector, a shuttle vector, and an integration vector.

[0021] In another aspect of the present invention, there is provided a genetically engineered host cell, wherein the host cell contains the vector described in any one of the above, or the polynucleotide described in any one of the above is integrated into the genome of the host cell.

[0022] In a preferred example, the host cell is a eukaryotic cell or a prokaryotic cell; preferably, the eukaryotic cell includes, but is not limited to, yeast cells, plant cells, fungal cells, insect cells, mold cells, mammalian cells; more preferably, the yeast cells include, but are not limited to, Saccharomyces cerevisiae cells or Pichia yeast cells (more preferably Saccharomyces cerevisiae cells); more preferably, the plant cells include, but are not limited to, carrot cells; preferably, the prokaryotic cells include, but are not limited to, Escherichia coli, Bacillus subtilis cells.

[0023] In another aspect of the present invention, there is provided a method for preparing the cytochrome P450 CYP716A53v2 mutant described in any one of the above, which comprises (i) culturing the host cell described above; (ii) collecting the culture containing the cytochrome P450 CYP716A53v2 mutant described above; (iii) isolating the cytochrome P450 CYP716A53v2 mutant from the culture. The method includes the above steps.

[0024] In another aspect of the present invention, there is provided a composition for catalyzing the production of protopanaxatriol from protopanaxadiol, which contains, in an effective amount, the cytochrome P450 CYP716A53v2 mutant described in any one of the above, or the host cell or its culture or lysate described above, and a food or industrially acceptable vector.

[0025] In a preferred example, the catalyst is a high-efficiency catalyst, and its catalytic efficiency is at least 10% higher than that of the wild type, preferably at least 20% higher, more preferably at least 30% higher, for example, 40% or more, 50% or more, 60% or more higher.

[0026] In another aspect of the present invention, there is provided the use of the cytochrome P450 CYP716A53v2 mutant according to any one of the above for catalyzing the production of protopanaxatriol from protopanaxadiol. Preferably, the cytochrome P450 CYP716A53v2 mutant increases one hydroxyl group at the C6 position of protopanaxadiol, thereby producing protopanaxatriol.

[0027] In another aspect of the present invention, there is provided the use of the above composition for catalyzing the production of protopanaxatriol from protopanaxadiol. Preferably, the cytochrome P450 CYP716A53v2 mutant increases one hydroxyl group at the C6 position of protopanaxadiol, thereby producing protopanaxatriol.

[0028] In another aspect of the present invention, there is provided a method for catalyzing the production of protopanaxatriol from protopanaxadiol, the method comprising treating protopanaxadiol with the cytochrome P450 CYP716A53v2 mutant or the above composition according to any one of the above. Preferably, the cytochrome P450 CYP716A53v2 mutant increases one hydroxyl group at the C6 position of protopanaxadiol, thereby producing protopanaxatriol.

[0029] In another aspect of the present invention, there is provided a kit for catalyzing the production of protopanaxatriol from protopanaxadiol, provided that it contains the cytochrome P450 CYP716A53v2 mutant or a combination of mutants; the above host cell; or the above composition.

[0030] Based on the content disclosed in this article, for those skilled in the art, other aspects of the present invention are self-evident.

Brief Description of the Drawings

[0031]

Figure 1

Embodiments for Carrying out the Invention

[0032] Through in-depth research, the inventor of the present invention constructed a large number of CYP716A53v2 mutants, studied the functions of the mutants, determined the amino acid sites related to enzyme catalytic activity, and carried out site-directed modification to obtain mutants with significantly improved enzyme catalytic activity.

[0033] The mutant of the present invention and the nucleic acid encoding the same

[0034] The inventor of the present invention constructed a mutant library of cytochrome P450 (CYP716A53v2) using the brewing yeast chassis cell ZW that synthesizes protopanaxadiol PPD: By transforming the brewing yeast chassis cell ZW with the randomly mutated CYP716A53v2 gene, a single copy was inserted into the yeast genome to construct a CYP716A53v2 yeast mutant library that synthesizes protopanaxatriol PPT. Based on the PPT production of the strains, the inventor determined the crucial amino acid sites that improve CYP716A53v2 activity. The present invention found that some mutants obtained by modifying the crucial sites of cytochrome P450 (CYP716A53v2) can improve the PPT production.

[0035] As used herein, the terms "mutant (protein)", "CYP716A53v2 mutant", and "mutant CYP716A53v2" are used interchangeably and all refer to a protein that non-naturally exists and catalyzes the production of protopanaxatriol from protopanaxadiol, and the mutant protein is a protein shown in SEQ ID NO:1 or an artificially modified protein based on the protein shown in SEQ ID NO:1 (including variants, derivatives, etc. with changed inactive sites), provided that 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 an enzyme activity that catalyzes the C6 hydroxylation of protopanaxadiol (PPD) to form protopanaxatriol (PPT).

[0036] The term "core amino acid" refers to a sequence based on SEQ ID NO:1 and having at least 80% homology with SEQ ID NO:1, such as 84%, 85%, 90%, 92%, 95%, 98%. At the corresponding sites, the specific amino acids are as described herein. For example, based on the sequence shown in SEQ ID NO:1, the core amino acids are: the amino acid at position 167 is V; the amino acid at position 451 is A; the amino acid at position 117 is S; the amino acid at position 208 is C; the amino acid at position 117 is S and the amino acid at position 208 is C; the amino acid at position 117 is S and the amino acid at position 451 is A; the amino acid at position 117 is S, the amino acid at position 208 is C, and the amino acid at position 451 is A; the amino acid at position 117 is S, the amino acid at position 167 is V, the amino acid at position 208 is C, and the amino acid at position 451 is A.

[0037] It should be understood that the amino acid numbers of the mutant proteins of the present invention are based on SEQ ID NO:1. When the homology between a specific mutant protein and the sequence shown in SEQ ID NO:1 reaches 80% or more, the amino acid numbers of the mutant protein may shift relative to the amino acid numbers of SEQ ID NO:1. For example, it may shift 1-5 positions to the N-terminus or C-terminus of the amino acid. For those skilled in the art, such a shift is within a reasonable range according to the general sequence alignment techniques in the art. Mutant proteins having the same or similar enzyme activity with a homology reaching 80% (such as 90%, 95%, 98%) due to the shift of amino acid numbers should not be excluded from the scope of the mutant proteins of the present invention.

[0038] The mutants (mutant proteins) of the present invention are synthetic proteins or recombinant proteins, that is, they may be chemically synthesized products or may be produced from prokaryotic or eukaryotic hosts (such as bacteria, yeast, plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the mutant proteins of the present invention may be glycosylated or non-glycosylated. The mutant proteins of the present invention may also contain or not contain the initiating methionine residue.

[0039] The present invention further includes fragments, derivatives, and analogs of the mutant proteins. As used herein, the terms "fragment", "derivative", and "analog" refer to proteins that substantially retain the same biological function or activity as the mutant proteins.

[0040] The mutant protein fragment, derivative or analog of the present invention is (i) a mutant protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a mutant protein having substituents on one or more amino acid residues; or (iii) a mutant protein formed by fusion of a mature mutant protein with another compound (e.g., a compound that extends the half-life of the mutant protein, such as polyethylene glycol); or (iv) a mutant protein formed by fusing an additional amino acid sequence to this mutant protein sequence (e.g., a leader sequence or a secretion sequence, or a sequence for purifying this mutant protein or the protein original sequence, or a fusion protein formed with an antigen IgG fragment); and may be. According to the teachings herein, these fragments, derivatives and analogs fall within the scope well known to those skilled in the art. In the present invention, conservative substituted amino acids are preferably produced by performing amino acid substitution according to Table 1.

[0041] Table 1

[0042] In addition, the mutant protein of the present invention can also be modified. Modification forms (generally without changing the primary structure) include chemically induced forms of the mutant protein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, e.g., mutant proteins produced by glycosylation modifications during the synthesis and processing of the mutant protein or in further processing steps. This modification can be achieved by exposing the mutant protein to enzymes that glycosylate the mutant protein (e.g., mammalian glycosylation enzymes or deglycosylation enzymes). Modification forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Mutant proteins whose anti-protein hydrolysis performance is improved or whose solubility performance is optimized by modification are also included.

[0043] The term "polynucleotide encoding a mutant protein" may include the polynucleotide encoding the mutant protein of the present invention, and may also include polynucleotides of additional coding and / or non-coding sequences.

[0044] The present invention also relates to variants of the above polynucleotides encoding polypeptides or fragments, analogs and derivatives of mutant proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, allelic variants can be substitutions, deletions, or insertions of one or more nucleotides, but are substitution forms of polynucleotides that do not substantially change the function of the encoded mutant protein.

[0045] The mutant protein and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.

[0046] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method. For the PCR amplification method, primers are designed according to the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by a general method known to those skilled in the art is used as a template to obtain the related sequences by amplification. When the sequence is long, it is often necessary to perform PCR amplification two or more times and ligate the amplified fragments in the correct order.

[0047] Once the related sequences are obtained, a large amount of related sequences can be obtained by recombinant methods. Usually, it is cloned into a vector, transfected into cells, and then the related sequences are separated and obtained from the propagated host cells by ordinary methods.

[0048] In addition, especially when the length of the fragment is short, the relevant sequences may be synthesized by artificial synthesis methods. Usually, first, a plurality of small fragments are synthesized, and then they are ligated to obtain a long sequence fragment.

[0049] Currently, the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention can be obtained by complete chemical synthesis. Then, the DNA sequence may be introduced into each existing DNA molecule (or vector) known in the art and cells. In addition, mutations may be introduced into the sequence of the protein of the present invention by chemical synthesis.

[0050] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the polynucleotide of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the PCR primers are appropriately selected according to the sequence information of the present invention disclosed herein and the RACE method (rapid amplification of cDNA ends) synthesized by ordinary methods is preferably used. The amplified DNA / RNA fragments may be isolated and purified by conventional methods such as gel electrophoresis.

[0051] Expression vectors and host cells

[0052] The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell genetically engineered 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.

[0053] By conventional recombinant DNA technology, recombinant mutant proteins can be expressed or produced using the polynucleotide sequence of the present invention. Generally, it includes the following steps: (1) Transform or transduce an appropriate host cell with the polynucleotide (or variant) encoding the protein of the present invention or a recombinant expression vector containing the polynucleotide; (2) Culture the host cells in an appropriate medium; (3) Isolate and purify the protein from the medium or cells.

[0054] In the present invention, a polynucleotide sequence encoding a mutant protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses such as mammalian cell viruses like adenovirus, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it is replicable and stable within the host. One of the important features of an expression vector is usually to contain an origin of replication, a promoter, a marker gene, and translation control elements.

[0055] Using methods well-known to those skilled in the art, an expression vector containing a DNA sequence encoding the mutant protein of the present invention and appropriate transcription / translation control signals can be constructed. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc. The DNA sequence can be operably linked to an appropriate promoter within the expression vector to direct mRNA synthesis. Representative examples of these promoters include the lac or trp promoter of Escherichia coli, the λ phage PL promoter, and for eukaryotic promoters, the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector further includes a ribosome binding site for translation initiation and a transcription terminator.

[0056] In addition, the expression vector preferably includes one or more selectable marker genes, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, green fluorescent protein (GFP), or tetracycline or ampicillin resistance for Escherichia coli, etc., to provide phenotypic traits for the selection of transformed host cells.

[0057] Vectors containing the above-mentioned appropriate DNA sequences together with appropriate promoters or control sequences are used for the transformation of appropriate host cells so as to be able to express proteins.

[0058] The host cell may be a prokaryotic cell such as a bacterial cell, or a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Representative examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium, fungal cells such as yeast, and plant cells such as carrot cells.

[0059] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, transcription is enhanced. An enhancer is a cis-acting element of DNA, usually about 10 to 300 base pairs in length, and acts on a promoter to enhance gene transcription. Examples include the 100-270 base pair SV40 enhancer on the late side of the replication origin, the polyomavirus enhancer on the late side of the replication origin, and the adenovirus enhancer.

[0060] Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0061] Transformation of host cells using recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of absorbing DNA are harvested after the exponential growth phase and can be treated by the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl 2 is to use. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as, for example, microinjection, electroporation, and liposome packaging.

[0062] The obtained transformant is cultured by a conventional method and expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used for culturing can be selected from various conventional media. Culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to an appropriate cell density, the promoter selected by an appropriate method (for example, temperature conversion or chemical induction) is induced, and the cells are recultured for a while.

[0063] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. 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 refolding treatment, treatment with a protein precipitant (salting-out method), centrifugation, cell disruption by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other liquid chromatography techniques and combinations of these methods.

[0064] Use

[0065] The CYP716A53v2 mutant of the present invention specifically acts on protopanaxadiol, adds a hydroxyl group to its C6 position, thereby generating protopanaxatriol, and its catalytic activity is higher than that of the wild-type CYP716A53v2. The said catalyst is a high-efficiency catalyst, and its catalytic efficiency is at least 10% higher than that of the wild type, preferably at least 20% higher, more preferably at least 30% higher, for example 40% or more, 50% or more, 60% or more higher.

Chemical formula

[0066] After obtaining the CYP716A53v2 mutant of the present invention, according to the disclosure of the present invention, those skilled in the art can conveniently apply the mutant of the present invention to exert a catalytic effect on protopanaxadiol as a substrate and obtain a technical effect superior to that of the wild-type CYP716A53v2.

[0067] When in use, especially in industrial production, the CYP716A53v2 mutant or its derivative polypeptide of the present invention can be immobilized on a solid-phase carrier to obtain an immobilized enzyme and used in an in vitro reaction with a substrate. The solid-phase carrier is, for example, microspheres, tubular bodies made of inorganic substances, etc. There are two types of methods for producing immobilized enzymes: physical methods and chemical methods. Physical methods include physical adsorption methods, embedding methods, etc. Chemical methods include binding methods and cross-linking methods. The binding method can be further divided into an ionic binding method and a covalent binding method. Any of the above methods for immobilizing enzymes can be applied to the present invention.

[0068] In any manner, in vitro production can be carried out using the CYP716A53v2 mutant of the present invention. When protopanaxadiol is present (as a substrate), it can react with the CYP716A53v2 mutant of the present invention produced on a large scale (which may be its extract (including crude extract) or fermentation broth, or may be the product after separation and purification), and protopanaxatriol can be obtained as a product.

[0069] In another preferred embodiment of the present invention, it is produced using a biosynthesis method. This generally includes: (1) providing an engineered cell characterized by including at least one anabolic pathway or production pathway containing protopanaxatriol (PPT), (2) in the engineered cell described in (1) above, expressing the CYP 716A53v2 mutant of the present invention, or replacing the wild-type CYP 716A53v2 in the metabolic pathway with the CYP 716A53v2 mutant of the present invention, and (3) culturing the engineered cell described in (2) to produce a protopanaxatriol product. In a more preferred embodiment, the method further includes a step of isolating and purifying the product from the culture of the engineered cell.

[0070] When produced using a biosynthesis method, a preferred embodiment of the present invention includes enhancing other compound metabolic pathways / production pathways in the protopanaxatriol (PPT) anabolic pathway in cells. As a precursor for the catalytic reaction of the present invention, it is also possible to provide more upstream substrates by enhancing the production of compounds in the upstream pathway of the anabolic pathway. It should be understood that several other methods for enhancing the protopanaxatriol (PPT) anabolic pathway may also be included in the present invention.

[0071] The CYP 716A53v2 mutant of the present invention can also be used in the production of a composition having a catalytic action. Those skilled in the art can determine the effective amount of the CYP 716A53v2 mutant in the composition according to the actual use of the composition.

[0072] The CYP 716A53v2 mutant described in the present invention, a composition containing the same, a cell expressing the same, etc. may be included in a kit in order to expand use or commercial use. Preferably, the kit may also include a medium or culture components suitable for culturing genetically engineered cells. Preferably, the kit also includes an instruction manual explaining the method of performing biosynthesis in order to guide those skilled in the art on the production by an appropriate method.

[0073] The present invention will be further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the present invention, but are merely illustrative of the present invention. The experimental methods that do not specify specific conditions in the following examples are usually carried out according to the normal conditions described in J. Sambrook et al., Guide to Molecular Cloning, Third Edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0074] Wild-type CYP716A53v2 protein sequence (SEQ ID NO: 1): MDLFISSQLLLLLVFCLFLFWNFKPSSQNKLPPGKTGWPIIGETLEFISCGQKGNPEKFVTQRMNKYSPDVFTTSLAGEKMVVFCGASGNKFIFSNENKLVVSWWPPAISKILTAT I PSVEKSKALRSLIVEFLKPEALHKFISVMDRTTRQHFEDKWNGSTEVKA F AMSESLTFELACWLLFSINDPVQVQKLSHLFEKVKAGLLS L PLNFPGTAFNRGIKAANLIRKELSVVIKQRRSDKLQTRKDLLSHVMLSNGEGEKFFSEMDIADVVLNLLIASHDTTSSAMGSVVYFLADHPHIYAKVLTEQMEIAKSKGAGELLSWEDIKRMKYSRNVINEAMRLVPPSQGGFKVVTSKFSYANFIIPKGWKIFWSVYSTHKDPKYFKNPEEFDPSRFEGDGPMPFTFIPFGGGPRMCPGSEFARLEVLIFMHHLVTNFKWEKVFPNEKIIY T PFPFPENGLPIRLSPCTL In the above sequence, the mutation sites are represented by bold underlines.

Example

[0075] Example 1: Obtaining a highly efficient cytochrome P450 mutant protein by random mutation

[0076] (1) Using pUC 57-synPPTS (a plasmid containing the CYP 716A53v2 coding gene) as a template, error-prone PCR was performed using primers SJ-F (SEQ ID NO:2) and SJ-R (SEQ ID NO:3). The error-prone PCR used the random mutation kit of Stratagene's GeneMorph II Random Mutagensis Kit. The PCR program was: 95°C for 2 min, 95°C for 10 s, 55°C for 15 s, 72°C for 1 min 30 s, a total of 24 cycles; 72°C for 10 min and then cooled to 10°C. The amount of pUC 57-synPPTS template used was 900 ng. After agarose gel electrophoresis of the PCR product, it was recovered to obtain the CYP 716A53v2 error-prone PCR product. SJ-F: atggatttgtttatttcttc (SEQ ID NO:2); SJ-R: ttacaatgtacatggagaca (SEQ ID NO:3).

[0077] (2) PCR reactions were performed using the primers and templates listed in Table 2 to amplify the target DNA fragments for strain construction. The PCR system was the 2×High-Fidelity Master Mix standard system of Tsingke's high-fidelity PCR enzyme I-5 (registered trademark). The PCR program was: 98°C for 2 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, a total of 30 cycles; 72°C for 10 min and then cooled to 10°C. After recovery by agarose gel electrophoresis, each PCR product was obtained. Both ends of the PCR product fragment were attached with sequences homologous to the adjacent both-end fragments approximately 70 bp before and after using PCR primers for homologous recombination in Saccharomyces cerevisiae.

[0078]

Table 2

[0079] (3) After mixing 100 ng of each of the above-mentioned product DNA fragments, they were transformed into competent Saccharomyces cerevisiae strain ZW (Wang, P. P. et al., Cell Discovery, 2019. 5(5)). After the transformation was completed, the strain was evenly spread on a YPD + 200 mg / L G418 antibiotic screening plate and statically cultured at 30 °C for 48 hours. All the clones were collected using toothpicks, transferred to a 96-well plate, and cultured with shaking at 30 °C for 24 h, then transferred to a new 96-well plate at a ratio of 1:100 and fermented for 96 h. Compound extraction: An equal volume of n-butanol solvent was added to the fermentation broth and extracted for 24 h. The upper organic phase was aspirated, and the yields and ratios of protopanaxadiol and protopanaxatriol of each transformant were measured by HPLC.

[0080] (4) After a large amount of screening work, the inventor obtained 4 clones in which the protopanaxatriol production PPT was improved by 20% and the ratio of protopanaxatriol / protopanaxadiol (PPT / PPD) was improved by 20% or more. Their numbers were SJ-1, SJ-2, SJ-3, and SJ-4, respectively. Using the genomes of the above 4 clones as templates, PCR was performed using primers SJ-F and SJ-R to obtain the cytochrome P450 fragments of each clone, and sequencing was performed to obtain each mutant protein sequence.

[0081] The sequence information and PPT production of each wild-type and mutant protein obtained above are shown in Table 3 and Figure 1:

Table 3

Example

[0082] Example 2. The mutation sites were incorporated to obtain a more efficient CYP716A53v2 mutant.

[0083] By the random mutation method of Example 1, four activity-improving sites of F167V, T451A, I117S, and L208C were obtained.

[0084] Based on the wild-type CYP716A53v2 gene, the above four mutation sites were combined in various ways to obtain a series of mutant genes of CYP716A53v2. By the methods shown in (2) and (3) of Example 1, the combined mutant genes of CYP716A53v2 were respectively transformed into ZW yeast competent cells, and a corresponding series of strains were constructed and fermented.

[0085] Fermentation method: For each mutant, six single clones were collected in a 96-well plate, cultured with shaking at 30 °C for 24 h, transferred to a new 96-well plate at a ratio of 1:100, and fermented for 96 h (yeast itself can produce hydroxyl donors). Compound extraction: An equal volume of n-butanol solvent was added to the fermentation broth to extract the compounds from the bacteria. Extraction was carried out for 24 h, and the upper organic phase was aspirated. The yields and ratios of protopanaxadiol and protopanaxatriol of each transformant were measured by HPLC.

[0086]

Table 4

Example

[0087] Example 3, using the cytochrome P450 mutant protein, protopanaxatriol was heterologously synthesized with high efficiency

[0088] In this example, using the cytochrome P450 mutant protein, protopanaxatriol was heterologously synthesized with high efficiency, and the specific method was as follows:

[0089] (1) PCR reactions were carried out using the primers and templates listed in Table 2 to amplify the target DNA fragments for strain construction. The PCR system was a 2×High-Fidelity Master Mix standard system with Tsingke's high-fidelity PCR enzyme I-5 (registered trademark). The PCR program was: 98°C for 2 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, for a total of 30 cycles; then 72°C for 10 min and cooled to 10°C. The PCR products were recovered by agarose gel electrophoresis to obtain each PCR product. Both ends of the PCR product fragments were attached with sequences homologous to the adjacent end fragments approximately 70 bp before and after for homologous recombination in Saccharomyces cerevisiae.

[0090] All of the above gene, homologous arm, and screening marker gene PCR fragments were mixed at 100 ng each, and then transformed into competent ZW of the Saccharomyces cerevisiae strain to obtain a recombinant Saccharomyces cerevisiae strain PPT-WT strain that produces protopanaxatriol.

[0091] (2) Similarly, instead of the wild-type CYP 716 A 53 v 2 gene, the mutant genes ZH-1, ZH-2, ZH-3, and ZH-4 were used as templates. The above PCR was performed to obtain each PCR fragment, which was then transformed into competent ZW of Saccharomyces cerevisiae to obtain recombinant Saccharomyces cerevisiae strains PPT-ZH-1 strain, PPT-ZH-2 strain, PPT-ZH-3 strain, and PPT-ZH-4 strain that produce protopanaxatriol and contain each mutant protein.

[0092] (3) Preparation of solid medium: Prepared medium: 1% yeast extract, 2% Bacto peptone, 2% D-glucose, 2% agar powder. Preparation of liquid medium: Prepared medium: 1% yeast extract, 2% Bacto peptone, 2% D-glucose.

[0093] (4) The recombinant brewing yeast strains PPT-ZH-1, PPT-ZH-2, PPT-ZH-3, and PPT-ZH-4 streaked on the solid medium plate were collected and cultured overnight with shaking in test tubes containing 5 mL of liquid medium (30 °C, 250 rpm, 16 h); centrifuged to collect the cells, transferred to a 50 mL Erlenmeyer flask with 10 mL of liquid medium, adjusted the OD 600 to 0.05, and cultured with shaking at 30 °C and 250 rpm to obtain fermentation products over 4 days. In this method, one parallel experiment is set up for each recombinant yeast simultaneously.

[0094] (5) Extraction and assay of protopanaxatriol: 100 μL of the fermentation broth was collected from 10 mL of the fermentation broth, the yeast was dissolved by shaking with Fastprep, an equal volume of n-butanol was added for extraction, and n-butanol was evaporated to dryness under vacuum conditions. After dissolving with 100 μL of methanol, the yield of the target product was measured by HPLC.

[0095] The protopanaxatriol of each recombinant brewing yeast strain obtained above is shown in Table 5 and Figure 1:

Table 5

[0096] All references mentioned in this application are hereby incorporated by reference as if each were individually cited. It should be understood that based on the above disclosure of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. A method for improving the catalytic activity of cytochrome P450 CYP716A53v2 by mutating, i.e., substituting, the amino acid sequence of cytochrome P450 CYP716A53v2, wherein the amino acid sequence number of cytochrome P450 CYP716A53v2 is SEQ ID NO: 1, corresponding to wild-type cytochrome P450 CYP716A53v2, the substitution is selected from the sites of the following group or combinations thereof: position 167, position 451, position 117, position 208, the substitution at position 167 is Val, the substitution at position 451 is Ala, the substitution at position 117 is Ser, and the substitution at position 208 is Cys, A method for improving the catalytic activity of cytochrome P450 CYP716A53v2, characterized by the above.

2. The amino acid sequence corresponds to wild-type cytochrome P450 CYP716A53v2, and the protein in which the site or combination of sites selected from the following group is substituted: position 167, position 451, position 117, position 208, the substitution at position 167 is Val, the substitution at position 451 is Ala, the substitution at position 117 is Ser, and the substitution at position 208 is Cys, The amino acid sequence number of cytochrome P450 CYP716A53v2 is SEQ ID NO:

1. A cytochrome P450 CYP716A53v2 mutant, characterized by the above.

3. The cytochrome P450 CYP716A53v2 mutant contains a protein selected from the following group: corresponding to wild-type cytochrome P450 CYP716A53v2, (1) the substitution at position 117 is Ser, and the substitution at position 451 is Ala; (2) the substitution at position 117 is Ser, and the substitution at position 208 is Cys, the substitution at position 167 is Val, and the substitution at position 451 is Ala; (3) the substitution at position 117 is Ser, and the substitution at position 208 is Cys; (4) the substitution at position 117 is Ser, and the substitution at position 208 is Cys, and the substitution at position 451 is Ala; (5) the substitution at position 167 is Val; (6) the substitution at position 451 is Ala; (7) the substitution at position 117 is Ser; (8) the substitution at position 208 is Cys; The cytochrome P450 CYP716A53v2 mutant according to Claim 2, characterized by the above. **Claim 4** An isolated polynucleotide encoding the cytochrome P450 CYP716A53v2 mutant according to any one of claims 2 to 3. **Claim 5** A vector comprising the polynucleotide according to claim 4. **Claim 6** A genetically engineered host cell comprising the vector according to claim 5 or having the polynucleotide according to claim 4 integrated into its genome. **Claim 7** The host cell according to claim 6, wherein the host cell is a eukaryotic cell or a prokaryotic cell, the eukaryotic cell includes yeast cells, plant cells, fungal cells, insect cells, mold cells, mammalian cells, and the prokaryotic cell includes Escherichia coli and Bacillus subtilis cells. **Claim 8** The host cell according to claim 7, wherein the yeast cell includes Saccharomyces cerevisiae cells or Pichia yeast cells, and the plant cell includes carrot cells. **Claim 9** (i) Culturing the host cell according to claim 6; (ii) Collecting a culture containing the cytochrome P450 CYP716A53v2 mutant according to any one of claims 2 to 3; (iii) Isolating the cytochrome P450 CYP716A53v2 mutant from the culture. A method for preparing the cytochrome P450 CYP716A53v2 mutant according to any one of claims 2 to 3, comprising the above steps. **Claim 10** The cytochrome P450 CYP716A53v2 mutant according to any one of claims 2 to 3; or the host cell according to claim 6 or its culture or lysate; and A food or industrially acceptable vector A composition for catalyzing the production of protopanaxatriol from protopanaxadiol, containing them in an effective amount. **Claim 11** Use of the cytochrome P450 CYP716A53v2 mutant according to any one of claims 2 to 3 or the composition according to claim 10 for catalyzing the production of protopanaxatriol from protopanaxadiol, wherein The cytochrome P450 CYP716A53v2 mutant increases one hydroxyl group at the C6 position of protopanaxadiol, thereby producing protopanaxatriol. Use of the cytochrome P450 CYP716A53v2 mutant or the composition, characterized by the above. **Claim 12** The cytochrome P450 CYP716A53v2 mutant according to any one of claims 2 to 3, or treating protopanaxadiol with the composition according to claim 10, comprising: A method for catalyzing the production of protopanaxatriol from protopanaxadiol, characterized in that the cytochrome P450 CYP716A53v2 mutant increases one hydroxyl group at the C6 position of protopanaxadiol, thereby generating protopanaxatriol.

13. The cytochrome P450 CYP716A53v2 mutant or combination of mutants according to any one of claims 2 to 3; The host cell according to claim 6; or The composition according to claim 10; A kit for catalyzing the production of protopanaxatriol from protopanaxadiol containing the same.

Images