WO2018133844A1 - 细胞色素p450突变蛋白及其应用 - Google Patents

细胞色素p450突变蛋白及其应用 Download PDF

Info

Publication number
WO2018133844A1
WO2018133844A1 PCT/CN2018/073453 CN2018073453W WO2018133844A1 WO 2018133844 A1 WO2018133844 A1 WO 2018133844A1 CN 2018073453 W CN2018073453 W CN 2018073453W WO 2018133844 A1 WO2018133844 A1 WO 2018133844A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutein
cytochrome
protopanaxadiol
ppd
seq
Prior art date
Application number
PCT/CN2018/073453
Other languages
English (en)
French (fr)
Inventor
周志华
王平平
严兴
程宜兴
杨成帅
Original Assignee
中国科学院上海生命科学研究院
宏冠生物药业有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海生命科学研究院, 宏冠生物药业有限公司 filed Critical 中国科学院上海生命科学研究院
Priority to KR1020197024319A priority Critical patent/KR102310518B1/ko
Priority to JP2019560442A priority patent/JP7090266B2/ja
Publication of WO2018133844A1 publication Critical patent/WO2018133844A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase

Definitions

  • the present invention relates to the field of biotechnology and plant biology, natural product medicine, and in particular, to a cytochrome P450 mutein and use thereof.
  • Ginsenoside is the main active substance in the genus Panax ginseng (such as ginseng, notoginseng, American ginseng, etc.). In recent years, some ginsenosides have also been found in the cucurbitaceae Gynostemma pentaphyllum. At present, domestic and foreign scientists have isolated at least 100 kinds of ginsenosides from plants such as ginseng and Gynostemma pentaphyllum. The content of these saponins in ginseng is very different. Some of the most effective triterpenoid saponins are extremely low in natural total saponins (also known as rare saponins), which are very expensive due to the high cost of extraction.
  • the drug Shenyi Capsule with ginsenoside Rg3 monomer as the main component can improve the symptoms of qi deficiency and improve the immune function of tumor patients.
  • Jinshen Capsule with ginsenoside Rh2 monomer as the main component is a health care medicine for improving immunity and enhancing disease resistance.
  • rare ginsenosides tend to have unique biological activity or more remarkable effects
  • the conventional preparation of rare ginsenosides is prepared by chemical hydrolysis, enzymatic hydrolysis and microbial hydrolysis of a large amount of saponins extracted from ginseng or notoginseng. Since the wild ginseng resources have been basically depleted, the ginseng total saponin resources are mainly derived from the artificial cultivation of ginseng or notoginseng, and the artificial cultivation has a long growth cycle (generally 5-7 years or more) and is subject to geographical restrictions. Frequently affected by pests and diseases, a large number of pesticides need to be applied.
  • ginseng or Panax notoginseng has serious continuous cropping obstacles (the ginseng or the Sanqi planting site needs to fallow for more than 5-15 years to overcome the continuous cropping obstacle), so the yield of ginsenosides, Quality and safety are facing challenges.
  • a single component saponin is prepared by using ginseng total saponin as a raw material, because a large amount of components in the total saponin cannot be converted into a target ginsenoside monomer (for example, ginseng triol saponin) cannot be utilized, and not only resources are caused.
  • the waste will also increase the extraction and purification costs.
  • yeast as the chassis, through the assembly and optimization of metabolic pathways, it has been realized to synthesize artemisinic acid or dihydroartemisinic acid with cheap monosaccharides, and then to produce artemisinin by one-step chemical conversion, which indicates the synthesis.
  • Biology has great potential for drug synthesis in natural products.
  • the yeast substrate cells are heterologously synthesized by synthetic biological methods to synthesize rare ginsenoside monomers.
  • the raw materials are cheap monosaccharides, and the preparation process is a safe and controllable fermentation process, avoiding any external pollution (for example, when the raw plants are artificially planted)
  • the pesticides therefore, the preparation of rare ginsenoside monomers by synthetic biological techniques not only has cost advantages, but also ensures the quality and safety of the finished product.
  • a variety of high-purity rare ginsenoside monomers are prepared by synthetic biological techniques for activity determination and clinical experiments, and promote the development of innovative drugs for rare ginsenosides.
  • the present invention provides a cytochrome P450 mutein which can produce a high PPD yield and a PPD/DM ratio by using the mutein.
  • a first aspect of the present invention provides a mutein of a cytochrome P450, the mutein is a non-native protein, and the mutein has catalytic activity for catalyzing the production of protopanaxadiol, and the mutein is in the wild A cytochrome P450 of the type SEQ ID NO.: 1 is mutated from one or more of the core amino acids associated with the catalytic activity of the enzyme selected from the group consisting of:
  • the mutein lacks one or more amino acids of SEQ ID NO.: 1 selected from the group consisting of:
  • the first methionine (M) The first methionine (M).
  • the mutein is deleted at positions 1-4 of SEQ ID NO.: 1, and the leucine (L) at position 18 is mutated.
  • the mutein is deleted at positions 1-4 of SEQ ID NO.: 1 and the leucine (L) at position 18 is mutated to isoleucine (I).
  • the 87th leucine (L) is mutated to isoleucine (I); and/or
  • the 235th lysine (K) is mutated to arginine (R); and/or
  • the 349th lysine (K) is mutated to arginine (R); and/or
  • the 231th asparagine (N) is mutated to tyrosine (Y); and/or
  • the 91th proline (P) is mutated to histidine (H); and/or
  • the 113th glutamine (Q) is mutated to arginine (R); and/or
  • the 18th is a mutation of leucine (L) to isoleucine (I).
  • the mutation is selected from the group consisting of L87I; K235R; K349R and V366I; N231Y and S285C; P91H; Q113R.
  • amino acid sequence of the mutant protein of the cytochrome P450 is set forth in SEQ ID NO.: 2-8.
  • the mutein is other than the mutation (eg, 87, 235, 349, 366, 231, 285, 91, 113, 18, and/or 1-4 amino acids)
  • the amino acid sequence is identical or substantially identical to the sequence shown in SEQ ID NO.: 1.
  • the substantially identical is that at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids are different, wherein The dissimilarity includes substitutions, deletions or additions of amino acids, and the muteins still have catalytic activity for catalyzing the production of protopanaxadiol.
  • sequence homology to the sequence of SEQ ID NO.: 1 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, optimally at least 98%, and homology ⁇ 485 / 486 or 99.79%.
  • mutant protein of cytochrome P450 catalyzes the reaction of dammaranediol (DM) to produce protopanaxadiol (PPD).
  • the mutein of the cytochrome P450 catalyzes the hydroxylation of the C12 position of the dammarane diol (DM) to form protopanaxadiol (PPD).
  • mutant protein of the cytochrome P450 catalyzes the following reaction:
  • the reaction has one or more characteristics selected from the group consisting of:
  • the pH of the reaction system is from 5.0 to 9.0, preferably from 7.0 to 8.0, more preferably from 7.4 to 7.5;
  • reaction temperature is 20-40 ° C, preferably 25-35 ° C, more preferably 26-33 ° C, optimally 30;
  • reaction time is from 0.5 h to 36 h, preferably from 2 h to 12 h, more preferably from 2 h to 3 h.
  • the mutant protein of cytochrome P450 catalyzes the catalytic activity of the crude ginseng diol (PPD) produced by dammarane diol (DM) to be 125 of wild-type P450 (SEQ ID NO.: 1) -250%.
  • the mutein of the cytochrome P450 has one or more characteristics selected from the group consisting of:
  • the ratio of proto-glycol diol yield/dammar diol (PPD/DM) catalyzed by the catalyzed cytochrome P450 protein is ⁇ 20%, preferably 23-250%, more preferably 25-250% or 30-200%.
  • the yield (mg/L) of the catalytically obtained protopanaxadiol (PPD) is ⁇ 300, preferably 303-600, more preferably 305-500, compared to the wild-type cytochrome P450 protein.
  • a second aspect of the invention provides a polynucleotide encoding the mutein of the first aspect of the invention.
  • polynucleotide is selected from the group consisting of:
  • nucleotide sequence has a homology of ⁇ 95% (preferably ⁇ 98%) to the sequence of any one of SEQ ID NO.: 15-21, and encodes SEQ ID NO.: 1 or 2-8 a polynucleotide of the indicated polypeptide;
  • the polynucleotide further comprises an auxiliary element selected from the group consisting of a signal peptide, a secreted peptide, a tag sequence (eg, 6His), or a flank of the ORF of the mutated protein of the cytochrome P450; combination.
  • an auxiliary element selected from the group consisting of a signal peptide, a secreted peptide, a tag sequence (eg, 6His), or a flank of the ORF of the mutated protein of the cytochrome P450; combination.
  • the polynucleotide is selected from the group consisting of a DNA sequence, an RNA sequence, or a combination thereof.
  • a third aspect of the invention provides a vector comprising the polynucleotide of the second aspect of the invention.
  • the vector comprises an expression vector, a shuttle vector, an integration vector.
  • a third aspect of the invention provides a host cell comprising the vector of the third aspect of the invention, or the polynucleotide of the second aspect of the invention integrated in the genome.
  • the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.
  • the host cell is a prokaryotic cell, such as E. coli.
  • the host cell is a ginseng cell.
  • a fifth aspect of the invention provides a method of producing a mutein of the cytochrome P450 of the first aspect of the invention, comprising the steps of:
  • the host cell of the fourth aspect of the invention is cultured under conditions suitable for expression to express a mutant protein of cytochrome P450;
  • the mutant protein of the cytochrome P450 is isolated.
  • a sixth aspect of the invention provides an enzyme preparation comprising the mutant protein of the cytochrome P450 of the first aspect of the invention.
  • the enzyme preparation comprises an injection, and/or a lyophilized preparation.
  • a seventh aspect of the invention provides a method for preparing protopanaxadiol, comprising the steps of:
  • the protopanaxadiol is isolated and purified.
  • reaction substrate is dammarane diol.
  • the catalytic reaction is carried out for a period of from 0.5 h to 36 h, preferably from 2 h to 12 h, more preferably from 2 h to 3 h.
  • the temperature of the catalytic reaction is 20 to 40 ° C, preferably 25 to 35 ° C, more preferably 26 to 33 ° C, most preferably 30 ° C. ;
  • the use of the mutein of the first aspect of the present invention wherein the mutein is used for catalyzing the formation of protopanaxadiol (PPD) by dammaranediol (DM), or is used A catalytic preparation for catalyzing the formation of protopanaxadiol (PPD) by catalyzing dammar diol (DM).
  • a ninth aspect of the invention provides the use of the mutein of the first aspect of the invention or the host cell of the invention, characterized in that it is used for the preparation of protopanaxadiol (PPD).
  • PPD protopanaxadiol
  • a tenth aspect of the invention provides a method for producing a transgenic plant, comprising the steps of: regenerating a host cell according to the fourth aspect of the invention into a plant, wherein the host cell is a plant cell.
  • the eleventh aspect of the present invention provides a mutant promoter sequence, wherein the promoter sequence has an increase in transcriptional activity of the P450 protein by at least 30% compared to the wild-type P450 promoter (SEQ ID NO.: 9) (eg, 40-100%).
  • sequence of the promoter is the sequence shown in any one of SEQ ID NO.: 10-13.
  • the mutant promoter is the promoter 1D1, which can significantly increase the expression level of the P450 protein;
  • the mutant promoter contains the following core nucleotides associated with transcriptional strength:
  • the 28th nucleotide T is deleted
  • the nucleotide at position 417 is G;
  • the nucleotide at position 445 is G;
  • the nucleotide at position 654 is A;
  • the nucleotide at position 655 is A;
  • nucleotide position number is based on the sequence shown in SEQ ID NO.: 9.
  • the mutated promoter 1D1 is associated with the following core nucleotides of transcriptional strength:
  • the nucleotide at position 417 is G;
  • the nucleotide at position 445 is G;
  • nucleotide position number is based on the sequence shown in SEQ ID NO.: 9.
  • mutant promoter 1D1 is identical or substantially identical to the sequence shown in SEQ ID NO.: 9 except for the nucleotides 417 and 445.
  • mutant promoter 1D1 has the sequence shown in SEQ ID NO.: 10.
  • the mutated promoter is promoter 9C1-2, having the following core nucleotides associated with transcriptional strength.
  • the 28th nucleotide T is deleted.
  • nucleotide position number is based on the sequence shown in SEQ ID NO.: 9.
  • mutant promoter 9C1-2 is identical or substantially identical to the sequence shown in SEQ ID NO.: 9 except for nucleotides 28.
  • mutant promoter 9C1-2 has the sequence shown in SEQ ID NO.:11.
  • the mutated promoter is promoter 11B5 having the following core nucleotides associated with transcriptional strength.
  • the nucleotide at position 654 is A.
  • nucleotide position number is based on the sequence shown in SEQ ID NO.: 9.
  • mutant promoter 11B5 is identical or substantially identical to the sequence shown in SEQ ID NO.: 9 except for nucleotide 654.
  • mutant promoter 11B5 has the sequence shown in SEQ ID NO.: 12.
  • the mutated promoter is the promoter 15F1 having the following core nucleotides associated with transcriptional strength:
  • the nucleotide at position 655 is A.
  • nucleotide position number is based on the sequence shown in SEQ ID NO.: 9.
  • mutant promoter 15F1 is identical or substantially identical to the sequence shown in SEQ ID NO.: 9 except for nucleotides 655.
  • mutant promoter 15F1 has the sequence shown in SEQ ID NO.: 13.
  • a twelfth aspect of the present invention provides an expression cassette comprising the above-described promoter of the present invention, and a nucleoside encoding the P450 mutein of the present invention, which is operably linked to the promoter, of the present invention Acid sequence.
  • Figure 1 shows a schematic representation of the construction of a recombinant S. cerevisiae strain WP8 producing damarene diol.
  • Figure 2 shows a histogram of PPD/DM of wild type and 1G4, 2D8, 3B9, 8G7, 9C1, 16F8 and 24A10 mutant strains.
  • Figure 3 shows an HPLC assay of recombinant S. cerevisiae strains producing protopanaxadiol.
  • Figure 4 shows a schematic representation of the yield of recombinant S. cerevisiae strains producing protopanaxadiol.
  • the present inventors have unexpectedly screened key amino acid sites which can significantly increase the catalytic activity of cytochrome P450 muteins through extensive screening.
  • the present inventors have found that the modification of key sites in cytochrome P450 CYP716A47 can significantly increase PPD production and PPD/DM ratio.
  • the present inventors have also found that deletion of the amino acid at position 1 to position 4 of the wild-type cytochrome P450 CYP716A47 and simultaneous mutation of one of the key sites (such as amino acid 18) can significantly improve the catalytic activity.
  • the ratio in PPD/DM can be increased to 125.6%.
  • the present inventors also constructed a Saccharomyces cerevisiae chassis cell WP8 which synthesizes dammarene diol DM, and obtained a mutant library of cytochrome P450 (CYP716A47) by using the Stratagene GeneMorph II Random Mutagenesis Kit random mutagenesis kit.
  • a CYP716A47 yeast mutant library was constructed by transforming the Saccharomyces cerevisiae substrate WP8 into a single copy inserted into the yeast genome and synthesizing the original ginseng diol PPD. On the basis of this, the present invention has been completed.
  • AxxB means that amino acid A at position xx becomes amino acid B, for example "L87I” indicates that amino acid L at position 87 is mutated to I, and so on.
  • Mutant protein of the invention and nucleic acid encoding the same
  • mutein As used herein, the terms "mutein”, “mutein of the invention”, “cytochrome P450 mutein of the invention” are used interchangeably and refer to a non-naturally occurring cytochrome P450 mutein, and the mutein is based on The protein of SEQ ID NO.: 1 is artificially engineered, wherein the mutant protein contains a core amino acid associated with catalytic activity of the enzyme, and at least one of the core amino acids is artificially engineered; and the invention
  • the mutein has an enzymatic activity that catalyzes the hydroxylation of C12 of dammarene diol DM to form protopanaxadiol PPD.
  • core amino acid refers to SEQ ID NO.: 1 and has a homology to SEQ ID NO.: 1 of at least 80%, such as 84%, 85%, 90%, 92%, 95%, 98%.
  • the corresponding site is a specific amino acid as described herein, such as the sequence based on SEQ ID NO.: 1, the core amino acid is:
  • Deleting 1, 2, 3, or 4 amino acids in positions 1-4, and the mutant protein obtained by mutating the above core amino acid or the mutant protein obtained by deleting the amino acids 1-4 has a catalytic dam
  • the C12 of the olefinic diol is hydroxylated to form the enzymatic activity of the protopanaxadiol PPD.
  • the core amino acid of the present invention is mutated as follows:
  • the 235th lysine (K) is mutated to arginine (R); and/or
  • the 349th lysine (K) is mutated to arginine (R); and/or
  • the 231th asparagine (N) is mutated to tyrosine (Y); and/or
  • the 91th proline (P) is mutated to histidine (H); and/or
  • the 113th glutamine (Q) is mutated to arginine (R); and/or
  • the 18th is a mutation of leucine (L) to isoleucine (I).
  • the amino acid numbering in the mutant protein of the present invention is based on SEQ ID NO.: 1.
  • the mutant protein may have a mismatch relative to the amino acid numbering of SEQ ID NO.: 1), such as a mismatch to the N-terminus or C-terminus of the amino acid, 1-5, using conventional sequence alignment techniques in the art, those skilled in the art It is generally understood that such misalignment is within a reasonable range and should not result in homology of 80% (eg, 90%, 95%, 98%) due to misalignment of amino acid numbers, with the same or similar production of proto-ginseng II.
  • the mutant protein of the alcohol PPD catalytic activity is not within the scope of the mutein of the present invention.
  • the muteins of the invention are synthetic or recombinant proteins, i.e., may be products of chemical synthesis, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants).
  • the muteins of the invention may be glycosylated or may be non-glycosylated, depending on the host used in the recombinant production protocol.
  • the muteins of the invention may or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the muteins.
  • fragment refers to a protein that substantially retains the same biological function or activity of the mutein.
  • the mutein fragment, derivative or analog of the present invention may be (i) a mutein having one or more conserved or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code, or (ii) a mutein having a substituent in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) a half-life compound, such as polyethylene glycol), a fusion protein formed by fusion, or (iv) a mutant protein formed by fused an additional amino acid sequence to the mutant protein sequence (such as a leader or secretion sequence or used to purify the mutant protein) Sequence or proprotein sequence, or fusion protein with the formation of an antigenic IgG fragment).
  • conservatively substituted amino acids are preferably produced by amino acid
  • the active mutant protein of the present invention has an enzymatic activity of catalyzing the hydroxylation of C12 of dammarene diol DM to form protopanaxadiol PPD.
  • the mutein is as set forth in SEQ ID NO.: 2-8. It will be understood that the muteins of the invention generally have a higher homology (identity) than the sequences set forth in SEQ ID NO.: 2-8, preferably the mutein and SEQ ID NO.:
  • the sequence of 2-8 has a homology of at least 80%, preferably at least 85% to 90%, more preferably at least 95%, most preferably at least 98%, optimally ⁇ 485/486 (99.79%).
  • the muteins of the invention may also be modified. Modifications (usually without altering the primary structure) include: chemically derived forms of the mutant protein, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of muteins or in further processing steps. Such modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are muteins that have been modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • polynucleotide encoding a mutein may be a polynucleotide comprising a mutein of the invention, or a polynucleotide further comprising an additional coding and/or non-coding sequence.
  • the present invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or muteins having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the encoded mutant protein thereof.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions (or stringent conditions).
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the muteins and polynucleotides of the invention are preferably provided in isolated form, and more preferably, purified to homogeneity.
  • the full length sequence of the polynucleotide of the present invention can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention.
  • RACE method RACE-cDNA end rapid amplification method
  • primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • wild-type cytochrome P450 refers to a naturally occurring, unengineered cytochrome P450 whose nucleotides can be obtained by genetic engineering techniques such as genome sequencing, polymerase chain reaction (PCR). Etc., the amino acid sequence thereof can be derived from a nucleotide sequence.
  • the amino acid sequence of the wild type cytochrome P450 is shown in SEQ ID NO.: 1.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the vectors of the invention or the mutant protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be utilized to express or produce recombinant muteins by conventional recombinant DNA techniques. Generally there are the following steps:
  • a polynucleotide sequence encoding a mutein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the mutein encoding DNA sequences of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Usable examples include a 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, a polyoma enhancer on the late side of the replication initiation site, and an adenovirus enhancer.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl2. Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated 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 such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present inventors have also found that mutations in one or more amino acids of other sites of the wild-type cytochrome P450 CYP716A47, such as the 235/349/366/231/285/91/113 isolocation, can also significantly enhance their catalysis.
  • the activity, specifically expressed in the PPD / DM ratio can be increased by 26.1-80.2%.
  • the present invention To screen a library of cytochrome P450 CYP716A47, the present invention first constructed a Saccharomyces cerevisiae chassis cell capable of producing the protopanaxadiol precursor compound damarene diol.
  • the dammarene diol synthase gene PgDDS is introduced into wild-type Saccharomyces cerevisiae, and the dammarane diol can be synthesized by using 2,3-epoxysqualene synthesized by Saccharomyces cerevisiae's own mevalonate pathway.
  • the Saccharomyces cerevisiae strain WP8 with high yield of dammarene diol was obtained by optimizing the synthetic dammarene diol synthesis pathway, including optimization of the rate-limiting step and optimization of precursor supply, as the Saccharomyces cerevisiae chassis cells, as shown in Figure 1. Shown.
  • cytochrome P450 gene sequence designated optPPDS, SEQ ID NO.: 14, encoding the amino acid sequence of SEQ ID NO.: 1 as a template
  • EP-1 5"-ATGGCTGCGGCCATGGTCTTAT-3
  • EP-2 5"-GTTATGTGGATGCAGATGGATT-3
  • the error-prone PCR was performed using the Stratagene GeneMorph II Random Mutagenesis Kit random mutagenesis kit.
  • the PCR program was: 95 ° C for 2 min; 95 ° C for 10 s, 55 ° C for 15 s, 72 ° C for 2 min, for a total of 28 cycles; 72 ° C for 10 min to 10 ° C, the template was used for 50 ng.
  • the PCR product was recovered by agarose gel electrophoresis. Cytochrome P450 error-prone PCR product.
  • the transformed product was uniformly coated on a YPD+200 mg/L G418 antibiotic screening plate, and cultured at 30 ° C for 2-3 days. All clones were picked with a toothpick and transferred to a 96-well plate, incubated at 30 °C for 1 day, and transferred to a new 96-well plate for fermentation for 4 days. An equal volume of n-butanol solvent was added to the fermentation broth for 1 h, and the upper organic phase was taken up for HPLC to determine the yield and ratio of each of the transformants damasene diol and protopanaxadiol.
  • mutant promoter sequences were further screened, and the promoter sequences of these mutations were beneficial to increase the expression of cytochrome P450 and thereby enhance the synthesis of proto-ginoldiol. Efficiency and yield.
  • PPD/DM proto-glycol diol production/dammar diol
  • SEQ ID NO. Remarks PPD/DM improvement GAL1 9 Wild type promoter 0 1D1 10 Mutation in SEQ ID NO.: 9: C417G, A445G 44.1% 9C1-2 11 Mutation in SEQ ID NO.: 9: T-bit deletion at position 28 80.1% 11B5 12 Mutation in SEQ ID NO.: 9: G654A 62.2% 15F1 13 Mutation in SEQ ID NO.: 9: G655A 44.1%
  • these mutated promoters can further increase the expression level of the wild-type P450 protein, thereby increasing the ratio of proto-glycol diol production/damaene diol. Furthermore, these mutated promoters can be further used in combination with the coding sequence of the P450 mutein of the present invention to further increase the ratio of protopanaxadiol production/dammar diol.
  • Saccharomyces cerevisiae genome as a template, the Saccharomyces cerevisiae strain WP8 was transformed with SEQ ID NO.: 14 by the method of Example 1, to obtain a recombinant Saccharomyces cerevisiae strain WP8-WT producing protopanaxadiol.
  • Disposition medium 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose), 2% agar powder.
  • Configure liquid medium Configure medium: 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose).
  • cytochrome P450 mutein of the present invention can significantly increase the yield of PPD (up to 490 mg/L) and the ratio of PPD/DM (up to 63%) compared to wild-type cytochrome P450.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nutrition Science (AREA)
  • Environmental Sciences (AREA)
  • Botany (AREA)
  • Physiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

本发明提供了一种细胞色素P450突变蛋白及其应用，具体地，本发明对细胞色素P450 CYP716A47中的关键位点进行改造后，可以显著提高PPD产量以及PPD/DM比例。

Description

细胞色素P450突变蛋白及其应用 技术领域
本发明涉及生物技术和植物生物学、天然产物药物领域，具体地，本发明涉及一种细胞色素P450突变蛋白及其应用。
背景技术
人参皂苷是五加科人参属植物(如人参、三七、西洋参等)中的主要活性物质，近年来在葫芦科植物绞股蓝中也发现一些人参皂苷。目前，国内外科学家已经从人参、绞股蓝等植物中分离出了至少100多种人参皂苷，这些皂苷在人参中的含量差别非常大。其中一些疗效显着的三萜皂苷在天然总皂苷中含量极低(亦被称为稀有皂苷)，由于提取的成本很高，所以价格就非常昂贵。目前多种皂苷已经用于临床，如以人参皂苷Rg3单体为主要成分的药物参一胶囊可改善肿瘤患者的气虚症状，提高机体免疫功能。以人参皂苷Rh2单体为主要成分的今幸胶囊是一种保健药品用于提高机体免疫力，增强抗病能力。
由于稀有人参皂苷往往具有独特生物活性或更显著疗效，传统制备稀有人参皂苷都是从人参或三七中提取的大量皂苷经过化学水解法、酶法水解和微生物法水解来制备。由于野生的人参资源已基本耗竭，人参总皂苷资源目前主要来源于人参或三七的人工栽培，而其人工栽培的生长周期长(一般需要5-7年以上)，并且受到地域的限制，还经常受到病虫害而需要施用大量的农药，所以，人参或三七的人工栽培有严重的连作障碍(人参或三七种植地需要休耕5-15年以上才能克服连作障碍)，所以人参皂苷的产量、品质及安全性都面临挑战。另一方面，以人参总皂苷为原料来制备单一成分的皂苷，因为总皂苷中还有大量的成份无法转化为目标人参皂苷单体(例如原人参三醇型皂苷)无法得到利用，不仅造成资源的浪费，还会增加抽提纯化成本。
合成生物学的发展为植物来源的天然产物的异源合成提供了新的机遇。以酵母为底盘，通过代谢途径的组装和优化，已经实现了用廉价的单糖来发酵合成青蒿酸或者双氢青蒿酸，继而再通过一步化学转化的方法生产青蒿素，这表明合成生物学在天然产物的药物合成方面具有的巨大潜力。利用酵母底盘细胞通过合成生物学方法异源合成稀有人参皂苷单体，原料为廉价的单糖，制备过程为安全性可调控的发酵过程，避免了任何外来污染(例如，原料植物人工种植时使用的农药)，因此，通过合成生物学技术制备稀有人参皂苷单体，不仅具有成本优势，而且，可以保证成品的品质与安全性。利用合成生物学技术制备足够量的各种高纯度稀有人参皂苷单体，用于活性测定及临床实验，促进稀有人参皂苷的创新药物研发。
利用合成生物学方法来人工合成具有药用活性的人参皂苷，首要需要解析与重构原人参二醇PPD的合成代谢途径。由于人参皂苷属于三萜化合物，植物中MVA和MEP代谢途径提供了萜类化合物的共同前体IPP和DMAPP，为三萜化合物前体角鲨烯和2,3-环氧角鲨烯的合成奠定了基础由于目前的细胞色素P450蛋白催化达玛烯二醇合成原人参二醇催化活性很低，在利用微生物细胞工厂生产PPD时是一个限速步骤，不仅限制了PPD产量的提高也导致了中间产物DM的积累。
因此，本领域需要对细胞色素P450进行更多的研究和改造，以获得更高效的细胞色素P450蛋白元件从而促进人参皂苷细胞工厂合成效率。
发明内容
本发明提供了一种细胞色素P450突变蛋白，通过采用所述的突变蛋白可以产生很高的PPD产量以及PPD/DM比例。
本发明的第一方面提供了一种细胞色素P450的突变蛋白，所述的突变蛋白为非天然蛋白，且所述突变蛋白具有催化生成原人参二醇的催化活性，并且所述突变蛋白在野生型的细胞色素P450的对应于SEQ ID NO.:1的选自下组的一个或多个与酶催化活性相关的核心氨基酸发生突变：
第91位脯氨酸(P)；
第87位亮氨酸(L)；
第235位赖氨酸(K)；
第349位赖氨酸(K)；
第366位缬氨酸(V)；
第231位天冬酰胺(N)；
第285位丝氨酸(S)；
第113位谷氨酰胺(Q)；
第18位亮氨酸(L)；和/或
缺失第1-4位中的1、2、3、或4个氨基酸。
在另一优选例中，所述突变蛋白缺失SEQ ID NO.:1的选自下组的一个或多个氨基酸：
第1位甲硫氨酸(M)；
第2位丙氨酸(A)；
第3位丙氨酸(A)；
第4位丙氨酸(A)。
在另一优选例中，所述的突变蛋白缺失SEQ ID NO.:1中1-4位，且第18位亮氨酸(L)发生突变。
在另一优选例中，所述的突变蛋白缺失SEQ ID NO.:1中1-4位，且第18位亮氨酸(L)突变为异亮氨酸(I)。
在另一优选例中，所述第87位亮氨酸(L)突变为异亮氨酸(I)；和/或
第235位赖氨酸(K)突变为精氨酸(R)；和/或
第349位赖氨酸(K)突变为精氨酸(R)；和/或
第366位缬氨酸(V)突变为异亮氨酸(I)；和/或
第231位天冬酰胺(N)突变为酪氨酸(Y)；和/或
第285位丝氨酸(S)突变为半胱氨酸(C)；和/或
第91位脯氨酸(P)突变为组氨酸(H)；和/或
第113位谷氨酰胺(Q)突变为精氨酸(R)；和/或
第18为亮氨酸(L)突变为异亮氨酸(I)。
在另一优选例中，所述的突变选自下组：L87I；K235R；K349R和V366I；N231Y和S285C；P91H；Q113R。
在另一优选例中，所述细胞色素P450的突变蛋白的氨基酸序列如SEQ ID NO.:2-8所示。
在另一优选例中，所述的突变蛋白除所述突变(如87、235、349、366、231、285、91、113位、18位、和/或1-4位氨基酸)外，其余的氨基酸序列与SEQ ID NO.:1所示的序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-20个，更佳地为1-10个、更佳地1-5个)氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加，且所述的突变蛋白仍具有催化生成原人参二醇的催化活性活性。
在另一优选例中，与SEQ ID NO.:1所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，最佳地至少为98％，且同源性≤485/486或99.79％。
在另一优选例中，所述细胞色素P450的突变蛋白催化达玛烯二醇(DM)反应生 成原人参二醇(PPD)。
在另一优选例中，所述细胞色素P450的突变蛋白催化达玛烯二醇(DM)的C12位发生羟基化，生成原人参二醇(PPD)。
在另一优选例中，所述的细胞色素P450的突变蛋白催化如下反应：
Figure PCTCN2018073453-appb-000001
在另一优选例中，所述反应具有选自下组的一个或多个特点：
(i)反应体系的pH为5.0-9.0，较佳地，7.0-8.0，更佳地，7.4-7.5；
(ii)反应温度为20-40℃，较佳地，25-35℃，更佳地，26-33℃，最佳地，30；
(iii)反应时间为0.5h-36h，较佳地，2h-12h，更佳地，2h-3h。
在另一优选例中，所述细胞色素P450的突变蛋白催化达玛烯二醇(DM)的生成原人参二醇(PPD)的催化活性为野生型P450(SEQ ID NO.:1)的125-250％。
在另一优选例中，所述细胞色素P450的突变蛋白具有选自下组的一个或多个特征：
(a)与野生型的细胞色素P450蛋白相比，催化获得的原人参二醇产量/达玛烯二醇(PPD/DM)的比例≥20％，较佳地23-250％，更佳地25-250％或30-200％。
(b)与野生型的细胞色素P450蛋白相比，催化获得的原人参二醇(PPD)的产量(mg/L)≥300，较佳地303-600，更佳地305-500。
本发明第二方面提供了一种多核苷酸，所述的多核苷酸编码本发明第一方面所述的突变蛋白。
在另一优选例中，所述多核苷酸选自下组：
(a)编码如SEQ ID NO.:2-8任一所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:15-21任一所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:15-21任一所示序列的同源性≥95％(较佳地≥98％)，且编码SEQ ID NO.:1或2-8任一所示多肽的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述的多核苷酸在细胞色素P450的突变蛋白的ORF的侧翼 还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的多核苷酸选自下组：DNA序列、RNA序列、或其组合。
本发明第三方面提供了一种载体，所述的载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
本发明第三方面提供了一种宿主细胞，所述的宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞或植物细胞。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述的宿主细胞为人参细胞。
本发明第五方面提供了一种产生本发明第一方面所述细胞色素P450的突变蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达出细胞色素P450的突变蛋白；和
分离所述细胞色素P450的突变蛋白。
本发明第六方面提供了一种酶制剂，所述酶制剂包含本发明第一方面所述的细胞色素P450的突变蛋白。
在另一优选例中，所述的酶制剂包括注射剂、和/或冻干制剂。
本发明第七方面提供了一种制备原人参二醇的方法，包括步骤：
(i)将本发明第一方面所述的细胞色素P450的突变蛋白与反应底物接触，进行催化反应，从而获得所述原人参二醇；和
(ii)任选地，分离并纯化所述原人参二醇。
在另一优选例中，所述反应底物为达玛烯二醇。
在另一优选例中，在步骤(i)中，所述催化反应的时间为0.5h-36h，较佳地，2h-12h，更佳地，2h-3h。
在另一优选例中，在步骤(i)中，所述催化反应的温度为20-40℃，较佳地，25-35℃，更佳地，26-33℃，最佳地，30℃；
本发明第八方面提供了一种本发明第一方面所述的突变蛋白的用途，所述的突变蛋白用于催化达玛烯二醇(DM)生成原人参二醇(PPD)，或被用于制备催化达 玛烯二醇(DM)生成原人参二醇(PPD)的催化制剂。
本发明第九方面提供了一种本发明第一方面所述的突变蛋白或本发明所述的宿主细胞的用途，其特征在于，用于制备原人参二醇(PPD)。
本发明第十方面提供了一种产生转基因植物的方法，其特征在于，包括步骤：将本发明第四方面所述的宿主细胞再生为植物，其中，所述的宿主细胞为植物细胞。
本发明第十一方面提供了一种突变的启动子序列，所述的启动子序列与野生型P450启动子(SEQ ID NO.:9)相比，P450蛋白的转录活性提高至少30％(如40-100％)。
在另一优选例中，所述的启动子的序列为SEQ ID NO.:10-13中任一所示的序列。
在另一优选例中，所述突变启动子为启动子1D1，可以显著提高P450蛋白的表达量；
所述突变启动子含有与转录强度相关的以下核心核苷酸：
第28位核苷酸T缺失；
第417位核苷酸为G；
第445位核苷酸为G；
第654位核苷酸为A；
第655位核苷酸为A；
其中，所述核苷酸位置编号基于SEQ ID NO.:9所示的序列。
在另一优选例中，所述突变的启动子1D1与转录强度相关的以下核心核苷酸：
第417位核苷酸为G；
第445位核苷酸为G；
其中，所述核苷酸位置编号基于SEQ ID NO.:9所示的序列。
在另一优选例中，所述的突变启动子1D1除了417和445位核苷酸外，其余核苷酸与SEQ ID NO.:9所示的序列相同或基本相同。
在另一优选例中，所述的突变启动子1D1具有SEQ ID NO.:10所示的序列。
在另一优选例中，所述突变的启动子为启动子9C1-2，具有与转录强度相关的以下核心核苷酸。
第28位核苷酸T缺失。
其中，所述核苷酸位置编号基于SEQ ID NO.:9所示的序列。
在另一优选例中，所述的突变启动子9C1-2除了28位核苷酸外，其余核苷酸与SEQ ID NO.:9所示的序列相同或基本相同。
在另一优选例中，所述的突变启动子9C1-2具有SEQ ID NO.:11所示的序列。
在另一优选例中，所述突变的启动子为启动子11B5，具有与转录强度相关的以下核心核苷酸。
第654位核苷酸为A。
其中，所述核苷酸位置编号基于SEQ ID NO.:9所示的序列。
在另一优选例中，所述的突变启动子11B5除了654位核苷酸外，其余核苷酸与SEQ ID NO.:9所示的序列相同或基本相同。
在另一优选例中，所述的突变启动子11B5具有SEQ ID NO.:12所示的序列。
在另一优选例中，所述突变的启动子为启动子15F1，具有与转录强度相关的以下核心核苷酸：
第655位核苷酸为A。
其中，所述核苷酸位置编号基于SEQ ID NO.:9所示的序列。
在另一优选例中，所述的突变启动子15F1除了655位核苷酸外，其余核苷酸与SEQ ID NO.:9所示的序列相同或基本相同。
在另一优选例中，所述的突变启动子15F1具有SEQ ID NO.:13所示的序列。
本发明第十二方面提供了一种表达盒，所述的表达盒含有本发明上述的启动子，以及与所述启动子操作性相连的本发明所述的编码本发明P450突变蛋白的核苷酸序列。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了生产达玛烯二醇的重组酿酒酵母菌株WP8的构建示意图。
图2显示了野生型及1G4、2D8、3B9、8G7、9C1、16F8和24A10突变体菌株PPD/DM柱状图。
图3显示了生产原人参二醇的重组酿酒酵母菌株HPLC检测图。
图4显示了生产原人参二醇的重组酿酒酵母菌株产量示意图。
具体实施方式
通过广泛而深入的研究，本发明人通过大量筛选，意外的筛到了可显著提高细胞色素P450突变蛋白催化活性的关键氨基酸位点。本发明发现，对细胞色素P450CYP716A47中的关键位点进行改造后，可以显著提高PPD产量以及PPD/DM比例。此外，本发明人还发现，野生型的细胞色素P450CYP716A47的第1位-第4位氨基酸缺失，并同时突变其中一个关键位点(如第18位氨基酸)，可显著提高其催化活性，具体表现在PPD/DM的比例可提高至125.6％。
此外，本发明人还构建了合成达玛烯二醇DM的酿酒酵母底盘细胞WP8，并通过选用Stratagene公司GeneMorph II Random Mutagenesis Kit随机突变试剂盒，获得了细胞色素P450(CYP716A47)的突变体库，并通过转化酿酒酵母底盘细胞WP8，构建了一个单拷贝插入酵母基因组并合成原人参二醇PPD的CYP716A47酵母突变体文库。在此基础上，完成了本发明。
术语
如本文所用，术语“AxxB”表示第xx位的氨基酸A变为氨基酸B，例如“L87I”表示第87位的氨基酸L突变为I，以此类推。
本发明突变蛋白及其编码核酸
如本文所用，术语“突变蛋白”、“本发明突变蛋白”、“本发明细胞色素P450突变蛋白”可互换使用，均指非天然存在的细胞色素P450突变蛋白，且所述突变蛋白为基于SEQ ID NO.:1所示蛋白进行人工改造的蛋白，其中，所述的突变蛋白含有与酶催化活性相关的核心氨基酸，且所述核心氨基酸中至少有一个是经过人工改造的；并且本发明突变蛋白具有催化达玛烯二醇DM的C12发生羟基化形成原人参二醇PPD的酶活性。
术语“核心氨基酸”指的是基于SEQ ID NO.:1，且与SEQ ID NO.:1同源性达至少80％，如84％、85％、90％、92％、95％、98％的序列中，相应位点是本文所述的特定氨基酸，如基于SEQ ID NO.:1所示的序列，核心氨基酸为：
第91位脯氨酸(P)；
第87位亮氨酸(L)；
第235位赖氨酸(K)；
第349位赖氨酸(K)；
第366位缬氨酸(V)；
第231位天冬酰胺(N)；
第285位丝氨酸(S)；
第113位谷氨酰胺(Q)；
第18位亮氨酸(L)；和/或
缺失第1-4位中的1、2、3、或4个氨基酸，且对上述核心氨基酸进行突变所得到的突变蛋白或缺失所述第1-4位氨基酸所得到的突变蛋白具有催化达玛烯二醇DM的C12发生羟基化形成原人参二醇PPD的酶活性。
优选地，在本发明中，对本发明的所述核心氨基酸进行如下突变：
第87位亮氨酸(L)突变为异亮氨酸(I)；和/或
第235位赖氨酸(K)突变为精氨酸(R)；和/或
第349位赖氨酸(K)突变为精氨酸(R)；和/或
第366位缬氨酸(V)突变为异亮氨酸(I)；和/或
第231位天冬酰胺(N)突变为酪氨酸(Y)；和/或
第285位丝氨酸(S)突变为半胱氨酸(C)；和/或
第91位脯氨酸(P)突变为组氨酸(H)；和/或
第113位谷氨酰胺(Q)突变为精氨酸(R)；和/或
第18为亮氨酸(L)突变为异亮氨酸(I)。
应理解，本发明突变蛋白中的氨基酸编号基于SEQ ID NO.:1作出，当某一具体突变蛋白与SEQ ID NO.:1所示序列的同源性达到80％或以上时，突变蛋白的氨基酸编号可能会有相对于SEQ ID NO.:1)的氨基酸编号的错位，如向氨基酸的N末端或C末端错位1-5位，而采用本领域常规的序列比对技术，本领域技术人员通常可以理解这样的错位是在合理范围内的，且不应当由于氨基酸编号的错位而使同源性达80％(如90％、95％、98％)的、具有相同或相似产生原人参二醇PPD催化活性的突变蛋白不在本发明突变蛋白的范围内。
本发明突变蛋白是合成蛋白或重组蛋白，即可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。根据重组生产方案所用的宿主，本发明的突变蛋白可以是糖基化的，或可以是非糖基化的。本发明的突变蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述突变蛋白的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持所述突变蛋白相同的生物学功能或活性的蛋白。
本发明的突变蛋白片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的突变蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的突变蛋白，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白半衰期的化合物，例如聚乙二醇)融合所形成的突变蛋白，或(iv)附 加的氨基酸序列融合到此突变蛋白序列而形成的突变蛋白(如前导序列或分泌序列或用来纯化此突变蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。本发明中，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
最初的残基 代表性的取代 优选的取代
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
本发明的活性突变蛋白具有催化达玛烯二醇DM的C12发生羟基化形成原人参二醇PPD的酶活性。
优选地，所述的突变蛋白如SEQ ID NO.:2-8所示。应理解，本发明突变蛋白与SEQ ID NO.:2-8所示的序列相比，通常具有较高的同源性(相同性)，优选地，所述的突变蛋白与SEQ ID NO.:2-8所示序列的同源性至少为80％，较佳地至少为85％-90％，更佳地至少为95％，最佳地至少为98％，最佳地，≥485/486(99.79％)。
此外，还可以对本发明突变蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的突变蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在突变蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的突变蛋白。这种修饰可以通过将突变蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨 酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的突变蛋白。
术语“编码突变蛋白的多核苷酸”可以是包括编码本发明突变蛋白的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或突变蛋白的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的突变蛋白的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的突变蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
野生型细胞色素P450
如本文所用，“野生型细胞色素P450”是指天然存在的、未经过人工改造的细胞色素P450，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述野生型细胞色素P450的氨基酸序列如SEQ ID NO.:1所示。
上述涉及的野生蛋白、本发明突变蛋白的序列信息如表2(见实施例)所示。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明突变蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明突变蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码突变蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明突变蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于 转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如人参细胞)。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的主要优点包括：
(i)经大量筛选和改造，本发明首次发现了细胞色素P450(CYP716A47)的催化活性位点及其启动子的关键位点，改造了相关位点后，能够显著提高细胞色素P450催化活性和表达量，并显著提高PPD产量以及PPD/DM比例。
(ii)本发明首次发现野生型的细胞色素P450CYP716A47的第1位-第4位氨基酸缺失，并同时突变其中一个关键位点(如第18位氨基酸)，可显著提高其催化活性，具体表现在PPD/DM的比例可提高至125.6％。
(iii)本发明还发现对野生型的细胞色素P450CYP716A47的其他位点如第235/349/366/231/285/91/113等位点的一个或几个氨基酸进行突变也可显著提高其催化活性，具体表现在PPD/DM的比例可提高26.1-80.2％。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
实施例1 生产达玛烯二醇的重组酿酒酵母菌株构建
为对细胞色素P450CYP716A47的文库进行筛选，本发明首先构建了一株可生产原人参二醇前体化合物达玛烯二醇的酿酒酵母底盘细胞。
向野生型酿酒酵母中导入达玛烯二醇合成酶基因PgDDS，利用酿酒酵母自身的甲羟戊酸途径合成的2,3-环氧角鲨烯可以合成达玛烯二醇。通过对人工构建的达玛烯二醇合成途径进行优化包括对合成限速步骤优化、前体供应优化等获得了高产达玛烯二醇的酿酒酵母菌株WP8，作为酿酒酵母底盘细胞，如图1所示。
实施例2 定向进化获得高效的细胞色素P450突变体蛋白
(1)以细胞色素P450基因序列(命名为optPPDS,SEQ ID NO.:14，编码SEQ ID NO.:1的氨基酸序列)为模板，利用引物EP-1(5”-ATGGCTGCGGCCATGGTCTTAT-3”(SEQ ID NO.:22))和EP-2(5”-GTTATGTGGATGCAGATGGATT-3”(SEQ ID NO.:23)进行易错PCR。所述易错PCR选用Stratagene公司GeneMorph II Random Mutagenesis Kit随机突变试剂盒。PCR程序为：95℃2min；95℃10s，55℃15s，72℃2min，共28个循环；72℃10min降至10℃，模板使用量为50ng。PCR产物经琼脂糖凝胶电泳后回收获得细胞色素P450易错PCR产物。
(2)以酿酒酵母基因组为模板，利用分子克隆常规方法中的醋酸锂转化方法将步骤(1)中的易错PCR产物导入实施例1中制备的酿酒酵母底盘细胞，获得转化产物(即转化子)。
(3)将转化产物均匀涂布于YPD+200mg/L G418抗生素筛选平板，30℃静置培养2-3天。将所有克隆用牙签挑取转接到96孔板中，30℃震荡培养1天，转接到一个新的96孔板中进行发酵4天。向发酵液中加入等体积的正丁醇溶剂萃取 1h，吸取上层有机相进行HPLC检测各个转化子达玛烯二醇和原人参二醇产量及其比例。
(4)经过对24块96孔板的筛选，获得原人参二醇产量/达玛烯二醇的比例(PPD/DM)提高20％以上的克隆共7个，编号分别为1G4、2D8、3B9、8G7、9C1、16F8和24A10。分别以各克隆的基因组为模板，利用引物EP-1和EP-2进行PCR获得各克隆的细胞色素P450片段，进行测序检测，从而获得各突变体的核苷酸序列和蛋白序列。
上述获得的各野生型和突变体蛋白序列信息及PPD/DM如图2和表2所示：
表2
  SEQ ID NO. 备注 PPD/DM提高
optPPDS 1 野生型 0
1G4 2 L87I 53.2％
2D8 3 K235R 57.7％
3B9 4 K349R,V366I 80.2％
8G7 5 N231Y,S285C 26.1％
9C1 6 P91H 84.20％
16F8 7 Q113R 30.6％
24A10 8 第1-4位氨基酸缺失，L18I 125.6％
(5)在对细胞色素P450突变体文库筛选的过程中还进一步筛选获得了一系列的突变启动子序列，这些突变的启动子序列有利于提高细胞色素P450的表达量从而提高原人参二醇合成效率和产量。获得原人参二醇产量/达玛烯二醇的比例(PPD/DM)提高20％以上的突变启动子有4个，编号分别为1D1、9C1-2、11B5和15F1。分别以1D1、9C1-2、11B5和15F1克隆的基因组为模板，利用引物进行PCR获得各克隆的启动子片段，进行测序检测获得突变启动子核苷酸序列。
上述获得的各野生型和突变启动子核苷酸序列信息及PPD/DM如表3所示：
表3不同启动子+野生型P450
  SEQ ID NO. 备注 PPD/DM提高
GAL1 9 野生型启动子 0
1D1 10 SEQ ID NO.:9中发生突变：C417G,A445G 44.1％
9C1-2 11 SEQ ID NO.:9中发生突变：第28位T缺失 80.1％
11B5 12 SEQ ID NO.:9中发生突变：G654A 62.2％
15F1 13 SEQ ID NO.:9中发生突变：G655A 44.1％
所述结果表明，这些突变的启动子可以进一步提高野生型P450蛋白的表达 量，进而提高原人参二醇产量/达玛烯二醇的比例。此外，这些突变的启动子还可以进一步与本发明的P450突变蛋白的编码序列联用，从而进一步提高原人参二醇产量/达玛烯二醇的比例。
实施例3 利用所述细胞色素P450突变体蛋白进行原人参二醇高效异源合成
(1)以酿酒酵母基因组为模板，用实施例1的方法，用SEQ ID NO.:14转化酿酒酵母菌株WP8，获得生产原人参二醇的重组酿酒酵母菌株WP8-WT。
(2)类似的分别以突变体基因1G4、2D8、3B9、8G7、9C1、16F8和24A10(核苷酸序列为SEQ ID NO.:15-21，相应的氨基酸序列为SEQ ID NO.:2-8)代替野生型optPPDS基因为模板。进行上述PCR获得各个PCR片段，分别转化酿酒酵母菌株WP8，获得含各突变体蛋白的生产原人参二醇的重组酿酒酵母菌株WP8-1G4、WP8-2D8、WP8-3B9、WP8-8G7、WP8-9C1、WP8-16F8和WP8-24A10。
(3)配置固体培养基：配置培养基：1％Yeast Extract(酵母膏)，2％Peptone(蛋白胨)，2％Dextrose(glucose)(葡萄糖)，2％琼脂粉。
配置液体培养基：配置培养基：1％Yeast Extract(酵母膏)，2％Peptone(蛋白胨)，2％Dextrose(glucose)(葡萄糖)。
挑取在固体培养基平板上划线的重组酿酒酵母菌WP8-1G4、WP8-2D8、WP8-3B9、WP8-8G7、WP8-9C1、WP8-16F8和WP8-24A10，分别于含有5mL液体培养基的试管震荡培养过夜(30℃，250rpm，16h)；离心收集菌体，转移至10mL液体培养基的50mL三角瓶中，调OD600至0.05，30℃，250rpm震荡培养4天得到发酵产物。本方法对每一株重组酵母同时设置一个平行实验。
达玛烯二醇及原人参二醇提取及检测：从10mL发酵液中吸取100μL发酵液，用Fastprep震荡裂解酵母，加入等体积的正丁醇抽提，而后在真空条件下使正丁醇蒸干。用100μL甲醇溶解后通过HPLC检测目的产物PPD和DM的产量(图3、图4和表4)。
上述获得的各重组酿酒酵母菌株原人参二醇和达玛烯二醇产量如表4所示：
表4
  PPD(mg/L) DM(mg/L) PPD/DM
WP8-WT 290 1058 27.42％
WP8-1G4 312 715 43.62％
WP8-2D8 316 699 45.23％
WP8-3B9 330 635 51.95％
WP8-8G7 335 925 36.22％
WP8-9C1 435 848 51.29％
WP8-16F8 386 1041 37.09％
WP8-24A10 490 777 63.08％
结果表明，与野生型的细胞色素P450相比，本发明的细胞色素P450突变蛋白可显著提高PPD的产量(最高可达490mg/L)和PPD/DM的比例(最高可达63％)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种细胞色素P450的突变蛋白，其特征在于，所述的突变蛋白为非天然蛋白，且所述突变蛋白具有催化生成原人参二醇的催化活性，并且所述突变蛋白在野生型的细胞色素P450的对应于SEQ ID NO.:1的选自下组的一个或多个与酶催化活性相关的核心氨基酸发生突变：
    第91位脯氨酸(P)；
    第87位亮氨酸(L)；
    第235位赖氨酸(K)；
    第349位赖氨酸(K)；
    第366位缬氨酸(V)；
    第231位天冬酰胺(N)；
    第285位丝氨酸(S)；
    第113位谷氨酰胺(Q)；
    第18位亮氨酸(L)；和/或
    缺失第1-4位中的1、2、3、或4个氨基酸。
  2. 一种多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的突变蛋白。
  3. 一种载体，其特征在于，所述的载体含有权利要求2所述的多核苷酸。
  4. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求3所述的载体，或其基因组中整合有权利要求2所述的多核苷酸。
  5. 一种产生权利要求1所述细胞色素P450的突变蛋白的方法，其特征在于，包括步骤：
    在适合表达的条件下，培养权利要求4所述的宿主细胞，从而表达出细胞色素P450的突变蛋白；和
    分离所述细胞色素P450的突变蛋白。
  6. 一种酶制剂，其特征在于，所述酶制剂包含权利要求1所述的细胞色素P450的突变蛋白。
  7. 一种制备原人参二醇的方法，其特征在于，包括步骤：
    (i)将权利要求1所述的细胞色素P450的突变蛋白与反应底物接触，进行 催化反应，从而获得所述原人参二醇；和
    (ii)任选地，分离并纯化所述原人参二醇。
  8. 一种权利要求1所述的突变蛋白的用途，其特征在于，所述的突变蛋白用于催化达玛烯二醇(DM)生成原人参二醇(PPD)，或被用于制备催化达玛烯二醇(DM)生成原人参二醇(PPD)的催化制剂。
  9. 一种权利要求1所述的突变蛋白或权利要求4所述的宿主细胞的用途，其特征在于，用于制备原人参二醇(PPD)。
  10. 一种产生转基因植物的方法，其特征在于，包括步骤：将权利要求4所述的宿主细胞再生为植物，其中，所述的宿主细胞为植物细胞。
PCT/CN2018/073453 2017-01-20 2018-01-19 细胞色素p450突变蛋白及其应用 WO2018133844A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1020197024319A KR102310518B1 (ko) 2017-01-20 2018-01-19 시토크롬 p450 돌연변이 단백질 및 이의 응용
JP2019560442A JP7090266B2 (ja) 2017-01-20 2018-01-19 シトクロムｐ４５０の突然変異タンパク質およびその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710057245.1A CN108330111B (zh) 2017-01-20 2017-01-20 细胞色素p450突变蛋白及其应用
CN201710057245.1 2017-01-20

Publications (1)

Publication Number Publication Date
WO2018133844A1 true WO2018133844A1 (zh) 2018-07-26

Family

ID=62908923

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/073453 WO2018133844A1 (zh) 2017-01-20 2018-01-19 细胞色素p450突变蛋白及其应用

Country Status (4)

Country Link
JP (1) JP7090266B2 (zh)
KR (1) KR102310518B1 (zh)
CN (1) CN108330111B (zh)
WO (1) WO2018133844A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109295027B (zh) * 2018-11-06 2020-12-29 浙江华睿生物技术有限公司 一种糖基转移酶突变体
CN109468287B (zh) * 2018-11-22 2020-10-16 浙江华睿生物技术有限公司 一种羟化酶突变体
CN114507646B (zh) * 2020-11-17 2023-11-10 生合万物(上海)生物科技有限公司 细胞色素p450突变体蛋白及其应用
CN114807065B (zh) * 2021-06-07 2023-06-09 山西农业大学 一种酶的突变体

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103045619A (zh) * 2011-12-19 2013-04-17 蔡剑平 包括293g＞t突变的cyp2c9基因片段、所编码的蛋白质片段及其应用
CN104894077A (zh) * 2014-03-06 2015-09-09 中国科学院上海生命科学研究院 烟酰胺腺嘌呤二核苷酸-细胞色素p450还原酶及其应用
WO2015167282A1 (en) * 2014-04-30 2015-11-05 Korea Advanced Institute Of Science And Technology A novel method for glycosylation of ginsenoside using a glycosyltransferase derived from panax ginseng

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101284707B1 (ko) * 2011-11-03 2013-07-23 강원대학교산학협력단 프로토파낙사디올 생합성 촉진용 조성물
KR101392987B1 (ko) * 2012-03-16 2014-05-09 건국대학교 산학협력단 고온성 베타-글루코시다아제를 이용한 희귀 진세노사이드 컴파운드 엠씨, 컴파운드 와이 및 아글리콘 프로토파낙사디올 생산
WO2016110512A1 (en) * 2015-01-06 2016-07-14 Dsm Ip Assets B.V. A crispr-cas system for a yeast host cell
KR101984957B1 (ko) * 2017-08-09 2019-06-04 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 효모의 단백질 접힘 기작(protein-folding machinery) 개량을 통한 진세노사이드 생산 증대

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103045619A (zh) * 2011-12-19 2013-04-17 蔡剑平 包括293g＞t突变的cyp2c9基因片段、所编码的蛋白质片段及其应用
CN104894077A (zh) * 2014-03-06 2015-09-09 中国科学院上海生命科学研究院 烟酰胺腺嘌呤二核苷酸-细胞色素p450还原酶及其应用
WO2015167282A1 (en) * 2014-04-30 2015-11-05 Korea Advanced Institute Of Science And Technology A novel method for glycosylation of ginsenoside using a glycosyltransferase derived from panax ginseng

Also Published As

Publication number Publication date
KR102310518B1 (ko) 2021-10-08
JP7090266B2 (ja) 2022-06-24
CN108330111A (zh) 2018-07-27
CN108330111B (zh) 2023-01-31
KR20190129840A (ko) 2019-11-20
JP2020513843A (ja) 2020-05-21

Similar Documents

Publication Publication Date Title
WO2018133844A1 (zh) 细胞色素p450突变蛋白及其应用
US9976167B2 (en) Group of glycosyltransferases and use thereof
KR102338008B1 (ko) 당사슬 연장에 촉매 작용하기 위한 한 그룹의 udp-글리코실트랜스퍼라제 및 이의 응용
WO2016119756A1 (zh) 糖基转移酶突变蛋白及其应用
CN110551701B (zh) 羰基还原酶突变体及其在环戊二酮类化合物还原中的应用
WO2018210208A1 (zh) 糖基转移酶、突变体及其应用
WO2015188742A2 (zh) 一组糖基转移酶及其应用
CN105087739A (zh) 一种新的制备稀有人参皂苷的催化体系及其应用
WO2022105729A1 (zh) 细胞色素p450突变体蛋白及其应用
CN112592912B (zh) 糖苷酶及其编码基因和它们的应用
JP2016154502A (ja) 酸化セスキテルペンの生産およびその利用
KR20240032944A (ko) 람노스가 고도로 특이적인 글리코실트랜스퍼라제 및 이의 응용
CN106318920B (zh) 黄酮-6-羟化酶及其在灯盏乙素合成中的应用
CN112831481B (zh) 糖基转移酶以及催化糖链延伸的方法
WO2021032159A1 (zh) 艾纳香单萜合酶BbTPS3及其相关生物材料与应用
CN108866142B (zh) 细胞色素p450、烟酰胺腺嘌呤二核苷酸-细胞色素p450还原酶及其应用
CN113444703B (zh) 催化糖链延伸的糖基转移酶突变体及其应用
CN111926000B (zh) 绞股蓝糖基转移酶及其应用
CN114807075B (zh) 糖基转移酶PpUGT73E5及其在重楼皂苷合成中的应用
CN108148871B (zh) 一种基因枪介导的雷公藤遗传转化方法
CN117866932A (zh) 氧化鲨烯环化酶NiOSC6和其编码基因在生物合成中的应用
CN118165972A (zh) 氧化鲨烯环化酶NiOSC4和其编码产物及其应用
CN117866933A (zh) 氧化鲨烯环化酶NiOSC5和其编码基因与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18742047

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019560442

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20197024319

Country of ref document: KR

Kind code of ref document: A

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM1205A DATED 28.10.2019)

122 Ep: pct application non-entry in european phase

Ref document number: 18742047

Country of ref document: EP

Kind code of ref document: A1