WO2015139599A1 - 新的贝壳烯酸-13α-羟化酶、其编码基因及其应用 - Google Patents

新的贝壳烯酸-13α-羟化酶、其编码基因及其应用 Download PDF

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WO2015139599A1
WO2015139599A1 PCT/CN2015/074344 CN2015074344W WO2015139599A1 WO 2015139599 A1 WO2015139599 A1 WO 2015139599A1 CN 2015074344 W CN2015074344 W CN 2015074344W WO 2015139599 A1 WO2015139599 A1 WO 2015139599A1
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hydroxylase
polypeptide
acid
stevioside
isenoic
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PCT/CN2015/074344
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English (en)
French (fr)
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王勇
熊智强
汪建峰
李诗渊
刘巧霞
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中国科学院上海生命科学研究院
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    • 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)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • A23L27/33—Artificial sweetening agents containing sugars or derivatives
    • A23L27/36—Terpene glycosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13079—Ent-kaurenoic acid oxidase (1.14.13.79)

Definitions

  • the invention relates to the field of molecular biology and bioengineering, and provides a novel stearyl source-derived aureic acid-13 ⁇ -hydroxylase (KAH) gene and application thereof in stevioside biosynthesis.
  • KAH stearyl source-derived aureic acid-13 ⁇ -hydroxylase
  • Stevioside is a diterpene glycoside compound from the genus Stevia Rebaudiana Bertoni. It has high sweetness and low heat energy. Its sweetness is 200-300 times that of sucrose. The calorific value is only 1/300 of sucrose. Stevia has been widely used as a herb and sugar substitute in South America and East Asia for hundreds of years. Regular consumption of stevioside can prevent high blood pressure, diabetes, obesity, heart disease, dental caries and other diseases. With the gradual recognition of the safety of stevia in countries such as Europe and the United States, stevia will have an increasingly large market as a new sweetener and functional food additive.
  • stevioside is mainly composed of a common precursor of quinones - 3 isoprene pyrophosphate (IPP) and 1 dimethyl propylene diphosphate (DMAPP) unit.
  • IPP isoprene pyrophosphate
  • DMAPP dimethyl propylene diphosphate
  • GGPPS Geranylgeranyl diphosphate synthase
  • CDPS ent-copalyl diphosphate synthase
  • KS kaorene synthase
  • the shellene is subjected to two steps of cytochrome P450 enzymes, namely, kaurene oxidase (KO) and 13 ⁇ -kaurenoic acid hydroxylase (KAH), respectively, in the C19 of the diterpene skeleton.
  • the C13 position forms a carboxyl group and a hydroxyl group to form an important intermediate steviol.
  • steviol is subjected to a four-step glycosylation reaction to form stevia rebaudioside A, as shown in FIG.
  • an isolated mandelonic acid-13 ⁇ -hydroxylase selected from the group consisting of
  • polypeptide having (a) a polypeptide function and having 90% or more (preferably 95% or more; more preferably 98% or more; more preferably 99% or more) amino acid sequence identity with the (a) polypeptide.
  • the isenoate-13 ⁇ -hydroxylase is derived from stevia.
  • an isolated polynucleotide encoding an isenoate-13a-hydroxylase as described.
  • nucleotide sequence providing the polynucleotide is set forth in SEQ ID NO: 1.
  • nucleotide sequence of the polynucleotide is set forth in SEQ ID NO: 5 (codon optimized sequence).
  • a recombinant vector comprising the polynucleotide of any of the foregoing is provided.
  • a host cell comprising the recombinant vector, or a polynucleotide in which the polynucleotide is integrated in the genome, is provided.
  • the host cell is a bacterial cell, a fungal cell or a plant cell;
  • the fungal cell comprises a yeast cell (Yeast), such as Pichia pastoris, Saccharomyces cerevisiae, lactic acid. Kluyveromyces (Saccharomyces cerecisiae) and the like.
  • the bacterial cells include: Enterobacter, such as Escherichia coli, Bacillus, such as Bacillus subtilis, Streptomyces, Streptomyces coelicolor (S.
  • Streptomyces coelicolor Streptomyces coelicolor
  • Streptomyces lividans Streptomyces avermitilis
  • Streptomyces venezuelae etc.
  • sugar multicellular Saccharopolyspora such as Saccharopolyspora erythraea.
  • a process for the preparation of the above-described econate-13 ⁇ -hydroxylase comprising: (a) cultivating said host cell; (b) isolating from the culture The isenoate-13 ⁇ -hydroxylase.
  • the use of the isenoate-13 ⁇ -hydroxylase for converting an isenoic acid to a stevioside preferably, in the presence of a cytochrome P450 redox protein
  • the lower mandelic acid is converted to stevioside.
  • the converting the isenoic acid to the stevioside is carried out in a plant cell or a non-plant cell; preferably, the non-plant cell comprises a bacterial cell, a fungal cell; the fungal cell Including yeast cells (Yeast), such as Pichia pastoris, Saccharomyces cerevisiae, Saccharomyces cerecisiae, and the like.
  • yeast cells such as Pichia pastoris, Saccharomyces cerevisiae, Saccharomyces cerecisiae, and the like.
  • the prokaryotic cells include: Enterobacter, such as Escherichia coli, Bacillus, such as Bacillus subtilis, Streptomyces, Streptomyces coelicolor ( Streptomyces coelicolor), Streptomyces lividans, Streptomyces avermitilis, Streptomyces venezuelae, etc., Saccharopolyspora, such as Saccharopolyspora Erythraea).
  • Enterobacter such as Escherichia coli
  • Bacillus such as Bacillus subtilis
  • Streptomyces Streptomyces coelicolor ( Streptomyces coelicolor)
  • Streptomyces lividans Streptomyces avermitilis
  • Streptomyces venezuelae etc.
  • Saccharopolyspora such as Saccharopolyspora Erythraea
  • a method of preparing stevioside comprising: converting the isenoic acid to stevioside using the isenoate-13 ⁇ -hydroxylase.
  • the method comprises:
  • the cytochrome P450 redox protein is expressed by the host cell of step (1).
  • the recombinant vector contained in the host cell described in the step (1) further comprises: a gene encoding a cytochrome P450 redox protein.
  • composition comprising a safe and effective amount of said olefinene Acid-13 ⁇ -hydroxylase and a food or industrially acceptable carrier.
  • FIG. 1 PCR amplification of the KAH gene fragment.
  • Lanes 1-4 are primer pairs 8-40-3US/8-40-4DS, 8-40-2DS/8-40-3US, 8-40-1US/8-40-4DS, 8-40-1US, respectively.
  • /8-40-2D Electropherogram of the amplified product obtained by PCR amplification.
  • FIG. 7 SDS-PAGE analysis of KAH S2 protein expression.
  • M protein molecular weight marker; 1: no induction of whole bacterial protein; 2: no induction of supernatant protein; 3: 16 ° C induced 5 h whole bacterial protein; 4: 16 ° C induced 5 h supernatant protein; 5: 16 ° C induced 16 h whole bacteria Protein; 6:16°C induced 16h supernatant protein; 7:28°C induced 5h whole protein; 8:28°C induced 5h supernatant protein; 9:28°C induced 16h whole protein; 10:28°C induced 16h supernatant protein.
  • FIG. 8 Biosynthesis of resting cells by biosynthesis of canrenic acid. a, biotransformation results of control strain DH10B (DE3); b, biotransformation results of strain DH10B (DE3) pSY183; c, detection results of Steviol Standard.
  • Figure 9 Biosynthesis of resting cell biosynthesis of stearic acid. a, molecular ion peak of the target peak of strain DH10B(DE3)pSY183; b, molecular ion peak of the target peak of Steviol Standard.
  • Figure 10 HPLC detection chromatogram of Pichia pastoris expression system KAHS2 activity assay.
  • KAH mandelic acid-13 ⁇ -hydroxylase
  • heterologous refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between proteins (or nucleic acids) from different sources and host cells. For example, if the combination of a nucleic acid and a host cell is generally not naturally occurring, the nucleic acid is heterologous to the host cell. A particular sequence is “heterologous” to the cell or organism into which it is inserted.
  • polypeptide of the invention As used herein, the terms “polypeptide of the invention”, “protein of the invention” or “KAH polypeptide (protein)” are used interchangeably and mean having the amino acid sequence of the mandelic acid-13 ⁇ -hydroxylase (SEQ ID NO: a protein or polypeptide of 2 or a variant or derivative thereof.
  • gene polynucleotide
  • KAH gene of the invention refers to a polynucleotide encoding the described isenoate-13 ⁇ -hydroxylase.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • the polypeptide of the invention may be a naturally purified product, a recombinantly expressed product or a chemically synthesized product.
  • fragments, derivatives and analogs of KAH are also included in the present invention.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the native KAH of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic IgG fragment).
  • a polypeptide having one or more conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • substituted amino acid residues It
  • KAH polypeptide includes a polypeptide of the sequence of SEQ ID NO: 2 having KAH activity, and also a variant form of the polypeptide encoded by the sequence of SEQ ID NO: 2 having the same function as the KAH polypeptide.
  • variants include, but are not limited to, one or more (usually 1-30, preferably 1-20, more preferably 1-10, optimally 1-5) amino acid deletions , insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • amino acids with similar or similar properties are used.
  • the function of the polypeptide is usually not altered.
  • the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the polypeptide; for example, only a portion of the domain expressing the polypeptide will also provide the same catalytic function as the intact polypeptide.
  • the term therefore also encompasses active fragments and active derivatives of KAH polypeptides.
  • the invention also provides analogs of KAH proteins or polypeptides.
  • the difference between these analogs and the natural KAH polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or both.
  • These polypeptides include natural or induced genetic variants.
  • KAH protein conservative variant polypeptide means up to 20, preferably up to 10, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of SEQ ID NO: 2. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1 or may be a degenerate variant.
  • a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence set forth in SEQ ID NO: 1.
  • Polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and Non-coding sequence.
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the full length sequence of the KAH nucleotide of the present invention or a fragment thereof can be usually 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.
  • 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.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vector or KAH protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • the polynucleotide sequences of the present invention can be utilized to express or produce recombinant KAH polypeptides by conventional recombinant DNA techniques. Generally, the following steps are carried out: (1) using a polynucleotide (or variant) encoding a KAH polypeptide of the present invention, or transforming or transducing a suitable sink with a recombinant expression vector containing the polynucleotide. Primary cells; (2) host cells cultured in a suitable medium; (3) isolated and purified proteins from the culture medium or cells.
  • a KAH polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, 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.
  • expression vectors containing the KAH encoding DNA sequence and appropriate transcriptional/translational control signals can be used to construct expression vectors containing the KAH encoding DNA sequence and appropriate transcriptional/translational 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.
  • 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.
  • 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.
  • prokaryotic cells such as Escherichia coli; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or animal cells of Bowes melanoma cells, and the like.
  • the host cell is a prokaryotic cell or a yeast cell.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • This patent reports a novel stevia-derived KAH gene, which is expressed in microbial cells, and undergoes biotransformation of the enoyl acid to steviol, which is the total synthesis pathway of stevioside in microbial cells.
  • the construction laid the foundation.
  • Recombinant KAH can be applied to the biotransformation of stevioside, including: conversion of the uric acid to stevioside.
  • a heterologous host such as a prokaryotic host or a yeast host
  • the conversion of the folic acid to the steviol is a rate-limiting step.
  • the KAH cloned by the present inventors is capable of achieving heterologous expression in microorganisms, it is apparent that this is of significant advancement.
  • a method of forming a biotransformation comprising: transforming the KAH into a host cell; and converting the isenoate to stevioside using KAH in the presence of cytochrome P450 redox protein (CPR).
  • CPR cytochrome P450 redox protein
  • CPR can be CPR of various species (such as stevia, Artemisia annua, Helminthosporium, Giardia lamblia, Arabidopsis, etc.), as long as it can function as a chaperone protein of KAH (helping to respond Electronic transfer in the process).
  • CPR can be CPR of various species (such as stevia, Artemisia annua, Helminthosporium, Giardia lamblia, Arabidopsis, etc.), as long as it can function as a chaperone protein of KAH (helping to respond Electronic transfer in the process).
  • the cytochrome P450 redox protein is expressed by the host cell described in the step (1).
  • a recombinant vector an expression cassette encoding the gene encoding the eicoate acid-13 ⁇ -hydroxylase and an expression cassette encoding the gene encoding the cytochrome P450 redox protein, transforming the host, and expressing the product for shell production Biotransformation of enoic acid to stevioside.
  • a host such as a yeast cell
  • the expression cassette encoding the gene encoding the isenoic acid-13 ⁇ -hydroxylase is transformed into the host by means of cells endogenously present.
  • Pigment P450 redox protein biotransformation of eicoate to stevioside.
  • the establishment of expression cassettes is currently a technique familiar to those skilled in the art. Therefore, it is easy for a person skilled in the art to carry out the establishment of an expression cassette after learning the enzyme to be selected.
  • the gene sequence encoding the enzyme can be inserted into a different expression cassette (such as an expression vector) or can be inserted into the same expression cassette as long as the enzyme can be efficiently expressed after being transferred into the cell.
  • the promoter or terminator required for the preparation of the expression cassette may be any suitable promoter or terminator and is not limited to those specifically recited in the present invention. Selection of a suitable promoter or terminator can be performed by those skilled in the art and can depend on the type of host cell. For example, when applied to yeast recombinant expression, the prior art has revealed some yeast promoters or terminators to facilitate selection.
  • suitable expression vectors are known in the art, so that it is easy to select a suitable expression vector as a backbone vector for cloning the gene, for example, when the cell is a bacterial cell, pET is used.
  • a series of expression vectors or a pSY series vector are used to recombinantly express each enzyme; when the cells are yeast cells, a pSY series expression vector is used.
  • the invention also provides a composition comprising an effective amount of a KAH polypeptide of the invention and a food or industrially acceptable carrier or excipient.
  • a carrier include, but are not limited to, water, buffers, glycerol, ethanol, and combinations thereof.
  • the present invention obtains a novel mandelic acid-13 ⁇ -hydroxylase (KAH) gene, which lays a foundation for further analysis of the stevioside biosynthesis pathway.
  • KAH mandelic acid-13 ⁇ -hydroxylase
  • the present invention realizes the active expression of the mandelic acid-13 ⁇ -hydroxylase (KAH) gene in a prokaryotic host, and successfully realizes the biotransformation of the isenoic acid to steviol, and is a plant cell metabolic engineering and stevia
  • KAH mandelic acid-13 ⁇ -hydroxylase
  • the above four PCR products were sequenced, and the sequence was spliced to obtain a full-length KAH sequence.
  • the primer KAH F/KAH R was then designed based on the full length KAH sequence.
  • KAH-XbaI F/KAH-BamHI R was amplified by PCR using Primestar DNA polymerase. The electrophoresis results of the amplified product are shown in Fig. 3.
  • the KAH PCR product was recovered by gel, and then the recovered KAH PCR product and the pET21a (+) plasmid (Novagen) were separately digested with XbaI and BamHI, and the two were ligated to construct plasmid pSY183, as shown in Fig. 4.
  • the inventors also optimized the KAH codon when used for expression of E. coli, and the optimized KAH gene sequence is SEQ ID NO: 5.
  • the black “ACTAGT” sequence represents the “SpeI” site.
  • the sequence “aataattttgtttaactttaagaaggagatatacat” represents the sequence between the XbaI and NdeI sites in the pET21a plasmid.
  • the cytochrome P450 redox protein (CPR, ABB88839) of stevia was selected and its gene sequence was codon optimized to obtain the gene sequence SEQ ID NO: 3 and named srcpr; its amino acid sequence encoding SrCPR SEQ ID NO: 4.
  • the optimized srcpr gene was cloned into the NdeI and BamHI restriction sites of pET21a (+) to obtain plasmid pET21a-srcpr (Fig. 5).
  • the plasmid pSY183 was digested with XbaI and HindIII
  • the plasmid pET21a-srcpr was digested with SpeI and HindIII to recover the KAH-containing gene.
  • the DNA fragment and the pET21a-srcpr vector were ligated to construct plasmid pSY198 (Fig. 6).
  • the plasmid pSY183 was transformed into BL21 (DE3), and after plating the plate, it was cultured overnight at 37 °C. Single colonies were picked and 2 ml of liquid LB medium (ampicillin 100 mg/L), 220 r/min, and cultured overnight at 37 ° C as a seed liquid. The seed solution was inoculated into a new 2 ml LB medium at a rate of 1%, 220 r/min, and cultured at 37 ° C until the OD600 was about 0.3-0.5. IPTG was added at a final concentration of 0.1 mM, and 220 r/min was placed at 16 ° C. After induction culture at 28 ° C for 5 h or 16 h, samples were collected and subjected to SDS-PAGE analysis.
  • Plasmid pSY198 After transformation of strain DH10B (DE3), a coating containing 100mg l -1 ampicillin resistance plates, 37 °C overnight. Pick a single colony in 2ml 100mg l -1 ampicillin-containing liquid LB medium, 37 °C overnight. Then, the cells were transferred to a new LB medium at a 1% inoculum, cultured at 37 ° C for 2 h to an OD 600 of about 0.6, IPTG was added to a final concentration of 0.1 mM, and culture was induced at 16 ° C for 16 h.
  • the cells were collected by centrifugation at 6000 g at 4 ° C, and washed twice with an equal volume of M9 salt/phosphate buffer system (4.2 mM Na 2 HPO 4 ; 2.2 mM KH 2 PO 4 ; 0.9 mM NaCl; 1.9 mM NH 4 Cl). Then, it was resuspended (M9 salt/phosphate buffer system + 50 mg l -1 yeast extract) by adding a certain volume of biotransformation buffer system, and the wet weight of the control cells was about 4 g l -1 after resuspension.
  • M9 salt/phosphate buffer system 4.2 mM Na 2 HPO 4 ; 2.2 mM KH 2 PO 4 ; 0.9 mM NaCl; 1.9 mM NH 4 Cl. Then, it was resuspended (M9 salt/phosphate buffer system + 50 mg l -1 yeast extract) by adding a certain volume of biotransformation buffer system, and the wet weight of the control
  • the KAHS2 gene was amplified by PCR using pSY220F and pSY220R as primers and pSY183 as a template. After the PCR product is recovered by gelation, it is digested with BglII/NotI, and then the digested product is recovered again, and it is linked to The pPIC3.5k vector (purchased from Invitorgen) treated with BamHI/NotI was used to obtain plasmid pSY220 (pPIC3.5k-KAHS2).
  • pSY220F (SEQ ID NO: 14): GGA AGATCT ATGAAATTCAAAAAGT TTTCTTGTACCCAC (underlined BamHI restriction site);
  • pSY220R (SEQ ID NO: 15): ATAAGAAT GCGGCCGC TTAGAGTTTG TGTAAAATCAAGTGAGCACC (underlined as NotI restriction site).
  • cytochrome P450 redox protein contained in Pichia can provide electron transport for exogenous P450 oxidase. Therefore, in this example, KAHS2 can be activated in Pichia pastoris without additional expression of heterologous CPR gene. expression.
  • the plasmid pSY220 was digested with SalI, and the linearized DNA fragment was recovered, which was then electroporated into Pichia pastoris strain KM71 (purchased from Invitrogen), and coated with MD plate (13.4 g/L yeast basic nitrogen source (YNB); 0.4 mg /L biotin; 20 g / L glucose), after 3 days of culture at 28 ° C, the recombinant strain was obtained by PCR.
  • MD plate (13.4 g/L yeast basic nitrogen source (YNB); 0.4 mg /L biotin; 20 g / L glucose
  • the recombinant strain was inoculated into 2 ml of YPD medium, cultured at 28 ° C, 250 r / min for 24 h, and then seeded with 1% inoculum, inoculated with 50 ml of BMGY (2% peptone, 1% yeast extract, 100 mM). Potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10 -5 % biotin, 0.5% glycerol) in a 500 ml shake flask of a medium, a total of two bottles, continued to culture for 24 h.
  • the bacterial solution was collected in a 50 ml centrifuge tube, a total of 2 tubes, 2000 r/min, centrifuged for 5 min, and the medium supernatant was discarded.
  • the cells were suspended and suspended in 5 ml of BMMY (2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10 -5 % biotin, 0.5% methanol). After the body suspensions were combined, a total of 10 ml was transferred to a 100 ml shake flask at 250 r/min, 22 ° C, and 50 ⁇ l of methanol was added every 24 hours to induce culture for 72 hours.
  • BMMY 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10 -5 % biotin, 0.5% methanol.
  • the cells were collected by centrifugation at 2000 g at 4 ° C, and washed twice with an equal volume of M9 salt/phosphate buffer system (4.2 mM Na 2 HPO 4 ; 2.2 mM KH 2 PO 4 ; 0.9 mM NaCl; 1.9 mM NH 4 Cl). Then, it was resuspended by adding a certain volume of biotransformation buffer system, and the wet weight of the control cells was about 4 g/L after resuspension.
  • M9 salt/phosphate buffer system 4.2 mM Na 2 HPO 4 ; 2.2 mM KH 2 PO 4 ; 0.9 mM NaCl; 1.9 mM NH 4 Cl.

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Abstract

提供了一种新的贝壳烯酸-13α-羟化酶、其编码基因及其应用。新的贝壳烯酸-13α-羟化酶自甜叶菊中首次克隆得到，并成功应用于甜菊糖的异源生物合成。

Description

一种新的贝壳烯酸-13α-羟化酶、其编码基因及其应用 技术领域
本发明涉及分子生物学与生物工程领域，提供了一种新的甜叶菊来源的贝壳烯酸-13α-羟化酶(KAH)基因及在甜菊糖苷生物合成中的应用。
背景技术
随着肥胖和高血压等心血管疾病对人类健康的威胁加剧，人类对新型甜味剂的需求越来越迫切。甜菊糖苷是一种产自菊科植物甜叶菊(Stevia Rebaudiana Bertoni)中的二萜类糖苷化合物，具有高甜度、低热能的特点，其甜度是蔗糖的200-300倍，热值仅为蔗糖的1/300。甜菊糖在南美和东亚地区被广泛地作为药草和代糖已经有几百年历史，经常食用甜菊糖可预防高血压、糖尿病、肥胖症、心脏病、龋齿等病症。随着欧美等国对甜菊糖安全性的逐渐认可，甜叶菊作为新型甜味剂和功能性食品添加剂将具有越来越大的市场。
目前有关甜菊糖苷生物合成途径研究相对比较清楚。作为典型的二萜类化合物，甜菊糖苷主要由萜类的通用前体——3个异戊烯焦磷酸(Isopentyl diphosphate，IPP)和1个二甲基丙烯基焦磷酸(Dimethylallyl diphosphate，DMAPP)单元经牻牛儿基牻牛儿基焦磷酸合成酶(Geranylgeranyl diphosphate synthase，GGPPS)，ent-柯巴基焦磷酸合成酶(ent-copalyl diphosphate synthase，CDPS)，贝壳烯合成酶(Kaurene synthase，KS)形成二萜骨架贝壳烯。随后贝壳烯经两步细胞色素P450酶，即贝壳烯氧化酶(Kaurene oxidase，KO)和贝壳烯酸-13α-羟化酶(13α-kaurenoic acid hydroxylase，KAH)，分别在二萜骨架的C19和C13位形成羧基和羟基，形成重要中间体甜菊糖醇。最后甜菊醇经四步糖基化反应，最终形成甜菊糖莱鲍迪甙A(Rebaudioside A)，如图1所示。
目前甜菊糖合成途径中所有酶的基因均已经获得了报道，并为植物细胞遗传改造提高甜菊糖产量和微生物异源合成甜菊糖苷的研究打下了基础。作为催化贝壳烯酸到甜菊醇的第二个细胞色素P450酶KAH，虽已经有多个甜叶菊来源的序列被报道，而且部分序列已经在植物中过表达以改善甜菊糖苷合成或酵母细胞中进行生物转化研究，但其目前仍是甜菊糖生物合成的限速步骤，而且仍然未有能够在原核宿主中活性表达KAH的报道。
发明内容
本发明的目的在于提供一种新的贝壳烯酸-13α-羟化酶、其编码基因及其应用。
在本发明的第一方面，提供一种分离的贝壳烯酸-13α-羟化酶，其选自下组：
(a)如SEQ ID NO:2氨基酸序列的多肽；
(b)将SEQ ID NO:2氨基酸序列经过一个或多个(如1-20个，较佳地1-10个；更佳地1-5个；更佳地1-3个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽功能的由(a)衍生的多肽；
(c)具有(a)多肽功能的SEQ ID NO:2的蛋白片段；或
(d)具有(a)多肽功能的且与(a)多肽具有90％以上(较佳地95％以上；更佳地98％以上；更佳地99％以上)氨基酸序列相同性的多肽。
在一个优选例中，所述的贝壳烯酸-13α-羟化酶来源于甜叶菊。
在本发明的第一方面，提供一种分离的多核苷酸，其编码如所述的贝壳烯酸-13α-羟化酶。
在一个优选例中，提供该多核苷酸的核苷酸序列如SEQ ID NO:1所示。
在另一优选例中，所述多核苷酸的核苷酸序列如SEQ ID NO:5所示(密码子优化序列)。
在本发明的第一方面，提供一种重组载体，它含有前面任一所述的多核苷酸。
在本发明的第一方面，提供一种宿主细胞，它含有所述的重组载体，或其基因组中整合有所述的多核苷酸。
在另一优选例中，所述的宿主细胞是细菌细胞、真菌细胞或植物细胞；所述的真菌细胞包括酵母细胞(Yeast)，如毕赤酵母(Pichiapastoris)、酿酒酵母(Saccharomyces cerevisiae)、乳酸克鲁维酵母(Saccharomyces cerecisiae)等。所述的细菌细胞包括：肠杆菌属(Enterobacter)，如大肠杆菌(Escherichia coli)，芽孢杆菌属(Bacillus)，如枯草芽孢杆菌(Bacillus subtilis)，链霉菌属(Streptomyces)，天蓝色链霉菌(Streptomyces coelicolor)、变铅青链霉菌(Streptomyces lividans)、阿维链霉菌(Streptomyces avermitilis)、委内瑞拉链霉菌(Streptomyces venezuelae)等，糖多胞 菌属(Saccharopolyspora)，如红霉糖多胞菌(Saccharopolyspora erythraea)。
在本发明的第一方面，提供一种所述的贝壳烯酸-13α-羟化酶的制备方法，该方法包含：(a)培养所述的宿主细胞；(b)从培养物中分离出所述的贝壳烯酸-13α-羟化酶。
在本发明的第一方面，提供所述的贝壳烯酸-13α-羟化酶的用途，用于将贝壳烯酸转化为甜菊糖醇；较佳地，在存在细胞色素P450氧化还原蛋白的情况下贝壳烯酸转化为甜菊糖醇。
在一个优选例中，所述的将贝壳烯酸转化为甜菊糖醇在植物细胞或非植物细胞中进行；较佳地，所述的非植物细胞包括细菌细胞、真菌细胞；所述的真菌细胞包括酵母细胞(Yeast)，如毕赤酵母(Pichiapastoris)、酿酒酵母(Saccharomyces cerevisiae)、乳酸克鲁维酵母(Saccharomyces cerecisiae)等。所述的原核细胞包括：肠杆菌属(Enterobacter)，如大肠杆菌(Escherichia coli)，芽孢杆菌属(Bacillus)，如枯草芽孢杆菌(Bacillus subtilis)，链霉菌属(Streptomyces)，天蓝色链霉菌(Streptomyces coelicolor)、变铅青链霉菌(Streptomyces lividans)、阿维链霉菌(Streptomyces avermitilis)、委内瑞拉链霉菌(Streptomyces venezuelae)等，糖多胞菌属(Saccharopolyspora)，如红霉糖多胞菌(Saccharopolyspora erythraea)。
在本发明的第一方面，提供一种制备甜菊糖醇的方法，该方法包含：利用所述的贝壳烯酸-13α-羟化酶将贝壳烯酸转化为甜菊糖醇。
在一个优选例中，所述方法包括：
(1)培养所述的宿主细胞，从而表达所述的贝壳烯酸-13α-羟化酶；
(2)在存在细胞色素P450氧化还原蛋白(CPR)的情况下，利用步骤(1)所述的贝壳烯酸-13α-羟化酶将贝壳烯酸转化为甜菊糖醇。
在另一优选例中，所述的细胞色素P450氧化还原蛋白由步骤(1)所述的宿主细胞表达。
在另一优选例中，步骤(1)所述的宿主细胞中包含的重组载体中还包括：细胞色素P450氧化还原蛋白的编码基因。
在本发明的另一方面，提供一种组合物，它含有安全有效量的所述的贝壳烯 酸-13α-羟化酶以及食品学或工业上可接受的载体。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、甜菊糖苷类化合物的生物合成途径。
图2、KAH基因片段的PCR扩增。泳道1-4分别是应用引物对8-40-3US/8-40-4DS，8-40-2DS/8-40-3US，8-40-1US/8-40-4DS，8-40-1US/8-40-2D进行PCR扩增获得的扩增产物的电泳图。
图3、KAH全长DNA的PCR扩增。
图4、质粒pSY183图谱。
图5、质粒pET21a-srcpr图谱。
图6、质粒pSY198图谱。
图7、KAH S2蛋白表达SDS-PAGE分析。M：蛋白分子量标记；1：未诱导全菌蛋白；2：未诱导上清蛋白；3：16℃诱导5h全菌蛋白；4：16℃诱导5h上清蛋白；5：16℃诱导16h全菌蛋白；6：16℃诱导16h上清蛋白；7：28℃诱导5h全菌蛋白；8：28℃诱导5h上清蛋白；9：28℃诱导16h全菌蛋白；10：28℃诱导16h上清蛋白。
图8、静息细胞生物转化贝壳烯酸合成甜菊醇质谱分析。a，对照菌株DH10B(DE3)的生物转化结果；b，菌株DH10B(DE3)pSY183的生物转化结果；c，甜菊糖醇标准品(Steviol Standard)的检测结果。
图9、静息细胞生物转化贝壳烯酸合成甜菊醇质谱峰分子离子峰。a，菌株DH10B(DE3)pSY183的目标峰的分子离子峰；b，甜菊糖醇标准品(Steviol Standard)的目标峰的分子离子峰。
图10、毕赤酵母表达系统KAHS2活性检测HPLC检测色谱图。
图11、毕赤酵母表达系统KAHS2活性MS检测结果。
具体实施方式
本发明人经过大量的研究和筛选，首次从甜叶菊中克隆到一种新的贝壳烯酸-13α-羟化酶(KAH)基因，并成功的将其应用于甜菊糖的异源生物合成。
如本文所用，所述的“异源”是指来自不同来源的两条或多条核酸或蛋白质序列之间的关系，或来自不同来源的蛋白(或核酸)与宿主细胞之间的关系。例如，如果核酸与宿主细胞的组合通常不是天然存在的，则核酸对于该宿主细胞来说是异源的。特定序列对于其所插入的细胞或生物体来说是“异源的”。
如本文所用，术语“本发明的多肽”、“本发明的蛋白”或“KAH多肽(蛋白)”可互换使用，都指具有贝壳烯酸-13α-羟化酶氨基酸序列(SEQ ID NO:2或其变异形式或衍生物)的蛋白或多肽。
如本文所用，术语“本发明的基因(多核苷酸)”或“KAH基因”指编码所述的贝壳烯酸-13α-羟化酶的多核苷酸。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
本发明的多肽可以是天然纯化的产物、重组表达的产物或是化学合成的产物。
所述的KAH的片段、衍生物和类似物也包含在本发明中。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的天然KAH相同的生物学功能或活性的多肽。本发明的多肽片段、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)被取代的多肽，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多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽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。
在本发明中，术语“KAH多肽”包括具有KAH活性的SEQ ID NO:2序列的多肽，也包括具有与KAH多肽相同功能的、SEQ ID NO:2序列编码多肽的的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-30个，较佳地1-20个，更佳地1-10个，最佳地1-5个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸 进行取代时，通常不会改变多肽功能。比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变多肽功能；又比如，仅表达该多肽的部分结构域也能获得和完整多肽同样的催化功能。因此该术语还包括KAH多肽的活性片段和活性衍生物。
发明还提供KAH蛋白或多肽的类似物。这些类似物与天然KAH多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。
在本发明中，“KAH蛋白保守性变异多肽”指与SEQ ID NO:2的氨基酸序列相比，有至多20个，较佳地至多10个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
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
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基 因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO:1所示的编码区序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:2的蛋白质，但与SEQ ID NO:1所示的编码区序列有差别的核酸序列。
编码SEQ ID NO:2的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，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多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。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多肽的功能。
本发明的KAH核苷酸全长序列或其片段通常可以用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。
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。应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。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。
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KAH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KAH多肽。一般来说有以下步骤：(1).用本发明的编码KAH多肽的多核苷酸(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，KAH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、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KAH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。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。
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：原核细胞如大肠杆菌；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等。作为本发明的优选方式，所述的宿主细胞是原核细胞或酵母细胞。
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。
本专利报道了一种新的甜叶菊来源的KAH基因，将其在微生物细胞中进行了活性表达，并进行了贝壳烯酸到甜菊醇的生物转化研究，为微生物细胞中甜菊糖苷全合成途径的构建打下了基础。
重组的KAH可被应用于甜菊糖醇的生物转化，包括：将贝壳烯酸转化为甜菊糖醇。现有技术中，很难实现在异源宿主(如原核宿主或酵母宿主)中活性表达KAH，使得贝壳烯酸向甜菊糖醇的转化成为限速步骤。而本发明人克隆到的所述KAH能够在微生物中实现异源表达，显然这是具有显著进步意义的。
在获得了本发明的KAH酶后，根据本发明的提示，本领域人员可以方便地应用该酶来实施贝壳烯酸向甜菊糖醇的生物转化。作为本发明的优选方式，还提 供了一种形成生物转化方法，该方法包括：将所述的KAH转化宿主细胞；在存在细胞色素P450氧化还原蛋白(CPR)的情况下，利用KAH将贝壳烯酸转化为甜菊糖醇。其中，CPR可以是各种物种来源的CPR(如甜叶菊、黄花蒿、暗球腔菌、藤仓赤霉、阿拉伯芥等来源)，只要其能发挥KAH的伴侣蛋白的功能(有助于反应过程中电子传递)。
作为本发明的优选方式，所述的细胞色素P450氧化还原蛋白由步骤(1)所述的宿主细胞表达。较佳地，在一个重组载体中同时包括：贝壳烯酸-13α-羟化酶的编码基因的表达盒和细胞色素P450氧化还原蛋白的编码基因的表达盒，转化宿主，表达产物用于进行贝壳烯酸向甜菊糖醇的生物转化。另一种方式，当宿主(如酵母细胞)内源存在细胞色素P450氧化还原蛋白时，将贝壳烯酸-13α-羟化酶的编码基因的表达盒转化该宿主，借助细胞内源存在的细胞色素P450氧化还原蛋白，进行贝壳烯酸向甜菊糖醇的生物转化。
表达盒的建立目前已经是本领域技术人员熟悉的技术。因此，在得知了所需选择的酶之后，本领域技术人员易于进行表达盒的建立。编码酶的基因序列可以被插入到不同的表达盒(如表达载体)中，也可以被插入到同一表达盒中，只要在转入到细胞后酶能够被有效地表达即可。制备表达盒所需的启动子或终止子可以是任何适用的启动子或终止子，并不限于本发明具体所记载的那些。适用的启动子或终止子的选择是本领域技术人员可以进行的，可以根据宿主细胞的种类而定。例如，当应用于酵母重组表达时，现有技术已经揭示了一些酵母启动子或终止子，从而易于作出选择。
针对原核细胞和真核细胞，本领域已知适合的表达载体是哪些，因此人们易于选择到合适的表达载体作为克隆编码基因的骨架载体，例如，当所述的细胞为细菌细胞时，采用pET系列表达载体或pSY系列载体来重组表达各酶；当所述的细胞为酵母细胞，采用pSY系列表达载体。
本发明还提供了一种组合物，它含有有效量的本发明的KAH多肽以及食品学上或工业上可接受的载体或赋形剂。这类载体包括(但并不限于)：水、缓冲液、甘油、乙醇、及其组合。
本发明的有益效果是：
(1)本发明获得了一种新的贝壳烯酸-13α-羟化酶(KAH)基因，为甜菊糖生物合成途径的进一步解析打下基础。
(2)本发明实现了贝壳烯酸-13α-羟化酶(KAH)基因在原核宿主中活性表达，成功地实现了贝壳烯酸到甜菊醇的生物转化，为植物细胞代谢工程改造和甜菊糖生物合成途径的重建打下基础。
以下实施例对本发明中克隆一种新的甜叶菊来源的贝壳烯酸-13α-羟化酶(KAH)基因，并以微生物大肠杆菌宿主进行了贝壳杉烯酸到甜菊醇的生物转化研究进行详细阐述。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，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、KAH基因的获得
1、甜叶菊mRNA的抽提
选取处于成熟期的甜叶菊叶片约100mg，放入研钵中，加入液氮研磨，将磨碎后的粉末转入1.5ml的离心管中，加入700μl的提取缓冲液(2％CTAB(W/V)，2％PVPK30(W/V)，100mM Tris-HCl(pH 8.0)，20mM Na2EDTA(pH 8.0)，1.4mM NaCl，2％β-巯基乙醇)，漩涡振荡混匀。50℃水浴20min，中间混合2-3次，使RNA充分析出。12000g离心10min，将上清吸至新的离心管中，加入等体积的酚：氯仿：异戊醇(25：24：1)，混匀后，12000g离心10min。吸取上清至离心管中后，用等体积氯仿再次重复抽提上清一次后。往上清中加入2倍体积的预冷无水乙醇，冰浴放置30min，12000g离心10min。弃上清后，往沉淀中加入75％乙醇再次洗涤，自然干燥后，加入60μl RNase-Free的水溶解，用于逆转录PCR扩增。
2、KAH基因的RT-PCR扩增和测序鉴定
以甜叶菊的mRNA为模板，采用宝生物工程(大连)有限公司的PrimeScriptTMRT-PCR试剂盒合成cDNA。参考美国专利US7927851，合成引物8-40-1US，8-40-2DS，8-40-3US和8-40-4DS。然后用PrimstarDNA聚合酶，以cDNA为模板，分别用引物对8-40-1US/8-40-2DS，8-40-1US/8-40-4DS，8-40-2DS/8-40-3US，8-40-3US/8-40-4DSPCR扩增KAH基因片段，扩增产物电泳结果如图2。对以上四种PCR产物进行测序，序列拼接后，获得了全长的KAH序列。然后根据全长 的KAH序列，设计引物KAH F/KAH R。以KAH-XbaI F/KAH-BamHI R为引物和cDNA为模板，用Primestar DNA聚合酶PCR扩增全长的KAH基因，扩增产物电泳结果如图3。胶回收KAH PCR产物，然后用XbaI和BamHI分别酶切回收后的KAH PCR产物与pET21a(+)质粒(Novagen)，将两者连接后构建质粒pSY183，如图4。
同时，本发明人还优化了当用于大肠杆菌表达时的KAH密码子，优化后的KAH基因序列如SEQ ID NO:5。
表1
Figure PCTCN2015074344-appb-000001
下划线为限制性内切酶位点，黑色粗体的“ACTAGT”序列代表“SpeI”位点。序列“aataattttgtttaactttaagaaggagatatacat”代表pET21a质粒中XbaI和NdeI位点之间的序列。
3、甜叶菊细胞色素P450氧化还原蛋白基因的密码子优化与合成
选取甜叶菊的细胞色素P450氧化还原蛋白(CPR，ABB88839)，对其基因序列进行密码子优化后，获得基因序列SEQ ID NO:3，并命名为srcpr；其编码SrCPR的氨基酸序列SEQ ID NO:4。将优化好的srcpr基因克隆到pET21a(+)的NdeI和BamHI酶切位点上，获得质粒pET21a-srcpr(图5)。然后用XbaI和HindIII双酶切质粒pSY183，用SpeI和HindIII酶切质粒pET21a-srcpr，分别回收含KAH基因 的DNA片段和pET21a-srcpr载体，将两者连接后构建质粒pSY198(图6)。
实施例2、KAH在大肠杆菌中的表达
将质粒pSY183转化BL21(DE3)，涂布平板后，37℃培养过夜。挑取单菌落与2ml液体LB培养基(氨苄青霉素100mg/L)，220r/min，37℃培养过夜后作为种子液。按1％接种量将种子液接种到新的2ml LB培养基中，220r/min，37℃培养至OD600约为0.3-0.5后，加入终浓度为0.1mM的IPTG，220r/min置于16℃或28℃诱导培养5h或者16h后，收集样品，进行SDS-PAGE分析。
结果如图7，可以看到，在16℃诱导16h时，KAH的表达非常明显，而且蛋白可溶性较好。此外，在28℃诱导时KAH也能够正常表达，但可溶性相比16℃诱导时稍差。
实施例3、利用原核表达的KAH进行贝壳烯酸到甜菊醇的生物转化
将质粒pSY198转化菌株DH10B(DE3)，涂布含100mg l-1氨苄青霉素抗性平板后，37℃培养过夜。挑取单克隆于2ml含100mg l-1氨苄青霉素液体LB培养基中，37℃培养过夜。然后以1％接种量转接到新的LB培养基中，37℃培养2h至OD600约为0.6，加入IPTG至终浓度0.1mM，16℃诱导培养16h。4℃，6000g离心收集菌体，用等体积的M9盐/磷酸缓冲体系(4.2mM Na2HPO4；2.2mM KH2PO4；0.9mM NaCl；1.9mM NH4Cl)洗涤两次。然后用加入一定体积的生物转化缓冲体系重悬(M9盐/磷酸缓冲体系+50mg l-1酵母抽提物)，重悬之后控制菌体湿重约为4g l-1。取以上的菌体悬液4ml，5×NADPH再生体系1ml(5mM NADP，25mM 6-磷酸葡萄糖和5Unit/ml的6-磷酸葡萄糖脱氢酶)，100g/L的贝壳烯酸母液30μl于100ml的摇瓶中，置于摇床上，28℃，150r/min反应16h。往反应液中加入等体积乙酸乙酯萃取，吸取有机相进行HPLC-MS分析。
结果如图8-9，可以看出，样品中成功的检测到了甜菊糖醇，而且其分子离子峰为317.2，与甜菊糖醇标准品(Steviol Standard)一致。
实施例4、利用酵母表达的KAH进行贝壳烯酸到甜菊醇的生物转化
1、酵母表达系统pSY220重组质粒的构建
以pSY220F和pSY220R为引物，以pSY183为模板PCR扩增KAHS2基因。PCR产物经胶回收后，用BglII/NotI酶切，然后再次回收酶切产物，将其连接到 用BamHI/NotI处理的pPIC3.5k载体(购自Invitorgen)上，获得质粒pSY220(pPIC3.5k-KAHS2)。
pSY220F(SEQ ID NO:14)：GGAAGATCTATGAAATTCAAAAAGT TTTCTTGTACCCAC(下划线为BamHI酶切位点)；
pSY220R(SEQ ID NO:15)：ATAAGAATGCGGCCGCTTAGAGTTTG TGTAAAATCAAGTGAGCACC(下划线为NotI酶切位点)。
2、重组毕赤酵母表达方法
毕赤酵母自身含有的细胞色素P450氧化还原蛋白就能够为外源P450氧化酶提供电子传递的作用，因此本实施例中不需要额外表达异源CPR基因就可以使KAHS2在毕赤酵母中进行活性表达。
将质粒pSY220用SalI酶切，回收线性化的DNA片段，然后将其电击转化毕赤酵母菌株KM71(购自Invitrogen)，涂布MD平板(13.4g/L酵母基本氮源(YNB)；0.4mg/L生物素；20g/L葡萄糖)，28℃培养3天后，经PCR验证获得重组菌株。将重组菌株接种到2ml YPD培养基中，28℃，250r/min，培养24h后，以其为种子，按1％接种量，接种到含有50ml BMGY(2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10-5％生物素，0.5％甘油)培养基的500ml摇瓶中，共两瓶，继续培养24h。用50ml离心管收集菌液，共2管，2000r/min，离心5min，弃尽培养基上清。分别用5mlBMMY(2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10-5％生物素，0.5％甲醇)培养基悬浮沉淀细胞，将菌体悬液合并后，共10ml，转移到100ml摇瓶中，250r/min，22℃，每24h添加50μl的甲醇，诱导培养72h。
3、活性检测
4℃，2000g离心收集菌体，用等体积的M9盐/磷酸缓冲体系(4.2mM Na2HPO4；2.2mM KH2PO4；0.9mM NaCl；1.9mM NH4Cl)洗涤两次。然后用加入一定体积的生物转化缓冲体系重悬，重悬之后控制菌体湿重约为4g/L。取以上的菌体悬液4ml，5×NADPH再生体系1ml(5mM NADP，25mM 6-磷酸葡萄糖和5Unit/ml的6-磷酸葡萄糖脱氢酶)，100g/L的贝壳烯酸母液30μl于100ml的摇瓶中，置于摇床上，28℃，150r/min反应16h。往反应液中加入等体积乙酸乙酯萃取，吸取有机相进行HPLC-MS分析。
毕赤酵母表达系统KAHS2活性检测HPLC检测色谱图如图10；MS检测结果如图11。由图可以看出，样品中成功的检测到了甜菊糖醇，而且其分子离子峰为317.2。
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 (13)

  1. 一种分离的贝壳烯酸-13α-羟化酶，其特征在于，其选自下组：
    (a)如SEQ ID NO:2氨基酸序列的多肽；
    (b)将SEQ ID NO:2氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽功能的由(a)衍生的多肽；
    (c)具有(a)多肽功能的SEQ ID NO:2的蛋白片段；或
    (d)具有(a)多肽功能的且与(a)多肽具有90％以上氨基酸序列相同性的多肽。
  2. 一种分离的多核苷酸，其特征在于，其编码如权利要求1所述的贝壳烯酸-13α-羟化酶。
  3. 如权利要求2所述的多核苷酸，其特征在于，该多核苷酸的核苷酸序列如SEQ ID NO:1或SEQ ID NO:5所示。
  4. 一种重组载体，其特征在于，它含有权利要求2-3任一所述的多核苷酸。
  5. 一种宿主细胞，其特征在于，它含有权利要求4所述的重组载体，或其基因组中整合有权利要求2-3任一所述的多核苷酸。
  6. 一种权利要求1所述的贝壳烯酸-13α-羟化酶的制备方法，其特征在于，该方法包含：
    (a)培养权利要求5所述的宿主细胞；
    (b)从培养物中分离出权利要求1所述的贝壳烯酸-13α-羟化酶。
  7. 权利要求1所述的贝壳烯酸-13α-羟化酶的用途，用于将贝壳烯酸转化为甜菊糖醇。
  8. 如权利要求7所述的用途，其特征在于，所述的将贝壳烯酸转化为甜菊糖醇在植物细胞或非植物细胞中进行；较佳地，所述的非植物细胞包括细菌细胞、真菌细胞；所述的真菌细胞包括酵母细胞(Yeast)，如毕赤酵母(Pichiapastoris)、酿 酒酵母(Saccharomyces cerevisiae)、乳酸克鲁维酵母(Saccharomyces cerecisiae)等。所述的细菌细胞包括：肠杆菌属(Enterobacter)，如大肠杆菌(Escherichia coli)，芽孢杆菌属(Bacillus)，如枯草芽孢杆菌(Bacillus subtilis)，链霉菌属(Streptomyces)，天蓝色链霉菌(Streptomyces coelicolor)、变铅青链霉菌(Streptomyces lividans)、阿维链霉菌(Streptomyces avermitilis)、委内瑞拉链霉菌(Streptomyces venezuelae)等，糖多胞菌属(Saccharopolyspora)，如红霉糖多胞菌(Saccharopolyspora erythraea)。
  9. 一种制备甜菊糖醇的方法，其特征在于，该方法包含：利用权利要求1所述的贝壳烯酸-13α-羟化酶将贝壳烯酸转化为甜菊糖醇。
  10. 如权利要求9所述的方法，其特征在于，所述方法包括：
    (1)培养权利要求5所述的宿主细胞，从而表达权利要求1所述的贝壳烯酸-13α-羟化酶；
    (2)在存在细胞色素P450氧化还原蛋白的情况下，利用步骤(1)所述的贝壳烯酸-13α-羟化酶将贝壳烯酸转化为甜菊糖醇。
  11. 如权利要求10所述的方法，其特征在于，所述的细胞色素P450氧化还原蛋白由步骤(1)所述的宿主细胞表达。
  12. 如权利要求11所述的方法，其特征在于，步骤(1)所述的宿主细胞中包含的重组载体中还包括：细胞色素P450氧化还原蛋白的编码基因。
  13. 一种组合物，其特征在于，它含有安全有效量的权利要求1所述的贝壳烯酸-13α-羟化酶以及食品学或工业上可接受的载体。
PCT/CN2015/074344 2014-03-20 2015-03-17 新的贝壳烯酸-13α-羟化酶、其编码基因及其应用 WO2015139599A1 (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017060318A3 (en) * 2015-10-05 2017-06-08 Dsm Ip Assets B.V. Kaurenoic acid hydroxylases
US10760085B2 (en) 2015-10-05 2020-09-01 Dsm Ip Assets B.V. Kaurenoic acid hydroxylases
WO2020227148A1 (en) * 2019-05-03 2020-11-12 Amyris, Inc. Kaurenoic acid 13-hydroxylase (kah) variants and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1897951A2 (en) * 2006-03-21 2008-03-12 Her Majesty the Queen in Right of Canada As represented by the Minister of Agriculture And Agri-Food Composition and methods for producing steviol and steviol glycosides
WO2012075030A1 (en) * 2010-11-30 2012-06-07 Massachusetts Institute Of Technology Microbial production of natural sweeteners, diterpenoid steviol glycosides
WO2013110673A1 (en) * 2012-01-23 2013-08-01 Dsm Ip Assets B.V. Diterpene production

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI523611B (zh) * 2009-08-12 2016-03-01 梅拉洛伊卡公司 抗氧化膳食補充組成物
KR101598935B1 (ko) * 2010-04-16 2016-03-03 씨제이제일제당(주) 레바우디오사이드 ａ의 생산 공정에서 발생하는 부산물을 재활용하여 고수득율의 레바우디오사이드 ａ를 제조하는 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1897951A2 (en) * 2006-03-21 2008-03-12 Her Majesty the Queen in Right of Canada As represented by the Minister of Agriculture And Agri-Food Composition and methods for producing steviol and steviol glycosides
WO2012075030A1 (en) * 2010-11-30 2012-06-07 Massachusetts Institute Of Technology Microbial production of natural sweeteners, diterpenoid steviol glycosides
WO2013110673A1 (en) * 2012-01-23 2013-08-01 Dsm Ip Assets B.V. Diterpene production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank [O] 13 June 2011 (2011-06-13), BRANDLE,J. AND RICHMAN,A.: "Sequence 3 from patent US 7927851", XP055224974, retrieved from ncbi Database accession no. GY221603.1 *
DATABASE GenBank [O] 8 July 1999 (1999-07-08), CABRERO,J.G., FREEMAN,G.J. AND REISER,H: "Mus musculus strain AKR/J CD48 antigen (Cd48) gene, partial cds", XP055224985, retrieved from ncbi Database accession no. AF064616.1 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017060318A3 (en) * 2015-10-05 2017-06-08 Dsm Ip Assets B.V. Kaurenoic acid hydroxylases
US10760085B2 (en) 2015-10-05 2020-09-01 Dsm Ip Assets B.V. Kaurenoic acid hydroxylases
US11203764B2 (en) 2015-10-05 2021-12-21 Dsm Ip Assets B.V. Kaurenoic acid hydroxylases
EP4043558A3 (en) * 2015-10-05 2022-11-16 DSM IP Assets B.V. Kaurenoic acid hydroxylases
WO2020227148A1 (en) * 2019-05-03 2020-11-12 Amyris, Inc. Kaurenoic acid 13-hydroxylase (kah) variants and uses thereof

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