CN101024840B - Nucleotide series of Pichia yeast delta9 fatty acid dehydro genase and use - Google Patents

Abstract

The invention relates to a nucleotide sequence of pichia yeast Delta9-fatiy acid desaturase separated from pichia yeast and its application. And it is a fraction, analogue and derivative which has the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence of the invention. And the invention comprises carrier connecting its nucleotide sequence with extrinsic adjusting sequence for functional expression, cell organisms containing the carrier, and final generations of organisms of this type; a method of generating unsaturated fatty acid with the above nucleotide sequence, or polypeptide sequence or carrier-containing cell organisms and the final generations of the organisms of this type; and using the nucleotide sequence shown in SEQ ID NO:1 as a probe for identifying related sequence.

Description

Pichia spp △ 9The nucleotide sequence of-fatty acid dehydrogenase and application thereof

[technical field]

The invention belongs to biological technical field and field of genetic engineering, relate to clone's Δ from one primary yeast-pichia spp (Pichia Pastoris) ⁹-fatty acid dehydrogenase gene specifically is the pichia spp Δ ⁹The nucleotide sequence of-fatty acid dehydrogenase and application thereof.This gene directly or with different expression vectors is connected, be transferred in bacterium, yeast, plant or the animal, utilize its coded delta ⁹-fatty acid dehydrogenase produces the methods and applications of unsaturated fatty acids.

[background technology]

Unsaturated fatty acids UFAs (unsaturated fatty acids) is the important component of cytolemma, and essential by biology growing.Δ ⁹-fatty acid dehydrogenase is a kind ofly unsaturated link(age) is introduced saturated fatty acid to form monounsaturated fatty acids---the film integral protein of Zoomeric acid (C16:1) and oleic acid (C18:1).It is the rate-limiting enzyme of unsaturated fatty acids route of synthesis.Many studies show that, the Δ of nearly all biology ⁹The expression of-fatty acid dehydrogenase is subjected to nutrition and other Effect of Environmental (V.M.McDonough in the growing environment, J.E.Stukey, C.E.Martin, Specificity ofunsaturated fatty acid-regulated expression of the Saccharomyces cerevisiae OLE1gene, J.Biol.Chem.267 (1992) 5931-5936).Up to the present, owing to separate and identify difficulty very big [MaKeon, 1981, Enzymology method, the 12141-12147 of embrane-associated protein; Wang etc., 1988, plant physiology biological chemistry, 26:777-792], therefore be difficult to obtain comprising Δ ⁹-fatty acid dehydrogenase is at the higher structure of interior various embrane-associated protein character desaturases, thereby for the biological function of fatty acid dehydrogenase, many laboratories have been adopted the gene that the clone is relevant from various organisms and have been changed in the various acceptors, observe the research method of the lipid acid change of acceptor in acceptor.At present, from different sourcess such as animal, plant, microorganism, be separated to the homologous sequence of many-lipid acid dehydrogenation gene, and proof has corresponding biological function.

The result of sequence alignment shows, coded delta ⁹The nucleotide sequence of-fatty acid dehydrogenase has the common constitutional features: have three Histidine conserved regions His I district HECGH, His II district HXXHH and His III district HVXHH, form structure [the Kyte et al. that strides film for 4 times, 1982, J.Mol.Biol.157:105-132], these all are to keep necessary [the Napier JA of dehydrogenase activity, Sayanova O, Stobart AK, et al., 1997, Biochem.J., 328:717-720].Because Δ ⁹To be first introduce enzymes of two keys to lipid acid to-fatty acid dehydrogenase, so it has play a part crucial in unsaturated fatty acids synthetic.The research Δ ⁹The expression of-fatty acid dehydrogenase and physiological action also become one of current focus.

[summary of the invention]

One of purpose of the present invention provides separated coding Δ from pichia spp ⁹The nucleotide sequence of-fatty acid dehydrogenase or the fragment of its nucleotide sequence, analogue or derivative.Clone's Δ from one primary yeast-pichia spp (Pichiapastoris) ⁹-fatty acid dehydrogenase gene specifically is the pichia spp Δ ⁹The nucleotide sequence of-fatty acid dehydrogenase and application thereof.

Two of purpose of the present invention provides the coded Δ of this nucleotide sequence ⁹-fatty acid dehydrogenase polypeptide or its fragment, analogue or derivative.

Three of purpose of the present invention provides and contains this gene nucleotide series and allos and regulate sequence and be connected, and carries out the recombinant vectors of functional expression.

Four of purpose of the present invention provides and contains this gene nucleotide series or this gene nucleotide series and allos and regulate host cell and the offspring thereof that recombinant vectors that sequence is connected transforms or transduces.

The method that five of purpose of the present invention provides that a kind of usefulness contains that this gene nucleotide series or this gene nucleotide series and allos regulate that recombinant vectors that sequence is connected transforms or the host cell of transduceing and progeny cell thereof produce corresponding unsaturated fatty acids.

Other aspects of the present invention are because disclosing of this paper technology is conspicuous to those skilled in the art.

First

The present invention is to provide a kind of isolating pichia spp Δ ⁹The nucleotide sequence of-fatty acid dehydrogenase is selected from:

(a) coding has the nucleotide sequence of the aminoacid sequence polypeptide shown in the SEQ ID NO:2;

(b) with (a) in nucleotide sequence complementary nucleotide sequence;

(c) at (a) or in aminoacid sequence that (b) limits or the nucleotide sequence through replacing, lack or adding one or several amino acid or Nucleotide and have a fatty acid dehydrogenase active by (a) or (b) polypeptides derived or nucleotide sequence.

Exactly, this nucleotide sequence has the nucleotide sequence shown in the SEQ ID NO:1.More accurately, this nucleotide sequence is to be selected from down a kind of in the group: (a) have the sequence of 1-1194 in the SEQ ID NO:1 sequence and (b) have the sequence of 1-397 among the SEQID NO:2.

Concrete As used herein, " isolating " are meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.For example, nucleotide sequence and polypeptide under the native state in the active somatic cell do not have separation and purification, but same nucleotide sequence or polypeptide as from native state with separating in other material that exists, then be separation and purification.

As used herein, " isolating nucleotide sequence " is meant and is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be with the DNA purification technique purifying of standard.

The invention provides a kind of new isolating nucleotide sequence---the nucleotide sequence of coding Pichia pastoris omega 3-fatty acid dehydrogenase, it is made up of the nucleotide sequence shown in the SEQ ID NO:1 basically, this sequence is long to be 1194bp, wherein 1bp-1194bp coded delta ⁹The open reading frame of-fatty acid dehydrogenase mature polypeptide SEQ ID NO:2.

The invention provides isolating nucleotide sequence, this nucleotide sequence is made up of the nucleotide sequence that coding has an active polypeptide of SEQ ID NO:2 aminoacid sequence substantially.Particularly, nucleotide sequence of the present invention has the nucleotide sequence of SEQ ID NO:1.

The nucleotide sequence of coding SEQ ID NO:2 active polypeptide comprises: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " coding pichia spp Δ ⁹The nucleotide sequence of-fatty acid dehydrogenase " be meant comprise coding pichia spp Δ- ⁹The nucleotide sequence of fatty acid dehydrogenase polypeptide and comprise additional code and/or noncoding nucleotide sequence.

Nucleotide sequence of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can with the identical or varient of degeneracy of coding region sequence shown in the SEQ ID NO:2.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ IDNO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The present invention also comprises coding pichia spp Δ ⁹The fragment of the nucleotide sequence of-fatty acid dehydrogenase, analogue or derivative.As used herein, term " fragment ", " analogue " are meant to encode with " derivative " and keep the natural identical biological function of the present invention or the nucleotide sequence of active polypeptide basically.

By the elaboration of this paper, such fragment, analogue or derivative are considered within those skilled in the art's ken.

The fragment of nucleotide sequence of the present invention, derived sequence or similar sequence can be that wherein one or more Nucleotide replace, the nucleotide sequence of disappearance, insertion, inversion, i.e. the artificial mutant of original separation sequence, and it also has required enzymatic functions.It can also be other gene Fusion sequence of described nucleotide sequence and fatty acid biological synthetic.

The allelic variant that derivative shown in SEQ ID NO:1 or functional derivatives are represented, it has at least 75% homology at the deutero-amino acid levels, preferred at least 80% homology, preferred especially 85% homology, very special preferably 90% homology.Described homology is based on complete amino acid fragment calculating.Shown in SEQ ID NO:2, homology is represented identity by described nucleotide sequence coded aminoacid sequence, and aminoacid sequence at least 70% is identical in other words.Described new nucleotide sequence has 65% homology at least on nucleic acid level, preferred at least 70% homology, preferred especially 75% homology, very special preferably 80% homology.

Derivative is also represented the homologue of sequence shown in the SEQ ID NO:1, as the sequence of eucaryon homologue, brachymemma, but still have required function, have this proteic enzymatic activity in other words, therefore, functionally equivalent comprise above-mentioned sequence natural variant and artificial sequence oligodeoxynucleotide.

Noncoding derivative also represents to can be used for suppressing the biosynthetic antisense DNA of described novel protein.Described antisense DNA belongs to no function derivative of the present invention.As not possessing the derivative of enzymatic activity.Available those skilled in the art knows other method of producing no function derivative has common inhibition, the use of ribozyme and intron.

The invention still further relates to and the nucleotide sequence of above-mentioned sequence hybridization (has at least 50% homology between two sequences, preferred at least 70% homology), and interfertile nucleotide sequence coded polypeptide has identical biological function with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, the length of " nucleic acid fragment " contain 10 Nucleotide at least, 20-30 Nucleotide at least preferably, and 50-60 Nucleotide at least especially preferably is very especially preferably more than at least 100 Nucleotide.Nucleotide fragments also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the separation nucleotide sequence coding.

Polypeptide among the present invention and nucleotide sequence preferably provide with isolating form, more preferably are purified to homogeneous.

Specific nucleotide sequence can obtain with several different methods among the present invention.For example, with hybridization technique separating nucleotide sequence well known in the art.These technology including, but not limited to: (1) with probe and genome or the hybridization of cDNA library to detect the homologous nucleotide sequence; (2) antibody screening of expression library is to detect the nucleotide sequence fragment of the clone with common structure feature.

Sequence dna fragment of the present invention also can obtain with following method: (1) separates double chain DNA sequence from genomic dna; (2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of dna sequence dna.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, also can obtain from commercial channels with test kit.And the construction cDNA library also is a usual method.When in conjunction with polymerase chain reaction technology (PCR), even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries, these methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or disappearance; (3) level of the transcript of Ce Dinging; (4) measure biologic activity by immunological technique, detect the protein product of genetic expression.Aforesaid method can singly be used, but also several different methods combined utilization.In (1) in the method, hybridize used probe and be any a part of homology with nucleotide sequence of the present invention, at least 10 Nucleotide of its length, preferably at least 30 Nucleotide, especially preferably at least 50 Nucleotide, very especially preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.The normally dna sequence dna of chemosynthesis on the basis of gene order information of the present invention of probe that gets used herein.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

The method of application round pcr DNA amplification/RNA (Saiki et al., 1985, Science 230:1350-1354) is optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can be according to nucleotide sequence information appropriate selection of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

Second

The invention provides isolating by above-mentioned nucleotide sequence encoded polypeptide, exactly, this polypeptide is to have the polypeptide of SEQ IDNO:2 aminoacid sequence or its to make amino acid variation be no more than 30% and have an active polypeptide of fatty acid dehydrogenase through replacing, lack or adding one or several amino acid.

The present invention also provides a kind of new peptide sequence one pichia spp Δ ⁹The aminoacid sequence of-fatty acid dehydrogenase, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology (for example, to produce in bacterium, yeast, higher plant, insect and the mammalian cell from protokaryon or eucaryon host.

The Δ that polypeptide of the present invention comprises ⁹The fragment of-fatty acid dehydrogenase, derivative and analogue.As used herein, term " fragment ", " analogue " are meant with " derivative " and keep natural identical biological function of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) a kind of like this, wherein one or more amino-acid residues are by conservative or non-conservative amino acid residues (preferably conservative amino acid residues) replacement, inversion, insertion or disappearance; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, and wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).

The 3rd

The invention provides by above-mentioned nucleotide sequence and plasmid, virus or the constructed recombinant vectors of vehicle expression vector.

Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carrier.The sequential element that can influence gene expression product includes replication origin, promotor, marker gene and translational control element.

Available method well-known to those having ordinary skill in the art makes up and contains coding pichia spp Δ ⁹The expression vector of the nucleotide sequence of-fatty acid dehydrogenase and suitable transcribing/translational control element.These methods comprise [Sambroook, et al., Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1989] such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described coding pichia spp Δ ⁹The nucleotide sequence of-fatty acid dehydrogenase can effectively be connected on the appropriate promotor of expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The P of lambda particles phage _LPromotor: eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and common nearly 10-300bp acts on promotor transcribing with enhancing gene.As the adenovirus enhanser.

The 4th

The invention provides a kind of by the host cell and the progeny cell thereof of above-mentioned nucleotide sequence or recombinant vectors conversion or transduction.Described host cell, it is bacterial cell or fungal cell or vegetable cell or zooblast, or the offspring of these host cells.Brewing yeast cell particularly.

Among the present invention, coding pichia spp Δ ⁹The nucleotide sequence of-fatty acid dehydrogenase or the recombinant vectors that contains this nucleotide sequence can transform or transduce into host cell, contain the genetically engineered host cell of this nucleotide sequence or recombinant vectors with formation.Term " host cell " refers to prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.The representative example of host cell has: intestinal bacteria; Fungal cell such as yeast; Vegetable cell such as rape, tobacco, soybean; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

Can carry out with method well-known to those having ordinary skill in the art with nucleotide sequence of the present invention or the recombinant vectors transformed host cell that contains nucleotide sequence.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be collected thalline in exponential phase of growth, uses CaCl ₂Method is handled, and used step is well known in the art.Also available MgCl ₂, methods such as electroporation are carried out.When the host is an eukaryote, can select methods such as DNA infection protocol, microinjection, electroporation, liposome packing for use.

The 5th

The invention provides a kind of usefulness contains this gene nucleotide series or this gene nucleotide series and allos and regulates that recombinant vectors that sequence is connected transforms or the method for transduction host cell and progeny cell generation unsaturated fatty acids thereof.

The invention still further relates to above-mentioned genetically modified host cell production, according to host cell, with those skilled in the art method growth or cultivation in common knowledge.Normally at 0-100 ℃, preferred 10-60 ℃, also want oxygen simultaneously such as microorganism cells.Contain carbon source in the substratum, as glucose, nitrogenous source, the form of organonitrogen normally, as yeast extract, amino acid, or salt, as ammonium sulfate, trace element as iron, magnesium salts, also has VITAMIN if necessary.The pH of the substratum value that can be maintained fixed in other words, is controlled in the training period or is not controlled during this period.Cultivation can batch culture, half discontinuous cultivation or cultured continuously form are carried out.After cultivating, collecting cell is smashed to pieces or directly use, extracts lipid acid by method well known to those skilled in the art from cell.

The invention still further relates to a kind of method for preparing unsaturated fatty acids, this method is achieved in that to have saturated or the unsaturated fatty acids triglyceride level is cultivated with SEQ ID NO:2.This method is preferably carried out under by the compound existence condition that can absorb or discharge the reductibility equivalent.Then, can release fat acid from triglyceride level.Aforesaid method preferably can synthesize the lipid acid with 9 two keys.

The invention still further relates to and use the method for preparing unsaturated fatty acids, and be applied to produce human food prods, animal-feed, makeup or medicine purposes.

[description of drawings]:

Fig. 1 shows pichia spp Δ of the present invention ⁹-fatty acid dehydrogenase and Ke Shi yeast Δ ⁹The amino acid sequence homology comparison diagram of-fatty acid dehydrogenase (BAB86330);

Fig. 2 is the yeast saccharomyces cerevisiae expression vector pYPPd9 that makes up;

Fig. 3 A is the gas chromatogram of C17:0 methyl ester standard substance;

Fig. 3 B is the gas chromatogram that contains the transgenic yeast of pYES2.0 empty carrier;

Fig. 3 C is the gas chromatogram that contains the transgenic yeast of recombinant plasmid pYPPd9.

[embodiment]

Embodiment 1

From pichia spp, separate Δ ⁹The nucleotide sequence of-fatty acid dehydrogenase

According to A. Adam Si grade (A. Adam Si etc., 2000, yeast genetics method experiment guide, 84-89.) method that provides, from cultivate 36 hours pichia spp thalline, extract total DNA, getting 7 μ g is that template is carried out the polymerase chain reaction.According to the Δ of being delivered ⁹The Histidine conserved regions I of-fatty acid dehydrogenase homologous sequence and III region amino acid sequence design degenerate primer (primer 1 and primer 2), DNA with said extracted is that template increases at the enterprising performing PCR of T-Gradient PCR instrument (Biometra company), and reaction the primer, component and amplification condition are as follows:

Primer 1:5 '-CA (TC) CA (TC) CG (ATGC) AC (ATGC) GA (TC) AC-3`

Primer 2: 5 '-TG (ATGC) CC (ATGC) CC (ATGC) GG (GA) TG (CT) TC-3`

Amplification condition: 94 ℃ of sex change 3min, use 94 ℃ of 1min again; 58 ℃ of 1min → 72 ℃ 1min carry out 3 circulations, then 94 ℃ of 1min; 52 ℃ of 1min → 72 ℃ 1min carry out 30 circulations, last 72 ℃ of 10min.The agarose gel electrophoresis detected result shows, amplification obtains the fragment of the about 800bp of size, reclaim with UNIQ-10 pillar PCR product purification test kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product), reclaiming the fragment subclone in sequencing vector pGEM-T (Promega company product); The connection product is transformed into uses CaCl ₂The bacillus coli DH 5 alpha that method is handled, overnight incubation on the LB solid medium that contains penbritin (final concentration is 100 μ g/ml); The white colony of growing on the picking flat board, access contains overnight incubation in the LB liquid nutrient medium of penbritin (final concentration is 60 μ g/ml), centrifugal collection thalline is pressed alkaline lysis [Sambroook, et al., 1989, Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory, New York, p19-21] extract plasmid, identify through NcoI and SacI double digestion and pcr amplification correct, order-checking (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).Sequencing result shows that the clip size that obtains that increases is 741bp, in the Genbank database, use Blast program (Basiclocal Alignment seatch tool) [Altschul SF et al by its amino acid sequence coded, 1997, Nucleic Acids Res.25:3389-3402] carry out the homology retrieval, result for retrieval shows that the homologous fragment the most similar to this fragment is Δ ⁹-fatty acid dehydrogenase, but and incomplete same, prove that institute's amplified fragments is the fragment of new potential dehydrogenase gene.

According to the partial sequence that obtained design gene-specific primer (primer 3 and primer 4), (ligation-mediated PCR LMPCR) obtains to comprise the 3` and the 5` end sequence of above-mentioned segmental gene to utilize the method for joint PCR.Extract the total DNA of cell earlier, with genome AatlI single endonuclease digestion, with Mortierella isabellina (Mortierella isabellina) Δ 6-fatty acid dehydrogenase open reading frame double digestion (AatlI﹠amp; PstI) gained fragment (731bp-1311bp) is as joint, enzyme cut the back genome spend the night with 1: 3 in molar ratio ratio of joint and be connected.To connect product is template, and respectively with primer 3 and 4, primer 3 and 5 carries out 3 ' downstream sequence and the amplification of 5 ' upstream sequence.

Primer 3:5`-GGGCAAACGA GAGAATGGGA AAGTA-3`,

Primer 4:5 '-GACAATGCCATCAAACAGGG 3`

Primer 5:5 '-CATGCGTCGTCCCAGAAATACTT

Template DNA 1 μ l, damping fluid (10 *) 5 μ l, dNTP (50 *) 1 μ l, primer 3 (10 μ mol/L) l μ l, primer 4/5 (10 μ mol/L) 1 μ l, Advantage2Polymeerase Mix 1 μ l, H2O 34.5 μ l amplification conditions: 95 ℃ of sex change 3min of elder generation enter 95 ℃ of 30sec; 60 ℃ of 1min, 72 ℃ of 3min carry out 30 circulations altogether, last 72 ℃ of 10min.With after 100 times of the pcr amplification product dilutions as template, adopt above-mentioned identical condition, respectively with primer 6 and 7, primer 6 and 8 carries out 3 ' downstream sequence and the amplification of 5 ' upstream sequence.

Primer 6:5`-GGTGCTCAAT CTGGTAGTTC AATCC-3`,

Primer 7:5`-CACGACAT TATCGAAACC CACGTCTGC-3`

Primer 8:5`-ATGACCTGTT GCCTTATGAT GC-3`

Order-checking, The sequencing results show the 3` amplification obtain from primer 7 sequences to joint sequence before the sequence information of 531bp altogether, and 5` obtains from primer 8 sequences to the sequence information of 865bp altogether the joint sequence.With sequence analysis software (DNAMANVersion4.0, Lynnon BioSoft) three fragments is spliced and analyze, obtain the nucleotide sequence shown in SEQ ID NO:1, wherein 379bp-1568bp (ATG----TAA) is the potential open reading frame, 397 amino acid of encoding.According to two end sequences, design gene-specific primer (primer 9 and primer 10) is by above-mentioned condition pcr amplification, order-checking, and the result of institute is with to splice the result consistent.

Primer 9:5 '-GACGTTACAGAGGCAAACGCAGTTG-3`

Primer 10:5 '-CACAATAACACCTAATAAAATTTCC`-3

Embodiment 2: the homology search of institute's separating nucleotide sequence

To infer Δ ⁹The aminoacid sequence of-fatty acid dehydrogenase carries out the homology search on Genbank, gained homologous sequence major part is a coded delta ⁹The sequence of-fatty acid dehydrogenase, wherein with come Ke Shi yeast (Saccharomyces kluyveri) homology the highest: homogeny 64%, this illustrates that the new coded enzyme of nucleotide sequence of the present invention has potential Δ ⁹The function of-fatty acid dehydrogenase.(Fig. 1)

Embodiment 3: the structure of yeast saccharomyces cerevisiae recombinant expression vector

According to coding region sequence shown in the SEQ ID NO:1, design a pair of gene specific amplimer (primer 11 and primer 12) and separate its potential open reading frame sequence:

Primer 11:5-GGCAAGCTTATGTCAAAAGTCACTGTTTCGGG-3`:

Primer 12:5`-GCCTCTAGATTAGGTATCC TTAGG-3`:

The 5` end black matrix of these two primers contains HindIII and XbaI enzyme cutting site respectively.Used amplification condition and reactive component are the same, and the sequencing result of amplified production shows consistent with the sequence of 379bp-1568bp shown in the SEQ ID NO:1.Get 50 μ l PCR products then and 1 μ l pYES2.0 carries out double digestion respectively, reclaim enzyme and cut big fragment, and with the T4 ligase enzyme in 4 degree refrigerator overnight connections.Connect product transformed into escherichia coli DH5 α, extract and the PCR screening positive clone by plasmid, and the evaluation of checking order.Plasmid construction the results are shown in accompanying drawing 2, the constructed Δ that contains ⁹The expression plasmid of yeast called after pYPPW3 of-fatty acid dehydrogenase gene.This plasmid is to be made up by the PCR product of pYES2.0 (Invitrogen company) and primer 11 and primer 12 to form.Plasmid and respectively behind HindIII and XbaI double digestion, electrophoresis reclaims purifying, connects through T4DNALigase, connects product transformed into escherichia coli DH5 α, the Screening and Identification matter example that goes out to recombinate, called after pYPPd9.

Embodiment 4: recombinant expression vector transformation receptor bacterium brewing yeast cell

Picking Wine brewing yeast strain Δ ⁹Deficient strain DTY-11A bacterium colony in 10ml YEPD liquid nutrient medium, 30 ℃ of shaking table incubated overnight, detect the OD of bacterium liquid ₆₀₀Value is got an amount of bacterium liquid and is diluted in 50ml, makes OD ₆₀₀Be 0.4; Continue to cultivate 2 behind 4h, the 2500rpm centrifugation cell, with the resuspended thalline of 40ml1 * TE, the 2500rpm centrifugation cell with 2ml1 * LiAc/0.5 * TE suspension cell, and is at room temperature placed 10min, and this cell is competent cell; Get the yeast competent cell that 100 μ l prepare, add 1 μ g recombinant plasmid pYPPW3 and 100 μ g sex change salmon sperm DNAs, mixing adds 700 μ l1 * LiAc/40%PEG-4000/1 * TE and mixings, in 30 ℃ of incubation 30min; Then, add 88 μ l DMSO mixings, heat shock 7min in 42 ℃ of water-baths; Centrifugal 10s sedimentation cell adds 1ml1 * TE suspension cell and recentrifuge sedimentation cell, adds 100 μ l re-suspended cells at last, be laid on SC-Ura/Leu (no uridylic entirely, no leucine) selects culture medium flat plate, put 30 ℃ and cultivate 48-72h, grow to transformant.

Embodiment 5: the abduction delivering of Yeast engineering bacteria

The positive transformant that occurs on the picking SC-Ura/Leu selection culture medium flat plate, be inoculated in 15ml SC-Ura/Leu and select substratum (containing 2% glucose), 28 ℃ are shaken the bacterium incubated overnight, inoculum size with 5% adds the 100mlSC-Ura/Leu substratum that contains 2% semi-lactosi, and add C17:0 as internal standard substance, 28 ℃ are continued to cultivate 72h; 2500rpm collects thalline, uses deionized water wash three times, and 50 ℃ of oven dry grind, and get the KOH-CH that 100mg adds 5ml 5% ₃OH solution, 70 ℃ of reaction 2-3h; Reaction finishes, and cool to room temperature is with the pH value to 2.0 of the hydrochloric acid conditioning solution of 6mol/L, adding 4ml 14%BF ₃-CH ₃OH, 70 ℃ of reaction 1.5h, synthesizing fatty acid methyl ester; Add saturated NaCl solution 10ml again, the concuss mixing, and transfer in the separating funnel, use 1: 4 chloroform of 8ml: hexane extracting twice, united extraction liquid; Add an amount of anhydrous Na ₂SO ₄Dry extraction liquid leaves standstill 1h, removes Na ₂SO ₄, the supernatant liquor that contains fatty acid methyl ester is dried up with nitrogen, return molten sample with the normal hexane of 200 μ L, the back is with the filtering with microporous membrane of 0.45mm.Obtain the total fatty acids of the positive yeast saccharomyces cerevisiae transformant of esterification

Embodiment 6: the lipid acid gas chromatographic analysis

Undertaken by following condition:

Instrument is Tianjin, island GC-7A, pillar: fused-silica capillary column, 0.32 * 30m, solid support: Dienthyeneglycol succinate (Poly-diethylene glycol succinate, DEGS) plated film thing: polyimide.Carrier gas: N ₂, linear speed: 10cm/s.Splitting ratio: 100: 1, the vaporizer temperature: 250 ℃, column temperature: 180 ℃, tail blew: 50ml/min, detector: flame ionization ditector.The C17:0 methyl esters of producing with Sigma company is standard substance, the sample of the methyl esterification of fatty acid of method for preparing, carries out GC and analyzes, and applied sample amount is 1 μ l; Analysis software: Anstar, the star color spectrum workstation of analysis.

Stratographic analysis the results are shown in accompanying drawing 3, and Fig. 3 A shows that C17:0 methyl ester standard substance, Fig. 3 B demonstration contain the transgenic yeast of pYES2.0 empty carrier and the gas chromatogram that Fig. 3 C shows the transgenic yeast that contains recombinant plasmid pYPPd9.Compare by retention time and to identify each peak with known fatty acid methyl ester standard substance.Retention time is that the peak of 11.12min correspondence is the pichia spp Δ among Fig. 3 C ⁹The C17:1 that-fatty acid dehydrogenase catalysis C17:0 produces.

Sequence table

SEQUENCE?LISTING

＜110〉Nankai University

＜120〉nucleotide sequence and the application thereof of pichia spp Δ 9-fatty acid dehydrogenase

<130> 2007.1.15

<160> 2

<170> PatentIn?version?3.1

<210> 1

<211> 1194

<212> DNA

＜213〉pichia spp (Pichia pastoris)

<400> 1

atgatcttgg?tttgcggttt?gcccctgttt?ggagctattg?caacgtatta?caaaccacct 60

accaaggaga?ccgttatact?gggagttgtt?ttgtacattc?tcggtggtct?gtcaattaca 120

gctggttacc?acagactgtg?gtctcataga?gcctattcgg?ctagaactcc?gttaaaggtt 180

ttatttgccc?tctttggagc?cggagctgtg?gagggttcca?tcaaatggtg?gtctcactct 240

cacagagttc?atcacagata?cacggacact?catagggccc?catatgatgc?cagaaaggga 300

ttctggtact?cccacatggg?ttggatgtta?accaagccaa?acccaagata?caaggcaaga 360

gccgatatct?cagacctcgc?tgatgactgg?attgtcagat?ttcaacacag?acactacctg 420

ttaattatga?ctttcatggc?ccttattttg?cccactatta?ttgccaagta?tttctgggac 480

gacgcatggg?gtggatttat?ctacggtggt?atcttcgaag?ttttcgtcat?tcaacaagcc 540

actttctgtg?ttaactctct?tgctcactgg?attggtgttc?aaccgttcga?tgacaggagg 600

acaccaagag?atcacttttt?gactgctatc?gtcaccttcg?gtgagggata?ccacaacttc 660

caccacgagt?tcccatcaga?ttacagaaac?gctctgaagt?ggtaccaata?cgatccaaca 720

aagattgtca?tttacttatc?atctaaggtt?ggattgtctt?acaatttgaa?gactttttct 780

gacaatgcca?tcaaacaggg?tttggtgcaa?caacagcaga?agaagctgga?ctccatgagg 840

gctcatctta?aetggggtac?tccattacaa?gacttgccag?tctgggataa?gtctgagttc 900

ttcgagaagt?Ctaaagataa?gaaaggtttg?gtcattattc?ctggtattgt?teataacgtt 960

gagaatttca?ttggtgaaca?tccaggtgga?gagaaacttg?tcaagggtgc?cctgggeaag 1020

gatgctacta?ctgctttcaa?tggaggtgtt?tatgcgcact?ccaatgcgge?ccacaactta 1080

ttagccacta?tgagagtggc?tgtcataagg?gatggtaacg?ccaatgcaaa?cactttctcc 1140

ttgcagaacg?agtttttgga?caagaaagcc?gccgctgcag?ctgcttcaaa?ttaa 1194

<210> 2

<211> 397

<212> PRT

＜213〉pichia spp (Pichia pastoris)

<400> 2

Met?Ile?Leu?Val?Cys?Gly?Leu?Pro?Leu?Phe?Gly?Ala?Ile?Ala?Thr?Tyr

1 5 10 15

Tyr?Lys?Pro?Pro?Thr?Lys?Glu?Thr?Val?Ile?Leu?Gly?Val?Val?Leu?Tyr

20 25 30

Ile?Leu?Gly?Gly?Leu?Ser?Ile?Thr?Ala?Gly?Tyr?His?Arg?Leu?Trp?Ser

35 40 45

His?Arg?Ala?Tyr?Ser?Ala?Arg?Thr?Pro?Leu?Lys?Val?Leu?Phe?Ala?Leu

50 55 60

Phe?Gly?Ala?Gly?Ala?Val?Glu?Gly?Ser?Ile?Lys?Trp?Trp?Ser?His?Ser

65 70 75 80

His?Arg?Val?His?His?Arg?Tyr?Thr?Asp?Thr?His?Arg?Ala?Pro?Tyr?Asp

85 90 95

Ala?Arg?Lys?Gly?Phe?Trp?Tyr?Ser?His?Met?Gly?Trp?Met?Leu?Thr?Lys

100 105 110

Pro?Asn?Pro?Arg?Tyr?Lys?Ala?Arg?Ala?Asp?Ile?Ser?Asp?Leu?Ala?Asp

115 120 125

Asp?Trp?Ile?Val?Arg?Phe?Gln?His?Arg?His?Tyr?Leu?Leu?Ile?Met?Thr

130 135 140

Phe?Met?Ala?Leu?Ile?Leu?Pro?Thr?Ile?Ile?Ala?Lys?Tyr?Phe?Trp?Asp

145 150 155 160

Asp?Ala?Trp?Gly?Gly?Phe?Ile?Tyr?Gly?Gly?Ile?Phe?Glu?Val?Phe?Val

165 170 175

Ile?Gln?Gln?Ala?Thr?Phe?Cys?Val?Asn?Ser?Leu?Ala?His?Trp?Ile?Gly

180 185 190

Val?Gln?Pro?Phe?Asp?Asp?Arg?Arg?Thr?Pro?Arg?Asp?His?Phe?Leu?Thr

195 200 205

Ala?Ile?Val?Thr?Phe?Gly?Glu?Gly?Tyr?His?Asn?Phe?His?His?Glu?Phe

210 215 220

Pro?Ser?Asp?Tyr?Arg?Asn?Ala?Leu?Lys?Trp?Tyr?Gln?Tyr?Asp?Pro?Thr

225 230 235 240

Lys?IIe?Val?lle?Tyr?Leu?Ser?Ser?Lys?Val?Gly?Leu?Ser?Tyr?Asn?Leu

245 250 255

Lys?Thr?Phe?Ser?Asp?Asn?Ala?Ile?Lys?His?Gly?Leu?Val?Gln?Gln?Gln

260 265 270

Gln?Lys?Lys?Leu?Asp?Ser?Met?Arg?Ala?His?Leu?Asn?Trp?Gly?Thr?Pro

275 280 285

Leu?Gln?Asp?Leu?Pro?Val?Trp?Asp?Lys?Ser?Glu?Phe?Phe?Glu?Lys?Ser

290 295 300

Lys?Asp?Lys?Lys?Gly?Leu?Val?Ile?Ile?Pro?Gly?Ile?Val?His?Asn?Val

305 310 315 320

Glu?Asn?Phe?Ile?Gly?Glu?His?Pro?Gly?Gly?Glu?Lys?Leu?Val?Lys?Gly

325 330 335

Ala?Leu?Gly?Lys?Asp?Ala?Thr?Thr?Ala?Phe?Asn?Gly?Gly?Val?Tyr?Ala

340 345 350

His?Ser?Asn?Ala?Ala?His?Asn?Leu?Leu?Ala?Thr?Met?Arg?Val?Ala?Val

355 360 365

Ile?Arg?Asp?Gly?Asn?Ala?Asn?Ala?Asn?Thr?Phe?Ser?Leu?Gln?Asn?Glu

370 375 380

Phe?Leu?Asp?Lys?Lys?Ala?Ala?Ala?Ala?Ala?Ala?Ser?Asn

385 390 395

Claims (8)

1. separated coding pichia spp Δ ⁹The nucleic acid of-fatty acid dehydrogenase, its nucleotide sequence is selected from:

(a) be encoded to the nucleotide sequence of the aminoacid sequence polypeptide shown in the SEQ ID NO:2;

(b) with (a) in nucleotide sequence complementary nucleotide sequence.

2. nucleic acid as claimed in claim 1 is the nucleotide sequence shown in the SEQ ID NO:1.

3. a peptide species is the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2.

4. recombinant expression vector, it is by the described nucleic acid of claim 1 and plasmid or the constructed recombinant vectors of virus.

5. recombinant expression vector as claimed in claim 4 is characterized in that it is pYPPd9.

6. genetically engineered host cell is selected from:

(a) host cell that transforms or transduce with the described nucleic acid of claim 1;

(b) host cell that transforms or transduce with the described recombinant expression vector of claim 4.

7. host cell as claimed in claim 6, it is bacterial cell or fungal cell or vegetable cell or zooblast, or the offspring of these host cells.

8. host cell as claimed in claim 6 is characterized in that it is a brewing yeast cell.

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