CN1978651B - Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use - Google Patents
Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use Download PDFInfo
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- CN1978651B CN1978651B CN2005101261584A CN200510126158A CN1978651B CN 1978651 B CN1978651 B CN 1978651B CN 2005101261584 A CN2005101261584 A CN 2005101261584A CN 200510126158 A CN200510126158 A CN 200510126158A CN 1978651 B CN1978651 B CN 1978651B
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Abstract
This invention involves the new encoding hydroxylase gene (pahB) from Penicillium chrysogenum, the gene encoding the peptide and containing the gene expression vector. It also demonstrated that the phenyl acetic acid can significantly induce the expression of pahB. The invention also provides a direction and target for increasing the production of penicillin from genitic modified Penicillium Chrysogenum, and is of great importance to the production of penicillin.
Description
Technical field
The present invention relates to new isolating polynucleotide, relate in particular to gene (pahB) from the new coding toluylic acid hydroxylase of Penicllium chrysogenum.
The invention still further relates to the polypeptide of this genes encoding.
The invention still further relates to and contain this expression carrier.
The present invention also provides the application of new gene in the penicillin yield that improves penicillium chrysogenum.
Background technology
With penicillin and cynnematin be the β-Nei Xiananleikangshengsu of representative as most important antibacterials, occupied 2/3 of anti-infectives market, the world, be that present known poisoning by antibiotic is minimum, kind is maximum, the class microbiotic that clinical application is the widest.
Penicillin is produced by filamentous fungus Penicllium chrysogenum (Penicillium chrysogenum), because the large market demand, people have dropped into huge energy to the improvement of penicillin production bacterium.Based on traditional mutagenesis screening technology, the production level of present industrial producing strain has reached quite high level.And the biosynthesizing mechanism that we disclose β-Nei Xiananleikangshengsu that develops into of Protocols in Molecular Biology provides help.
Studies show that; the biosynthesizing step of penicillin is by three precursor amino acid; α-An Jijiersuan, halfcystine, Xie Ansuan begin; become the LLD-ACV tripeptides through three peptide synthetases (ACVS) catalyzing and condensing; cyclisation forms isopenicillin N under the effect of isopenicillin N synthetic enzyme, and finally at acetyl-CoA: the side chain exchange obtains penicillin under 6-amino-penicillanic acid acyltransferase (AAT) catalysis.The encoding gene of three key enzymes that biosynthesizing is relevant is all cloned (Ingolia ﹠amp; Queener, Med.Res.Rev.9:245-264,1989; Aharonowitz, et al., Ann.Rev.Microbiol.46:461-495,1992).
In the industrial production of penicillin, (Phenylacetic acid PA) is used as the side chain precursor of penicillin G to toluylic acid.During the fermentation, toluylic acid at first is activated and is the coenzyme A thioester, again under the AAT effect through transacylation, substitute the L-alpha-amino group hexanedioyl side chain of isopenicillin N, form penicillin G.Because AAT has the substrate scope of broad, therefore adding toluylic acid during the fermentation can promote penicillin G.Because AAT has the substrate scope of broad, therefore add the biosynthesizing that toluylic acid can promote penicillin G during the fermentation.As the side chain precursor of penicillin G, its utilising efficiency depend on to a great extent it toxicity and Penicillium notatum to its oxidation capacity.Toluylic acid occupies suitable proportion in production cost, the utilising efficiency that improves toluylic acid is a most important content in the penicillin strain improved, process.
At Pseudomonas fluorescens (Kunita, N., Med.J.Osaka Univ.3:703-708,1955), Aspergillus niger (Kluyver, A.J.﹠amp; J.C.M.van Zijp., Antonie vanLeeuwenhoek17:47-55,1951), Nocardia salmonicolor (Sariaslani, F.S.et al.Biochem.J.140:31-45,1974), and Aspergillus nidulans (Mingot, J.M.et al.J.Biol.Chem.274,14,545 14550,1999), Penicillium chrysogenum (Wisconsin Q-176 (Isono, M.J., Agric.Chem.Jpn.27:297-301,1953; Nishikida, T., J.Antibiot.4:299-300,1951) etc. in the microorganism, the metabolism of toluylic acid is that 2 hydroxylations form 2-OHPA, form homogentisic acid (homogentisic acid) then successively, maleoyl etheric acid (maleylacetoacetic acid) fumarylacetoacetic acid (fumarylacetoacetic acid) is hydrolyzed into fumaric acid at last and etheric acid enters tricarboxylic acid cycle.
The Aspergillus nidulans (Aspergillus nidulans) that produces one of bacterium as penicillin can be grown on toluylic acid is the substratum of sole carbon source, 2 hydroxylations of the metabolic the first step of its toluylic acid are by the catalysis of a P450 monooxygenase, this enzyme is by the phacA genes encoding, the inactivation of this gene has caused penicillin yield to improve 5 times, make the metabolic capacity of toluylic acid partly lose (Mingot simultaneously, J.M.et al.J.Biol.Chem.274,14,545 14550,1999).In Penicllium chrysogenum, corresponding P450 monooxygenase is by the pahA genes encoding.The consistence of PahA and PhacA aminoacid sequence is 84%.Studies show that the pahA expression of gene is induced by toluylic acid, its expression level then becomes negative correlation with the penicillin production ability of bacterial classification.Traditional mutagenesis and separation screening cause the toluylic acid oxidation capacity of this enzyme in the high yield Penicllium chrysogenum significantly to descend the raising of penicillin production ability.Sequential analysis find L → F sudden change of 181 generations of superior strain PahA sequence be the reason that causes this enzyme function to reduce (
-S á iz, M. et al.J.Bacteriol.183,54655471,2001).
However, under the industrial fermentation condition, still can detect the existence of a considerable amount of 2-HPAAs (2-OHPA) in the fermented liquid, though this show in industrial strain, still have the side chain precursor of considerable part to be fallen by metabolism.The sudden change of the special oxidation of right and wrong, PahA only part has been lost its oxidation activity, has still been existed other oxidation mechanism still unknown in Penicllium chrysogenum.The oxidation mechanism of further investigation toluylic acid is found new toluylic acid oxidation key gene, can be Penicillium notatum and produce the target spot that the seed selection of bacterial classification provides genetic modification, to improve output, the reduction fermentation costs is significant.
Summary of the invention
The gene of the coding Penicllium chrysogenum toluylic acid hydroxylase that one object of the present invention is to provide new is named and is pahB.
Another object of the present invention is to provide the said gene encoded polypeptides.
A further object of the present invention is to provide the expression vector that contains the said gene sequence.
According to an aspect of the present invention, the gene of coding Penicllium chrysogenum paddy toluylic acid hydroxylase derives from penicillium chrysogenum (Penicillin chrysogenum), names to be pahB.This gene ORF is long to be 1551bp, 517 the amino acid whose albumen of encoding, with PahA (516aa, AAF21759), PhacA (518aa, CAB43093) length is close, the supposition molecular weight is 57.7kDa.The consistence of its aminoacid sequence and PahA, PhacA is respectively 44% and 42%.In its 442-451 position be the conservative halfcystine protoheme-iron part signal sequence-YGMGYRMCAG-of P450 protein family ([FW]-[SGNH]-x-[GD]-F}-[RKHPT]-P}-C-[LIVMFAP]-[GAD], Prosite database entry PS00086), (YGAGSRMCAG-), (YGAGSRMCAG-) the same, phenylalanine residue (F) is replaced by tyrosine (Y) PhacA with PahA.Gene expression research shows that the expression of pahB equally obviously is subjected to inducing of toluylic acid.
Specifically, the invention provides new isolating polynucleotide, it contains one of following sequence:
(1) sequence of the 43rd～1593 Nucleotide of sequence shown in the SEQ ID No:1 in the sequence table or SEQ ID NO:1;
(2) polynucleotide sequence of protein sequence shown in the SEQ ID No:2 in the code sequence tabulation.
The above-mentioned polynucleotide that relate to also comprise replacement, disappearance and insert variant and allelic variant, splice variant, fragment, derivative etc., wherein can be by replacing, lack, insert or one or more Nucleotide of deriving.Preferred these polynucleotide are polynucleotide that those codings have the toluylic acid hydroxylase of biologic activity.
It will be understood by those skilled in the art that, above-mentioned isolating polynucleotide comprise that also the sequence of the 43rd～1593 Nucleotide of sequence shown in those and the SEQID NO.1 or SEQ ID NO:1 has the sequence of higher homology, and for example homology is greater than 90% even 95% even 98% sequence; Also comprise those under rigorous condition can with the sequence of the sequence hybridization of the 43rd～1593 Nucleotide of sequence shown in the SEQ ID NO.1 or SEQ ID NO:1; Perhaps can with above-mentioned sequence complementary sequence.
According to a further aspect in the invention, new polypeptide is provided, it contains the coded aminoacid sequence of above-mentioned nucleotide sequence, promptly contain the sequence shown in the SEQ ID NO.2, proteins encoded is the toluylic acid hydroxylase of Penicllium chrysogenum (Penicillium chrysogenum), its have the protein of the amino acid residue sequence of SEQ ID No:2 in the sequence table or with the amino acid residue sequence of SEQ ID No:2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical with SEQ ID No:2 active by SEQID No:2 deutero-protein.
According to a further aspect in the invention, the present invention also provides the recombinant vectors that comprises one or more above-mentioned polynucleotide, and the genetically engineered host cell that comprises the carrier of above-mentioned polynucleotide.In preferred embodiments, this recombinant vectors comprises above-mentioned polynucleotide, and its coding contains the polypeptide of SEQ ID NO:2, and it contains the polynucleotide shown in the sequence of the 43rd～1593 Nucleotide of the sequence of SEQ ID NO:1 or SEQ ID NO:1.
The present invention also relates to comprise the host cell of any above-mentioned recombinant vectors.After carrier construction, can insert all or part of carrier to the host cell that is fit to, so that amplification and/or expression of polypeptides.Host cell can be prokaryotic organism host cell (for example E.coli) or eukaryote host cell (for example filamentous fungal cells, yeast cell, insect cell, or vertebrate cells).
The present invention also provides the new application of gene on raising Penicllium chrysogenum penicillin yield, because pahB is the key gene of toluylic acid catabolic pathway, therefore can improve the utilising efficiency of the toluylic acid in the penicillin production technology by the expression that reduces pahB, thereby improve the output of penicillin.
The present invention successfully separates the gene that has obtained new coding Penicllium chrysogenum toluylic acid hydroxylase from Penicllium chrysogenum, and proved that toluylic acid can significantly induce this expression of gene, thereby the present invention provides direction and target spot for utilizing genetic engineering means to transform penicillium chrysogenum raising penicillin yield, and is significant for the production of penicillin.
Brief description of drawings
Fig. 1 is the structure iron of pahB gene.
The embodiment of invention
Below in conjunction with accompanying drawing,, describe the present invention in detail by description to preferred embodiment of the present invention.
The reagent material of Shi Yonging is commercially available analytical reagent if no special instructions below, available from Tianjin reagent company.
[embodiment 1]: pahB gene clone
Penicillium chrysogenum Wis54-1255 (ATCC28089) is cultivated 24h in YPD (1% yeast extract, 2% peptone, 2% glucose) substratum, filter and receive mycelia, get the wet mycelia liquid N of 0.1g
2Grind, (Invitrogen company, 15596-026) the total RNA of extracting Penicllium chrysogenum is dissolved in the total RNA of gained Penicllium chrysogenum in the DEPC water, preserves standby down for-70 ℃ to add 1ml Trizol reagent.
Pollute for removing possible DNA, get the total RNA 5 μ g of Penicllium chrysogenum, in 100 μ l reaction systems, add 5 μ l RQ1 RNase-Free DNase (Promega company, M6101), 10 μ l RQ1 RNase-FreeDNase reaction buffers (10 *), 37 ℃ are incubated 30 fens, use phenol: the chloroform extracting, ethanol sedimentation is dissolved in 10 μ l DEPC water.Get the RNA sample that 5 μ l remove DNA, with random primer (RandomHexamers, Promega company C1181) carry out reverse transcription, obtain cDNA, used ThermoScript II be SuperScriptTM II RNase H-(Invitrogen company, 18064-022).
With 5 '-tatatatcaacgtcaagatctatc-3 ' and 5 '-taccccaaatagataccaccaatg-3 ' is primer, is that template is carried out pcr amplification with cDNA, and the PCR condition is: 94 ℃, and 5 minutes; 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 2 minutes-40 the circulation; 72 ℃ 7 minutes; 4 ℃ of preservations.With RT-PCR product cloning and sequencing according to a conventional method, sequencing result shows that the RT-PCR product has the nucleotide sequence of SEQ ID NO.1 in the sequence table, and length is 1629bp.By sequential analysis and GenBank database retrieval, show this sequence open reading frame (Open Reading Frame, ORF) be from 5 ' end the 43rd to the 1593rd bit base, length is 1551bp.15bp place before ORF initiator codon ATG, same-phase has terminator codon TAA, illustrates that thus we have obtained the full length sequence of this gene, names to be pahB.
517 amino acid of the segmental reading frame coding of this gene ORF, the supposition molecular weight is 57.7kDa, shown in SEQ ID NO.2 in the sequence table.This aminoacid sequence with Penicllium chrysogenum toluylic acid hydroxylase PahA (516aa, AAF21759), (518aa, CAB43093) length is close for Aspergillus nidulans toluylic acid hydroxylase PhacA.The consistence of its aminoacid sequence and PahA, PhacA is respectively 44% and 42%.。In its 442-451 position be the conservative halfcystine protoheme-iron part signal sequence-YGMGYRMCAG-of P450 protein family ([FW]-[SGNH]-x-[GD]-F}-[RKHPT]-P}-C-[LIVMFAP]-[GAD], Prosite database entry PS00086), (YGAGSRMCAG-), (YGAGSRMCAG-) the same, phenylalanine residue (F) is replaced by tyrosine (Y) PhacA with PahA.
The genomic dna of while with the Wis54-1255 bacterial strain of extraction is that masterplate carries out pcr amplification, primer and reaction conditions are the same, obtain the amplified production of 1860bp, and itself and RT-PCR product sequence are compared, confirm that the pahB gene has 3 introns, length is respectively 97bp, 67bp, 67bp.
[embodiment 2]: the pahB expression of gene is subjected to inducing of toluylic acid
Penicillium chrysogenum Wis54-1255 (ATCC28089) is pressed 2 * 10
6Conidium/ml is inoculated into seed culture medium, cultivates 24 hours down at 26 ℃, and the seed culture based formulas is (g/l): glucose, 30; Lactose, 10; Cottonseed meal, 10; Yeast powder, 10; Corn steep liquor, 10; (NH
4)
2SO
4, 2; KH
2PO
4, 1; CaCO
3, 5; PH5.8.Then inoculum is inoculated into fermention medium with 5% inoculum size, cultivates down for 26 ℃.Fermention medium is (g/l): lactose, 120; Cottonseed meal, 30; Corn steep liquor, 20; (NH
4)
2SO
4, 5; KH
2PO
4, 1; K
2SO
4, 5; CaCO
3, 10; PH6.5.
Experiment is divided into 2 groups, normally adds the toluylic acid precursor one group of every day, and another group does not add toluylic acid for contrast, collects thalline after 72 hours and extracts total RNA, and extraction and treatment process are seen embodiment 1.
Make real-time quantitative PCR detection pahB expression of gene with SYBR Green reagent, instrument is ABIPRISM 7000 quantitative PCR instrument.According to the cDNA sequence of pahB gene, utilize Primer Express software design quantification PCR primer, AS006:5 '-GCTGAACAGTGAGGAGTTGAC-3 '
AA006：5′-CGCAAGAACCTTCGACAGAG-3′。With Penicllium chrysogenum γ-actin gene (AF056975) is interior mark, act1:5 '-CTCGCTGAGCGTGGTTACAC-3 '
act2：5′-TTGATGTCACGGACGATTTCA-3′。It is synthetic that primer is given birth to the worker by Shanghai.Quantitative PCR reagent is
Premix Ex Tag
TMTest kit (precious biotechnology (Dalian) company limited).Reaction conditions is: 95 ℃, and 1 minute; 95 ℃, 15 seconds, 60 ℃, 1 minute, 40 circulations.C as a result to quantitative PCR
TValue converts, and adopts
Method (Livak K.J.and T.D.SchmittgenMethods 25:402-408,2001).As a result, under the situation of not adding toluylic acid, the expression level of pahB is very low, and toluylic acid can significantly be induced the pahB expression of gene, is 1 with the expression amount that does not add toluylic acid, and the expression of pahB has improved 580 times (following tables) when then adding toluylic acid.
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, as long as do not break away from spirit of the present invention, all should belong to the defined scope of claim of the present invention.
SEQUENCE?LISTING
＜110〉North China Pharmacuetical Group New Drug Research ﹠ Development Co., Ltd
＜120〉gene of coding penicillium chrysogenum phenylacetic acid hydroxylase and application thereof
<130>05P101406
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＜213〉penicillium chrysogenum (Penicillin chrysogenum)
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tatatatcaa?cgtcaagatc?tatcccataa?ccatccagca?cc?atg?gct?atc?gcc 54
Met?Ala?Ile?Ala
1
gct?gcc?ctg?gcg?tcg?cta?caa?ggc?agt?gtg?atc?gag?tat?ccc?tac?cac 102
Ala?Ala?Leu?Ala?Ser?Leu?Gln?Gly?Ser?Val?Ile?Glu?Tyr?Pro?Tyr?His
5 10 15 20
tca?cta?gcc?gcc?gca?gcc?gtc?ctc?gcc?ccc?atc?ctc?tat?gtc?atc?ctt 150
Ser?Leu?Ala?Ala?Ala?Ala?Val?Leu?Ala?Pro?Ile?Leu?Tyr?Val?Ile?Leu
25 30 35
aat?gag?ttc?att?cgt?gat?gcc?gcc?cgc?gtc?aag?ggt?atg?aag?ggt?ccg 198
Asn?Glu?Phe?Ile?Arg?Asp?Ala?Ala?Arg?Val?Lys?Gly?Met?Lys?Gly?Pro
40 45 50
cgc?gga?ttg?ccg?ttg?att?gga?aac?ctg?gcc?cag?att?cgc?aaa?gat?gcc 246
Arg?Gly?Leu?Pro?Leu?Ile?Gly?Asn?Leu?Ala?Gln?Ile?Arg?Lys?Asp?Ala
55 60 65
gcg?gag?cag?tat?cgc?ctc?tgg?tcg?aaa?aag?cac?ggc?gct?gtg?tat?cag 294
Ala?Glu?Gln?Tyr?Arg?Leu?Trp?Ser?Lys?Lys?His?Gly?Ala?Val?Tyr?Gln
70 75 80
att?cag?ctg?ggg?aac?atc?ccc?gtc?gtt?gtg?gtc?aac?tct?gct?gcg?tcg 342
Ile?Gln?Leu?Gly?Asn?Ile?Pro?Val?Val?Val?Val?Asn?Ser?Ala?Ala?Ser
85 90 95 100
gca?aaa?gtg?ctt?ttc?ggt?cag?aat?gcg?cag?gct?ttg?agc?tct?agg?ccc 390
Ala?Lys?Val?Leu?Phe?Gly?Gln?Asn?Ala?Gln?Ala?Leu?Ser?Ser?Arg?Pro
105 110 115
gaa?act?tat?act?ttc?cat?aag?att?gtt?tcc?aat?act?ccc?gga?aca?act 438
Glu?Thr?Tyr?Thr?Phe?His?Lys?Ile?Val?Ser?Asn?Thr?Pro?Gly?Thr?Thr
120 125 130
atc?ggc?acc?tcg?cca?tat?agc?gac?tct?ctg?aag?aga?cgc?aga?aag?ggc 486
Ile?Gly?Thr?Ser?Pro?Tyr?Ser?Asp?Ser?Leu?Lys?Arg?Arg?Arg?Lys?Gly
135 140 145
gcc?gcg?tcg?gct?ctc?aac?cgg?ccc?tct?gtc?gat?acg?tac?gtg?tca?cat 534
Ala?Ala?Ser?Ala?Leu?Asn?Arg?Pro?Ser?Val?Asp?Thr?Tyr?Val?Ser?His
150 155 160
ttg?gac?gtc?gag?tca?aag?gcc?ttc?gtg?gaa?gag?ctg?tac?aga?tat?gga 582
Leu?Asp?Val?Glu?Ser?Lys?Ala?Phe?Val?Glu?Glu?Leu?Tyr?Arg?Tyr?Gly
165 170 175 180
aac?cac?ggg?aag?aca?ccc?gtc?gat?ccc?atg?gcc?atg?atc?cag?cgt?ctg 630
Asn?His?Gly?Lys?Thr?Pro?Val?Asp?Pro?Met?Ala?Met?Ile?Gln?Arg?Leu
185 190 195
agt?ttg?agt?ttg?gca?ttg?act?ctg?aac?tgg?ggc?gtg?cgt?atc?gct?tca 678
Ser?Leu?Ser?Leu?Ala?Leu?Thr?Leu?Asn?Trp?Gly?Val?Arg?Ile?Ala?Ser
200 205 210
cag?gaa?gag?gaa?ttg?ttc?gat?gag?atc?acc?gag?gtg?gag?gaa?gag?atc 726
Gln?Glu?Glu?Glu?Leu?Phe?Asp?Glu?Ile?Thr?Glu?Val?Glu?Glu?Glu?Ile
215 220 225
agt?cgg?ttc?cga?agc?acc?acc?gga?aac?ctg?cag?gat?tat?atc?cct?ctt 774
Ser?Arg?Phe?Arg?Ser?Thr?Thr?Gly?Asn?Leu?Gln?Asp?Tyr?Ile?Pro?Leu
230 235 240
ctg?cgg?ttg?aac?ccg?ttt?agc?tcc?aac?tcg?aag?aaa?gcc?gcc?gag?atg 822
Leu?Arg?Leu?Asn?Pro?Phe?Ser?Ser?Asn?Ser?Lys?Lys?Ala?Ala?Glu?Met
245 250 255 260
aga?agg?cgt?cgg?gac?aag?tac?ctc?ggt?gcg?ctt?aat?agt?gac?ttg?gat 870
Arg?Arg?Arg?Arg?Asp?Lys?Tyr?Leu?Gly?Ala?Leu?Asn?Ser?Asp?Leu?Asp
265 270 275
gat?cgc?atg?gca?aag?ggg?aca?cat?aag?cct?tgt?att?cag?gcc?aac?gtt 918
Asp?Arg?Met?Ala?Lys?Gly?Thr?His?Lys?Pro?Cys?Ile?Gln?Ala?Asn?Val
280 285 290
att?atg?gat?aag?gaa?gct?aag?ctg?aac?agt?gag?gag?ttg?acg?tcg?att 966
Ile?Met?Asp?Lys?Glu?Ala?Lys?Leu?Asn?Ser?Glu?Glu?Leu?Thr?Ser?Ile
295 300 305
agc?ttg?acc?atg?ctt?tct?ggt?ggc?ctt?gac?act?gtc?acc?act?ttg?gtt 1014
Ser?Leu?Thr?Met?Leu?Ser?Gly?Gly?Leu?Asp?Thr?Val?Thr?Thr?Leu?Val
310 315 320
gcg?tgg?agt?ttg?ggt?ttg?ctg?gct?caa?cgt?cct?gat?att?cag?gat?cgg 1062
Ala?Trp?Ser?Leu?Gly?Leu?Leu?Ala?Gln?Arg?Pro?Asp?Ile?Gln?Asp?Arg
325 330 335 340
gcg?gcc?aag?gct?atc?cag?gag?atg?tat?ggc?gag?gat?gat?ccc?atg?tgc 1110
Ala?Ala?Lys?Ala?Ile?Gln?Glu?Met?Tyr?Gly?Glu?Asp?Asp?Pro?Met?Cys
345 350 355
tct?cct?gac?gat?gat?cag?agg?tgt?gcg?tac?att?gca?gct?ctg?gtc?aag 1158
Ser?Pro?Asp?Asp?Asp?Gln?Arg?Cys?Ala?Tyr?Ile?Ala?Ala?Leu?Val?Lys
360 365 370
gaa?tgt?ctt?cga?ttc?ttc?act?gta?ctg?cga?tta?gca?ctt?cct?cgc?act 1206
Glu?Cys?Leu?Arg?Phe?Phe?Thr?Val?Leu?Arg?Leu?Ala?Leu?Pro?Arg?Thr
375 380 385
tca?atc?aaa?gat?atc?acc?tat?aag?gga?atc?acc?att?ccc?aag?ggc?aca 1254
Ser?Ile?Lys?Asp?Ile?Thr?Tyr?Lys?Gly?Ile?Thr?Ile?Pro?Lys?Gly?Thr
390 395 400
gtg?ttc?ttt?ttg?aat?gcc?tgg?gcc?tgc?aac?atg?gac?ccc?gac?gtg?tgg 1302
Val?Phe?Phe?Leu?Asn?Ala?Trp?Ala?Cys?Asn?Met?Asp?Pro?Asp?Val?Trp
405 410 415 420
act?gac?ccg?gat?gac?ttc?cgt?ccc?gag?cga?tgg?ctc?gag?cag?ccc?gac 1350
Thr?Asp?Pro?Asp?Asp?Phe?Arg?Pro?Glu?Arg?Trp?Leu?Glu?Gln?Pro?Asp
425 430 435
gcg?cct?ctc?ttt?acg?tat?ggt?atg?ggt?tac?cgg?atg?tgc?gcc?ggg?tea 1398
Ala?Pro?Leu?Phe?Thr?Tyr?Gly?Met?Gly?Tyr?Arg?Met?Cys?Ala?Gly?Ser
440 445 450
ctg?cta?gcc?aat?cgt?gag?ctc?tac?ctg?gtc?ttc?atc?cgc?acc?ctc?aac 1446
Leu?Leu?Ala?Asn?Arg?Glu?Leu?Tyr?Leu?Val?Phe?Ile?Arg?Thr?Leu?Asn
455 460 465
agc?ttc?cgc?ctc?gaa?cct?cat?gac?aaa?aca?gac?tgt?cac?cct?ctc?cgg 1494
Ser?Phe?Arg?Leu?Glu?Pro?His?Asp?Lys?Thr?Asp?Cys?His?Pro?Leu?Arg
470 475 480
ggt?aac?tcg?gat?ccg?act?agt?ctg?gtg?gcc?att?cct?caa?aaa?tat?aag 1542
Gly?Asn?Ser?Asp?Pro?Thr?Ser?Leu?Val?Ala?Ile?Pro?Gln?Lys?Tyr?Lys
485 490 495 500
gtc?cga?ttt?gtg?ccg?aag?aat?gaa?aag?gct?ctg?tcg?aag?gtt?ctt?gcg 1590
Val?Arg?Phe?Val?Pro?Lys?Asn?Glu?Lys?Ala?Leu?Ser?Lys?Val?Leu?Ala
505 510 515
cag?tgaatggtgc?cacattggtg?gtatctattt?ggggta 1629
Gln
<210>2
<211>517
<212>PRT
＜213〉penicillium chrysogenum (Penicillin chrysogenum)
<400>2
Met?Ala?Ile?Ala?Ala?Ala?Leu?Ala?Ser?Leu?Gln?Gly?Ser?Val?Ile?Glu
1 5 10 15
Tyr?Pro?Tyr?His?Ser?Leu?Ala?Ala?Ala?Ala?Val?Leu?Ala?Pro?Ile?Leu
20 25 30
Tyr?Val?Ile?Leu?Ash?Glu?Phe?Ile?Arg?Asp?Ala?Ala?Arg?Val?Lys?Gly
35 40 45
Met?Lys?Gly?Pro?Arg?Gly?Leu?Pro?Leu?Ile?Gly?Asn?Leu?Ala?Gln?Ile
50 55 60
Arg?Lys?Asp?Ala?Ala?Glu?Gln?Tyr?Arg?Leu?Trp?Ser?Lys?Lys?His?Gly
65 70 75 80
Ala?Val?Tyr?Gln?Ile?Gln?Leu?Gly?Asn?Ile?Pro?Val?Val?Val?Val?Asn
85 90 95
Ser?Ala?Ala?Ser?Ala?Lys?Val?Leu?Phe?Gly?Gln?Asn?Ala?Gln?Ala?Leu
100 105 110
Ser?Ser?Arg?Pro?Glu?Thr?Tyr?Thr?Phe?His?Lys?Ile?Val?Ser?Asn?Thr
115 120 125
Pro?Gly?Thr?Thr?Ile?Gly?Thr?Ser?Pro?Tyr?Ser?Asp?Ser?Leu?Lys?Arg
130 135 140
Arg?Arg?Lys?Gly?Ala?Ala?Ser?Ala?Leu?Asn?Arg?Pro?Ser?Val?Asp?Thr
145 150 155 160
Tyr?Val?Ser?His?Leu?Asp?Val?Glu?Ser?Lys?Ala?Phe?Val?Glu?Glu?Leu
165 170 175
Tyr?Arg?Tyr?Gly?Asn?His?Gly?Lys?Thr?Pro?Val?Asp?Pro?Met?Ala?Met
180 185 190
Ile?Gln?Arg?Leu?Ser?Leu?Ser?Leu?Ala?Leu?Thr?Leu?Asn?Trp?Gly?Val
195 200 205
Arg?Ile?Ala?Ser?Gln?Glu?Glu?Glu?Leu?Phe?Asp?Glu?Ile?Thr?Glu?Val
210 215 220
Glu?Glu?Glu?Ile?Ser?Arg?Phe?Arg?Ser?Thr?Thr?Gly?Asn?Leu?Gln?Asp
225 230 235 240
Tyr?Ile?Pro?Leu?Leu?Arg?Leu?Asn?Pro?Phe?Ser?Ser?Asn?Ser?Lys?Lys
245 250 255
Ala?Ala?Glu?Met?Arg?Arg?Arg?Arg?Asp?Lys?Tyr?Leu?Gly?Ala?Leu?Asn
260 265 270
Ser?Asp?Leu?Asp?Asp?Arg?Met?Ala?Lys?Gly?Thr?His?Lys?Pro?Cys?Ile
275 280 285
Gln?Ala?Asn?Val?Ile?Met?Asp?Lys?Glu?Ala?Lys?Leu?Asn?Ser?Glu?Glu
290 295 300
Leu?Thr?Ser?Ile?Ser?Leu?Thr?Met?Leu?Ser?Gly?Gly?Leu?Asp?Thr?Val
305 310 315 320
Thr?Thr?Leu?Val?Ala?Trp?Ser?Leu?Gly?Leu?Leu?Ala?Gln?Arg?Pro?Asp
325 330 335
Ile?Gln?Asp?Arg?Ala?Ala?Lys?Ala?Ile?Gln?Glu?Met?Tyr?Gly?Glu?Asp
340 345 350
Asp?Pro?Met?Cys?Ser?Pro?Asp?Asp?Asp?Gln?Arg?Cys?Ala?Tyr?Ile?Ala
355 360 365
Ala?Leu?Val Lys?Glu?Cys?Leu?Arg?Phe?Phe?Thr?Val?Leu?Arg?Leu?Ala
370 375 380
Leu?Pro?Arg?Thr?Ser?Ile?Lys?Asp?Ile?Thr?Tyr?Lys?Gly?Ile?Thr?Ile
385 390 395 400
Pro?Lys?Gly?Thr?Val?Phe?Phe?Leu?Asn?Ala?Trp?Ala?Cys?Asn?Met?Asp
405 410 415
Pro?Asp?Val?Trp?Thr?Asp?Pro?Asp?Asp?Phe?Arg?Pro?Glu?Arg?Trp?Leu
420 425 430
Glu?Gln?Pro?Asp?Ala?Pro?Leu?Phe?Thr?Tyr?Gly?Met?Gly?Tyr?Arg?Met
435 440 445
Cys?Ala?Gly?Ser?Leu?Leu?Ala?Asn?Arg?Glu?Leu?Tyr?Leu?Val?Phe?Ile
450 455 460
Arg?Thr?Leu?Asn?Ser?Phe?Arg?Leu?Glu?Pro?His?Asp?Lys?Thr?Asp?Cys
465 470 475 480
His?Pro?Leu?Arg?Gly?Asn?Ser?Asp?Pro?Thr?Ser?Leu?Val?Ala?Ile?Pro
485 490 495
Gln?Lys?Tyr?Lys?Val?Arg?Phe?Val?Pro?Lys?Asn?Glu?Lys?Ala?Leu?Ser
500 505 510
Lys?Val?Leu?Ala?Gln
515
Claims (8)
1. isolating polynucleotide, it is the polynucleotide sequence of the protein sequence shown in the coding SEQ ID No:2.
2. described polynucleotide of claim 1, it is the sequence of the 43rd～1593 Nucleotide of sequence shown in the SEQ ID No:1 or SEQ IDNO:1.
3. isolating polynucleotide, it is and claim 1 or the complete complementary nucleotide sequence of 2 described polynucleotide.
4. by the polypeptide of claim 1 or 2 described polynucleotide encodings.
5. the described polypeptide of claim 4, it has the sequence shown in the SEQ ID NO.2, has the toluylic acid hydroxylase activity.
6. the expression vector that contains claim 1 or 2 described polynucleotide.
7. the host cell that contains the described expression vector of claim 6.
8. claim 1 or the 2 described isolating polynucleotide application in the penicillin yield that improves penicillium chrysogenum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005101261584A CN1978651B (en) | 2005-11-30 | 2005-11-30 | Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005101261584A CN1978651B (en) | 2005-11-30 | 2005-11-30 | Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1978651A CN1978651A (en) | 2007-06-13 |
| CN1978651B true CN1978651B (en) | 2010-11-17 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2005101261584A Expired - Fee Related CN1978651B (en) | 2005-11-30 | 2005-11-30 | Gene for coding penicillium chrysogenum phenylacetic acid hydroxylase and its use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1978651B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299060A (en) * | 2017-06-12 | 2017-10-27 | 华北制药河北华民药业有限责任公司 | A kind of Penicillin high-producing strain and its screening technique and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1226932A (en) * | 1997-04-18 | 1999-08-25 | 抗生素有限公司 | Process for inactivation of genes which code for enzymes for catabolism of phenyl acetate, plasmids involved in such process and strains transformed therewith |
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2005
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1226932A (en) * | 1997-04-18 | 1999-08-25 | 抗生素有限公司 | Process for inactivation of genes which code for enzymes for catabolism of phenyl acetate, plasmids involved in such process and strains transformed therewith |
Non-Patent Citations (4)
| Title |
|---|
| 任志红等.产黄青霉工业生产菌种基因报告系统的构建及启动子效率的评价.菌物学报24 3.2005,24(3),第376-384页. |
| 任志红等.产黄青霉工业生产菌种基因报告系统的构建及启动子效率的评价.菌物学报24 3.2005,24(3),第376-384页. * |
| 王富强等.产黄青霉转化载体pPIPKA的构建及青霉素工业生产菌株的转化研究.菌物学报23 1.2004,23(1),第66-72页. |
| 王富强等.产黄青霉转化载体pPIPKA的构建及青霉素工业生产菌株的转化研究.菌物学报23 1.2004,23(1),第66-72页. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107299060A (en) * | 2017-06-12 | 2017-10-27 | 华北制药河北华民药业有限责任公司 | A kind of Penicillin high-producing strain and its screening technique and application |
| CN107299060B (en) * | 2017-06-12 | 2020-08-11 | 华北制药股份有限公司 | Penicillin high-yield strain and screening method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1978651A (en) | 2007-06-13 |
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