Functional analyses of cytochrome P450 genes responsible for the early steps of brassicicene C biosynthesis

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 19, 2009, Pages 5640-5643

  Hashimoto, Makoto ^a   Higuchi, Yusuke ^b   Takahashi, Shunji ^c   Osada, Hiroyuki ^c   Sakaki, Toshiyuki ^a   Toyomasu, Tomonobu ^d   Sassa, Takeshi ^d   Kato, Nobuo ^b   Dairi, Tohru ^a  

^a TOYAMA PREFECTURAL UNIVERSITY   (Japan)

^b OSAKA UNIVERSITY   (Japan)

^c RIKEN   (Japan)

^d YAMAGATA UNIVERSITY   (Japan)

Author keywords

Biosynthesis; Brassicicene C; Cytochrome P450; Diterpene

Indexed keywords

CYTOCHROME P450; FUSICOCCADIENE; PLANT GLYCOSIDE; UNCLASSIFIED DRUG;

ALTERNARIA; ARTICLE; BIOSYNTHESIS; FUNGAL STRAIN; GENE IDENTIFICATION; GENETIC ANALYSIS; HYDROXYLATION; MOLECULAR CLONING; NONHUMAN; SACCHAROMYCES CEREVISIAE;

ALTERNARIA; CYTOCHROME P-450 ENZYME SYSTEM; DITERPENES; HYDROXYLATION; MULTIGENE FAMILY; RECOMBINANT PROTEINS;

ALTERNARIA BRASSICICOLA; SACCHAROMYCES CEREVISIAE;

EID: 70049096072     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2009.08.026     Document Type: Article

References (32)

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19. The cDNA sequences of orf1, orf2, orf5, orf7, orf11, and AbP450Red from A. brassicicola ATCC 96836 was deposited at GenBank under Accession No. AB506078, AB506079, AB506080, AB506081, AB506082, and AB506083, respectively.
20. 18 The following primers were used: AbP450Red, 5′-ACGCGTCGACATGGCACAACTCGATACCTTGGACA-3′ and 5′-CGGGGTACCTCATGACCAGACGTCCTCTTGGTAT-3′; orf1, 5′-ATAAGAAT GCGGCCGCATGGAGATGGCTACAACCTTTACA-3′ and 5′-CGCGGATCCAGATCTACTCCTTGTTTTTTCTAACGATT-3′; orf2, 5′-ATAAGAATGCGGCCGCATGGAATCTGCTCAAATACATTCCA-3′ and 5′-CGCGGATCCAGATCTAGGCGGTCTCCTTCGCTACCTTTGT-3′; orf5, 5′-ATAAGAATGCGGCCGCATGCTCGAAGGCACGCTTCAGGACT-3′ and 5′-CCGGAATTCAGATCTACTCATACAGTGCCTCAATTCGGA-3′; orf7, 5′-ATAAGAATGCGGCCGCATGGCTTCCATACTATGGACAACT-3′ and 5′-CCGGAATTCAGATCTATTTCGTTCTCGGAGCGAAACGCA-3′; orf11, 5′-ATAAGAATGCGGCCGCATGATTTTTCTTGCTCCCTTCGAAT-3′ and 5′-CGCGGATCCAGATCTTAGGTGCTCGATATAGGAGAATA-3′. The orf1, orf5, and orf7 genes were cloned in the NotI-BglII site of pESC-URA to construct pESC-URA-orf1, pESC-URA-orf5 and pESC-URA-orf7, respectively. The AbP450Red gene was then inserted into the SalI-KpnI sites of these plasmids to yield pESC-URA-orf1 + AbP450Red, pESC-URA-orf5 + AbP450Red and pESC-URA-orf7 + AbP450Red, respectively. To construct pESC-URA-orf2 + AbP450Red and pESC-URA-orf11 + AbP450Red, the AbP450Red gene was first inserted into pESC-URA, followed by insertion of the orf2 and orf11 genes into the NotI-BglII sites.
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25. Procedures used for maintenance and transformation of S. cerevisiae were essentially the same as those described in the manufacturer′s protocols. SGI medium consisted of 0.1% Bacto casamino acids, 0.7% yeast nitrogen base without amino acids, and 2% glucose. Transformed yeast cells were inoculated into the liquid SGI medium and cultured at 28 °C for 30 h on a reciprocal shaker (120 strokes/min). Then, 2 mL of culture was inoculated in 100 mL of the same medium and was cultivated for 48 h at the same condition. After the check of glucose assumption by urinalysis reagent strip, 2% galactose was fed to the culture and cultivation was continued for 60-108 h. The culture broth was extracted with equal volume of acetone and 1.5 volume of pentane by stirring for 1.5 h at room temperature. The pentane extract was then concentrated in vacuo and subjected to GC-MS and LC-MS analysis.
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27. 26 as a template and the following primers; 5′-CGGGATCCGCTAGCCGTGGAATATTTCGGATATCCTT-3′ and 5′-CCGCTCGAGCTAGCACTAGTGGACGGATTACAACAGGTATTGT-3′. The amplified products were cloned into the NheI-SpeI sites of pESC-TRP to construct pESC-TRP-ADH. Then, the orf8 fragment was inserted into the EcoRI-SalI sites of pESC-TRP-ADH to yield pESC-TRP-orf8-ADH.
28. The orf1 gene with the ADH1 promoter and terminator was cloned into the NheI-SpeI sites of pESC-URA to construct pESC-URA-orf1-ADH. Then, the AbP450Red gene with ADH1 promoter and terminator was inserted into the NheI site of pESC-URA-orf1-ADH to yield pESC-URA-orf1-ADH + AbP450Red-ADH.
29. The orf2, orf5, orf7, and orf11genes were cloned into the NotI-BglII sites of pESC-LEU to construct pESC-LEU-orf2, pESC-LEU-orf5, pESC-LEU-orf7, and pESC-LEU-orf11, respectively.
30. The pentane extracts were analyzed under the following conditions: column; Mightysil RP-18 (250 × 4.6 mm, Merck), column temperature; 40 °C, detection at 205 nm, flow rate; 1.0 mL/min, 40% acetonitrile for 0-5 min, a linear gradient from 40% to 100% for an additional 40 min.
31. The pentane extracts were fractionated with preparative HPLC under the following conditions: column; Mightysil RP-18 (250 × 20 mm, Merck), column temperature; 40 °C, detection at 205 nm, flow rate; 5.0 mL/min, 40% acetonitrile for 0-5 min, a linear gradient from 40% to 100% for an additional 40 min. The fractionated materials (2.7 mg) were further purified with the same column by an isocratic elution of 40% acetonitrile to obtain the purified FD 8β,16-diol.
32. + 327.2300, found 327.2278.