Cloning and heterologous expression of the epothilone gene cluster

Science

Volumn 287, Issue 5453, 2000, Pages 640-642

  Tang, Li ^a   Shah, Sanjay ^a   Chung, Loleta ^a   Carney, John ^a   Katz, Leonard ^a   Khosla, Chaitan ^a   Julien, Bryan ^a  

^a KOSAN BIOSCIENCES INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EPOTHILONE A; EPOTHILONE B; POLYKETIDE; SYNTHETASE;

ARTICLE; BIOSYNTHESIS; DNA SEQUENCE; GENE CLUSTER; GENE EXPRESSION SYSTEM; GENE STRUCTURE; MOLECULAR CLONING; MYXOCOCCALES; NONHUMAN; PRIORITY JOURNAL; PROTEIN STRUCTURE; STREPTOMYCES COELICOLOR;

BACTERIAL PROTEINS; CLONING, MOLECULAR; COSMIDS; CYTOCHROME P-450 ENZYME SYSTEM; EPOTHILONES; EPOXY COMPOUNDS; FERMENTATION; GENES, BACTERIAL; MOLECULAR SEQUENCE DATA; MULTIENZYME COMPLEXES; MULTIGENE FAMILY; MYXOCOCCALES; OPERON; OXIDOREDUCTASES; RECOMBINANT PROTEINS; STREPTOMYCES; THIAZOLES; TRANSFORMATION, GENETIC;

MYXOCOCCALES; POLYANGIUM CELLULOSUM; PROKARYOTA; SORANGIUM; STREPTOMYCES COELICOLOR; UNCULTURED ACTINOMYCETE;

EID: 0034723333     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5453.640     Document Type: Article

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21. Coexpression of the three DEBS genes from multi-plasmids produced active PKS in Streptomyces [Q. Xue, G. Ashley, C. R. Hutchinson, D. V. Santi, Proc. Natl. Acad. Sci. U.S.A. 96, 11740 (1999)].
22. Plasmid pKOSO39-124R was constructed from an intermediate plasmid pKOS039-124 that contained an eryKS5 linker sequence at the 5′ end of the epoA gene-coding sequence. The eryKS5 linker coding sequences were cloned as an ∼0.4-kb Pac I-Bgl II restriction fragment from plasmid pKOS10-153 into pKOS039-98 to construct plasmid pKOS039-117. The coding sequences for the eryKS5 linker were linked to those for the epothilone loading domain by inserting the ∼8.7-kb Eco RI-Xba I restriction fragment from cosmid pKOS35-70.1A2 into Eco RI-Xba I-digested plasmid pLitmus28. The ∼3.4-kb Bsa BI-Not I and ∼3.7-kb Not I-Hind III restriction fragments from the resulting plasmid were inserted into Bsa BI-Hind III-digested plasmid pKOS039-117 to construct plasmid pKOS039-120. The ∼7-kb Pac I-Xba I restriction fragment of plasmid pKOS039-120 was inserted into plasmid pKAO18′ to construct plasmid pKOS039-123. Plasmid pKOS039-124 vector was constructed by ligating the ∼34-kb Xba I-Avr II restriction fragment of cosmid pKOS35-70.1A2 with the ∼21.1-kb Avr II-Xba I restriction fragment of pKOS039-123. The eryKS5 linker sequences were then replaced by epoA gene-coding sequences. To amplify by PCR coding sequences from the epoA gene-coding sequence, two oligonucleotides primers were used: N39-73, 5′-GCTTAATTAAGGAGGACACATATGCCCGTCGTGGCGGATCGTCC-3′; and N39-74, 5′-GCGGATCCTCGAATCACCGCCAATATC-3′. The template DNA was derived from cosmid pKOS35-70.8A3. The ∼0.8-kb PCR product was digested with restriction enzymes Pac I and Bam HI and then ligated with the ∼2.4-kb Bam HI-Not I and the ∼6.4-kb Pac I-Not I restriction fragments of plasmid pKOS039-120 to construct plasmid pKOS039-136. Then, the ∼5 kb Pac I-Avr II restriction fragment of plasmid pKOS039-136 was ligated with the ∼50 kb Pac I-Avr II restriction fragment of plasmid pKOS039-124 to construct the expression plasmid pKOS039-124R.
23. Plasmid pKOS039-126R was constructed from intermediate plasmid pKOS039-126 that contained an eryKS5 linker sequence at the 5′ end of the epoE gene-coding sequence. The coding sequences for module 7 were linked from cosmids pKOS35-70.4 and pKOS35-79.85 by cloning the ∼6.9-kb Bgl II-Not I restriction fragment of pKOS35-70.4 and the ∼5.9-kb Not I-Hind III restriction fragment of pKOS35-79.85 into Bgl II-Hind III-digested plasmid pLitmus28 to construct plasmid pKOS039-119. The ∼12-kb Nde I-Nhe I restriction fragment of cosmid pKOS35-79.85 was cloned into Nde I-Xba I-digested plasmid pKOS039-119 to construct plasmid pKOS039-122. To fuse the eryKS5 linker coding sequences with the coding sequences for module 7, the ∼1-kb Bsa BI-Bgl II restriction fragment derived from cosmid pKOS35-70.4 was cloned into Bsa BI-Bcl I-digested plasmid pKOS039-117 to construct plasmid pKOS039-121. The ∼21.5-kb Avr II restriction fragment from plasmid pKOS039-122 was cloned into Avr II-Xba I-digested plasmid pKOS039-121 to construct plasmid pKOS039-125. The ∼21.8-kb Pac I-Eco RI restriction fragment of plasmid pKOS039-125 was ligated with the ∼9-kb Pac I-Eco RI restriction fragment of plasmid pKOS039-44 to construct pKOS039-126. The eryKS5 sequences were then replaced by epoE sequences as follows. To amplify by PCR sequences from the epoE gene-coding sequence, two oligonucleotide primers were used: N39-67A, 5′-GCTTAATTAAGGAGGACACATATGACCGACCGAGAAGGCCAGCTC-CTGGA-3′; and N39-68, 5′-GGACCTAGGCGGGATGCCGGCGTCT-3′. The template DNA was derived from cosmid pKOS35-70.1A2. The ∼0.4-kb amplification product was digested with restriction enzymes Pac I and Avr II and ligated with the ∼29.5-kb Pac I-Avr II restriction fragment of plasmid pKOS039-126 to construct plasmid pKOS039-126R.
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25. 2O), 318.3, and 306.4. Epothilone B eluted at 10.5 min and displayed a molecular ion at m/z 508.4 (M+H). Authentic samples of epothilone A and B derived from S. cellulosum SMP44 were used as reference compounds.
26. L. Tang et al., unpublished results.
27. 6 were constructed by PCR. Soluble EpoK was obtained in BL21 (DE3) harboring pREP4-groELS encoding GroEL and GroES [P. Caspers, M. Stieger, P. Burn, Cell Mol. Biol. 40, 635 (1994)]. Purification was performed on a Ni-nitrilotriacetic acid His-Bind resin as recommended (Novagen).
28. max = 250 nm), evaporative light scattering detector and MS detection. Epothilone D eluted at 11.6 min and epothilone B at 9.3 min. The liquid chromatography-MS spectra were obtained by using an atmospheric pressure chemical ionization source with orifice and ring voltages set at 20 and 250 V, respectively, at a mass resolution of 1 amu. Under these conditions, epothilone D shows an [M+H] at m/z 493, with observed fragments at 405 and 304; epothilone B shows an [M+H] at m/z 509, with observed fragments at 491 and 320.
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31. We are grateful to S. Danishefsky for providing desoxyepothilone B used in the EpoK assay. We thank M. Betlach and G. Ashley for suggestions, H. Tsuruta and R. Goldman for technical assistance, and T. Omba for encouragement. Partially funded by Small Business Innovative Research grant 1 R43 CA79228-01. The epothilone gene cluster sequence has been deposited in GenBank with the accession number AF217189.