Heterologous production of epothilone C and D in Escherichia coli

被引:117
作者
Mutka, SC [1 ]
Carney, JR [1 ]
Liu, YQ [1 ]
Kennedy, J [1 ]
机构
[1] Kosan Biosci Inc, Hayward, CA 94545 USA
关键词
D O I
10.1021/bi052075r
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster, epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.
引用
收藏
页码:1321 / 1330
页数:10
相关论文
共 34 条
[1]   The complete genome sequence of Escherichia coli K-12 [J].
Blattner, FR ;
Plunkett, G ;
Bloch, CA ;
Perna, NT ;
Burland, V ;
Riley, M ;
ColladoVides, J ;
Glasner, JD ;
Rode, CK ;
Mayhew, GF ;
Gregor, J ;
Davis, NW ;
Kirkpatrick, HA ;
Goeden, MA ;
Rose, DJ ;
Mau, B ;
Shao, Y .
SCIENCE, 1997, 277 (5331) :1453-+
[2]   Precursor-directed biosynthesis of epothilone in Escherichia coli [J].
Boddy, CN ;
Hotta, K ;
Tse, ML ;
Watts, RE ;
Khosla, C .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2004, 126 (24) :7436-7437
[3]  
BOLLAG DM, 1995, CANCER RES, V55, P2325
[4]   Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit [J].
Chen, HW ;
O'Connor, S ;
Cane, DE ;
Walsh, CT .
CHEMISTRY & BIOLOGY, 2001, 8 (09) :899-912
[5]   The biosynthesis of the aromatic myxobacterial electron transport inhibitor stigmatellin is directed by a novel type of modular polyketide synthase [J].
Gaitatzis, N ;
Silakowski, B ;
Kunze, B ;
Nordsiek, G ;
Blöcker, H ;
Höfle, G ;
Müller, R .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (15) :13082-13090
[6]   Epothilons A and B: Antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria) - Production, physico-chemical and biological properties [J].
Gerth, K ;
Bedorf, N ;
Hofle, G ;
Irschik, H ;
Reichenbach, H .
JOURNAL OF ANTIBIOTICS, 1996, 49 (06) :560-563
[7]   ISOLATION OF LACTOSE PERMEASE MUTANTS WHICH RECOGNIZE ARABINOSE [J].
GOSWITZ, VC ;
BROOKER, RJ .
MEMBRANE BIOCHEMISTRY, 1993, 10 (01) :61-70
[8]   NEW METHOD FOR GENERATING DELETIONS AND GENE REPLACEMENTS IN ESCHERICHIA-COLI [J].
HAMILTON, CM ;
ALDEA, M ;
WASHBURN, BK ;
BABITZKE, P ;
KUSHNER, SR .
JOURNAL OF BACTERIOLOGY, 1989, 171 (09) :4617-4622
[9]   DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis [J].
Hoover, DM ;
Lubkowski, J .
NUCLEIC ACIDS RESEARCH, 2002, 30 (10) :e43
[10]   Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase [J].
Jacobsen, JR ;
Hutchinson, CR ;
Cane, DE ;
Khosla, C .
SCIENCE, 1997, 277 (5324) :367-369