This proposal requests continuation of our study of the biochemistry and genetics of anthracycline antibiotic biosynthesis for a five year period. During this time we will (i) isolate and study the properties of the socalled """""""" aromatic polyketide synthases"""""""" and their structural genes, (ii) define the genetic and biochemical organization of anthracycline antibiotic biosynthetic pathways with special emphasis on their regulation at DNA, RNA and protein levels, and (iii) explore the possibilities for expressing Streptomyces genes in other actinomycetes and creating new anthracycline antibiotics by genetic engineering. The major project during this period will be a study of tetracenomycin C, and antitumor antibiotic produced by Streptomyces glaucescens. We will complete the cloning of the tcm genes then dissect the tcm gene cluster by standard techniques individual tcm genes. Their structures will be studied by nucleotide sequencing, S1 mapping, and other methods to locate the regulatory regions and transcriptional/translational start/stop sites. This information and the results of Northern hybridizations will be used to identify the transcriptional units within the tcm gene cluster. The enzymes catalyzing the formation of the aromatic polyketide intermediates of TcmC biosythesis will be overproduced by inserting their genes in suitable expression vectors to facilitate the study of their physical and biochemical properties. In this way we will establish information about the regulation of TcmC biosynthesis at the DNA, RNA and protein levels. Subsidiary projects will involve cloning the genes for the production of elloramycin, an analog of TcmC; using the tcm genes as probes for genes governing related biochemical pathways in Streptomyces; expressing the tcm genes in other actinomycetes; and exploring the possibilities for the construction of hybrid anthracyclines by genetic engineering.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA035381-05
Application #
3172998
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-09-01
Project End
1991-05-31
Budget Start
1987-06-01
Budget End
1988-05-31
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Pharmacy
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Huang, Yong; Wendt-Pienkowski, Evelyn; Shen, Ben (2006) A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus. J Biol Chem 281:29660-8
Wendt-Pienkowski, Evelyn; Huang, Yong; Zhang, Jian et al. (2005) Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus. J Am Chem Soc 127:16442-52
Wohlert, S E; Wendt-Pienkowski, E; Bao, W et al. (2001) Production of aromatic minimal polyketides by the daunorubicin polyketide synthase genes reveals the incompatibility of the heterologous DpsY and JadI cyclases. J Nat Prod 64:1077-80
Bao, W; Sheldon, P J; Wendt-Pienkowski, E et al. (1999) The Streptomyces peucetius dpsC gene determines the choice of starter unit in biosynthesis of the daunorubicin polyketide. J Bacteriol 181:4690-5
Bao, W; Sheldon, P J; Hutchinson, C R (1999) Purification and properties of the Streptomyces peucetius DpsC beta-ketoacyl:acyl carrier protein synthase III that specifies the propionate-starter unit for type II polyketide biosynthesis. Biochemistry 38:9752-7
Kendrew, S G; Katayama, K; Deutsch, E et al. (1999) DnrD cyclase involved in the biosynthesis of doxorubicin: purification and characterization of the recombinant enzyme. Biochemistry 38:4794-9
Bao, W; Wendt-Pienkowski, E; Hutchinson, C R (1998) Reconstitution of the iterative type II polyketide synthase for tetracenomycin F2 biosynthesis. Biochemistry 37:8132-8
Seow, K T; Meurer, G; Gerlitz, M et al. (1997) A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms. J Bacteriol 179:7360-8
Shen, B; Hutchinson, C R (1996) Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase. Proc Natl Acad Sci U S A 93:6600-4
Decker, H; Rohr, J; Motamedi, H et al. (1995) Identification of Streptomyces olivaceus Tu 2353 genes involved in the production of the polyketide elloramycin. Gene 166:121-6

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