As a model of secondary metabolism, I propose to continue a study of the biochemistry and genetics of the production of erythromycin A (erythro'A), a metabolite of Saccharopolyspora erythraea that is classified chemically as a macrolide and has broad spectrum antiinfective activity. Information about the biosynthesis of macrolides, like erythro'A, can be used to increase the production of known drugs and to make new drugs by genetic engineering. This use is an attractive prospect for macrolides because many of them have medically valuable antibiotic activity. Since the principal portion of macrolides is made by the polyketide pathway, which represents hundreds of different structural types, it is likely that numerous biochemical perturbations are possible. This chemical diversity could make the search for new drugs by genetic engineering especially fruitful. Antibiotic production is a characteristic of actinomycetes that has attracted considerable attention. Genetic studies are likely to uncover significant new information because secondary metabolism, a unique characteristic of the slow growth (stationary) phase of laboratory cultures, is quite unlike the primary metabolism of rapidly growing cells that has been the focal point of most studies of prokaryotic genetics. Moreover, the possibility of using the macrolide PKS genes and genes that regulate macrolide antibiotic production to construct strains that overproduce secondary metabolites is very promising; thus, information about the regulatory mechanisms should have wide-spread utility in biotechnology. Our goals for the next four years, listed in the priority in which they will be pursued, are as follows. 1) Complete characterization of the ery structural and regulatory genes. This goal will be reached through: (a) Cloning and analysis of other genes required for the action of 6- deoxyerythronolide B and erythromycin D hydroxylases. (b) Cloning and analysis of the TDP-4-keto-6-deoxy-D-glucose epimerase and reductase genes involved in TDP-L-mycarose formation. (c) Characterization of other ery genes in the region upstream of eryA1 by sequence analysis and gene disruption and replacement expts, in collaboration with researchers at Abbott Laboratories. (d) Identification of the genes controlled by eryC1 or other regulatory genes found in the ery cluster and exploration of the molecular mechanisms involved. 2. Investigation of metabolic sources of propionate, the main erythronolide building block. This goal will be reached through: (a) Cloning and analysis of valine dehydrogenase (vdh) and threonine dehydratase (tdt) genes. (b) Analysis of the regulation of vdh and tdt expression in relation to their role in amino acid utilization. (c) Determination of the effect of inactivation of the vdh and tdt genes on erythromycin production.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Wisconsin Madison
Schools of Pharmacy
United States
Zip Code
Wohlert, S; Lomovskaya, N; Kulowski, K et al. (2001) Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans. Chem Biol 8:681-700
Zhang, Y X; Denoya, C D; Skinner, D D et al. (1999) Genes encoding acyl-CoA dehydrogenase (AcdH) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched-chain fatty acids and macrolide antibiotic production. Microbiology 145 ( Pt 9):2323-34
Olano, C; Lomovskaya, N; Fonstein, L et al. (1999) A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of epsilon-rhodomycinone to rhodomycin D. Chem Biol 6:845-55
Madduri, K; Kennedy, J; Rivola, G et al. (1998) Production of the antitumor drug epirubicin (4'-epidoxorubicin) and its precursor by a genetically engineered strain of Streptomyces peucetius. Nat Biotechnol 16:69-74
Jacobsen, J R; Hutchinson, C R; Cane, D E et al. (1997) Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science 277:367-9
Zhang, Y X; Tang, L; Hutchinson, C R (1996) Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor. J Bacteriol 178:490-5
Gallo, M A; Ward, J; Hutchinson, C R (1996) The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshift mutation that is suppressed by another locus outside of the daunorubicin-production gene cluster. Microbiology 142 ( Pt 2):269-75
Rodriguez, A M; Olano, C; Mendez, C et al. (1995) A cytochrome P450-like gene possibly involved in oleandomycin biosynthesis by Streptomyces antibioticus. FEMS Microbiol Lett 127:117-20
Zotchev, S B; Schrempf, H; Hutchinson, C R (1995) Identification of a methyl-specific restriction system mediated by a conjugative element from Streptomyces bambergiensis. J Bacteriol 177:4809-12
Tang, L; Zhang, Y X; Hutchinson, C R (1994) The genetic basis of precursor supply for the biosynthesis of macrolide and polyether antibiotics. Ann N Y Acad Sci 721:105-16

Showing the most recent 10 out of 25 publications