9729528 Kevin A. Reynolds In streptomycetes variations of the shikimic acid pathway provide building blocks for numerous important natural products. Cyclohexanecarboxylic acid (CHC) is derived by a reductive biosynthetic process from shikimic acid and is found in numerous antibiotics such as ansatrienin and asukamycin. CHC is also used in directed biosynthetic fermentations to prepare doramectin (an analog of avermectin), an important antiparasitic veterinary product. Dihydroxycyclohexanecarboxylic acid (DHCHC) is also derived from shikimic acid and is used as a starter unit in the biosynthesis of the immunosuppressants FK506, rapamycin and ascomycin. This project involves an analysis of the CHC, DHCHC, and shikimic acid pathways in Streptomyces collinus and Streptomyces hygroscopicus. The specific objectives are as follow: 1) To continue an analysis of the unique, individual enzymes involved in CHC biosynthesis in S. collinus and to determine if the genes encoding this pathway are clustered. These genes will be used to engineer the complete CHC pathway into a blocked mutant of S. avermitilis, creating a new mutant that can produce doramectin without addition of CHC to the fermentation broth. In the long term coexpression of these genes (or those involved in DHCHC biosynthesis) with hybrid polyketide synthases may provide approaches for production of novel polyketides. 2) To clone and sequence genes encoding dehydroquinate dehydratase, dehydroshikimate dehydrogenase, and a deoxy-arabino-heptulosonate 7-phosphate synthase, in a rapamycin-producing strain of Streptomyces hygroscopicus. Disruption of these genes will then be used to determine their role in providing shikimic acid for both primary metabolism and DHCHC biosynthesis. In the long term these studies will provide a) an understanding of the regulation of the shikimic acid pathway in organisms that produce shikimate-derived natural products, and b) genetic approaches to increase the yields of these products in fermentation process es. Blocked mutants generated in this study will also be used to obtain rapamycin analogs by directed biosynthetic experiments. 3) Time permitting to initiate the purification of an enzyme involved in DHCHC biosynthesis in a rapamycin producing strain of S. hygroscopicus. This project represents an excellent opportunity for graduate students and postdoctoral fellows to obtain broad-based training in enzymology, molecular biology, and organic chemistry. It is through such interdisciplinary approaches that secondary metabolic pathways can be understood and harnessed to create additional diversity in natural product biosynthetic processes.
