Biosynthesis of 5-hydroxy-4-oxonorvaline and aspartate family amino acids in Streptomyces akiyoshiensis.
Date
1994
Authors
Le, Yunzheng.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
Cultures of Streptomyces akiyoshiensis accumulate 5-hydroxy4-oxonorvaline (HON), which is an antifungal antibiotic and a chiral synthon biosynthesized from aspartate and acetate. To develop the potential for economic production of HON by fermentation, links between the biosynthesis of HON and aspartate family amino acids have been investigated.
Bioassays for HON based on its antifungal activity were developed and used to screening NTG-treated spores for mutants blocked in the biosynthesis of HON. Cross-feeding between pairs of blocked mutants identified four co-synthetic groups of mutants. One consisted of regulatory and/or non-pathway mutants. The remaining three groups, which were blocked in the biosynthetic pathway, were placed in biosynthetic sequence. Since blocked mutants were not auxotrophic, aspartate pathway auxotrophs were isolated by screening NTG-treated spores; putative mutants in ask, hsd and lysA were obtained. Their phenotypes indicated that the HON pathway does not require the steps after homoserine dehydrogenase and diaminopimelate decarboxylase, but probably includes aspartate kinase and aspartate semialdehyde dehydrogenase. To examine this further, asd from S. akiyoshiensis was cloned by complementing a mutation in E. coli. The gene was localized to a 2.2-kb fragment, which was completely sequenced. The sequence data suggested that the gene organization for aspartate kinase and aspartate semialdehyde dehydrogenase in S. akiyoshiensis is different from that in other high %G+C Gram-positive bacteria. To facilitate molecular genetic analysis procedures for DNA transformation in S. akiyoshiensis were developed.
Thesis (Ph.D.)--Dalhousie University (Canada), 1994.
Bioassays for HON based on its antifungal activity were developed and used to screening NTG-treated spores for mutants blocked in the biosynthesis of HON. Cross-feeding between pairs of blocked mutants identified four co-synthetic groups of mutants. One consisted of regulatory and/or non-pathway mutants. The remaining three groups, which were blocked in the biosynthetic pathway, were placed in biosynthetic sequence. Since blocked mutants were not auxotrophic, aspartate pathway auxotrophs were isolated by screening NTG-treated spores; putative mutants in ask, hsd and lysA were obtained. Their phenotypes indicated that the HON pathway does not require the steps after homoserine dehydrogenase and diaminopimelate decarboxylase, but probably includes aspartate kinase and aspartate semialdehyde dehydrogenase. To examine this further, asd from S. akiyoshiensis was cloned by complementing a mutation in E. coli. The gene was localized to a 2.2-kb fragment, which was completely sequenced. The sequence data suggested that the gene organization for aspartate kinase and aspartate semialdehyde dehydrogenase in S. akiyoshiensis is different from that in other high %G+C Gram-positive bacteria. To facilitate molecular genetic analysis procedures for DNA transformation in S. akiyoshiensis were developed.
Thesis (Ph.D.)--Dalhousie University (Canada), 1994.
Keywords
Biology, Microbiology., Chemistry, Biochemistry.