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Metabolic Studies of Albomycin Biosynthesis

Kulkarni, Aditya S.
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1439979936

Abstract Details

2015, Doctor of Philosophy (PhD), Ohio University, Biological Sciences (Arts and Sciences).
Albomycin is a sulfur containing metabolite produced by Streptomcyes sp. ATCC 700974. It is a structurally unique molecule with potent antibiotic activity. Unfortunately, it is produced in low amounts and this is thought to be the stumbling block for its commercial development. Cytosine is thought to be a precursor for albomycin production and experiments were done to increase intracellular cytosine to possibly increase albomycin production. Isolation of a codA mutant was attempted. Feeding cytosine in the growth medium and overexpressing a blsM gene (which generates cytosine) were found to have no effect on albomycin production. Experiments were also done to gain insights into the sulfur source utilized for albomycin biosynthesis by Streptomyces sp. ATCC 700974. Addition of iron and propargylglycine altered intracellular homocysteine and cysteine levels and albomycin production suggesting homocysteine as a precursor. Based on these results, sulfur amino acid metabolism of the albomycin producing strain was manipulated by overexpressing select pathways to observe effects on albomycin production. Albomycin production increased with overexpression of pathways producing homocysteine, namely the transsulfuration and direct sulfhydrylation pathways. No effect was observed on expression of the reverse transsulfuration pathway. Overexpression of the genes in the active methyl cycle leading to homocysteine was also found to increase albomycin production. AbmD is proposed to be the first enzyme of the albomycin biosynthetic pathway in Streptomyces sp. ATCC 700974 and to utilize cysteine or homocysteine as one of its substrates. Enzyme assays using purified AbmD demonstrated that homocysteine was utilized along with a co-substrate. Five pathway genes directing homocysteine biosynthesis along with the abmD gene were integrated into the Streptomcyes sp. ATCC 700974 genome to produce the strain SAK9. Albomycin production by SAK9 was found to be four times the wild type strain in glycerol containing media. These results suggest homocysteine is the precursor for albomycin biosynthesis. 13C labeling experiments conducted by feeding labeled cysteine and methionine were inconclusive. The seleno-amino acids selenomethionine and selenocystine were not incorporated into albomycin. Future directions for albomycin biosynthesis research are discussed.
Donald Holzschu, Ph.D (Advisor)
Erin Murphy, Ph.D (Committee Member)
Tomohiko Sugiyama, Ph.D (Committee Member)
Martin Tuck, Ph.D (Committee Member)
Mark McMills, Ph.D (Committee Member)
232 p.
Biochemistry; Microbiology; Molecular Biology
Albomycin; sulfur metabolism; secondary metabolite biosynthesis; metabolic engineering; Streptomyces

Recommended Citations

Citations

  • Kulkarni, A. S. (2015). Metabolic Studies of Albomycin Biosynthesis [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1439979936

    APA Style (7th edition)

  • Kulkarni, Aditya. Metabolic Studies of Albomycin Biosynthesis. 2015. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1439979936.

    MLA Style (8th edition)

  • Kulkarni, Aditya. "Metabolic Studies of Albomycin Biosynthesis." Doctoral dissertation, Ohio University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1439979936

    Chicago Manual of Style (17th edition)

ohiou1439979936
474
© 2015, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.