Azinomycins A and B are antitumor compounds isolated from soil bacteria, Streptomyces sahachiroi. The azinomycin structure contains an unusual aziridine [1,2a] pyrrolidine ring and an epoxyvaline moiety which are used in forming DNA cross-links. The biosynthetic gene cluster of azinomycin B has been characterized but the biosynthetic routes to these intermediates and overall architecture of this compound is not well understood. This work investigates the role of aziW, a lysW homologue in azinomycin biosynthesis. Gene replacement of aziW by homologous exchange abolished the production of azinomycin thereby implicating the gene in azinomycin biosynthesis. Complementation of aziW in the mutant strain, however, did not restore production of this natural product as observed by HPLC analysis, which suggests that aziW as well as genes downstream could be responsible for lack of azinomycin production. Sequence analysis of aziW suggests co-transcription with the downstream genes which are homologous to lysine biosynthetic genes in T. thermophilus. The mRNA expression profile of the genes downstream of aziW exhibited a down-regulation or transcription termination for these genes which appear to be in the same operon. The impact is stronger the further away the gene is from aziW. Negative lysine auxotrophy test showed that this gene is not involved in lysine biosynthhesis but is crucial to azinomycin biosynthesis. This dissertation also discusses the investigation of the role of ORF10, a dehydrogenase which is located at the terminal end of the gene cluster. Gene disruption of ORF10 had no effect in azinomycin production but established the boundary of the azinomycin gene cluster. AziC9 was charaterized as an authentic cytochrome P450 through gene expression in E.coli, purification, carbon monoxide binding and activity assay with a putative substrate.