Bleomycin is a hybrid polyketide/non-ribosomal peptide that possesses anti-tumor properties. This work is aimed at characterizing the predicted Asp/Asn hydroxylase ORF1 encoded in bleomycin’s gene cluster. Interestingly, despite the presence of this predicted hydroxylase, there does not appear to be a hydroxylated Asp/Asn in the structure of bleomycin. We cloned the ORF1 gene and overexpressed the corresponding protein in E. coli. Various experiments utilizing high performance liquid chromatography (HPLC) to detect possible hydroxylation catalyzed by ORF1 were performed. Ultimately, results from the HPLC experiments did not demonstrate the predicted hydroxylation of Asp or Asn, suggesting that the isolated protein was inactive, or the ORF1 gene was misannotated. Future work will be focused on re-expressing the protein in active form.

Cloning and expression of the predicted aspartate hydroxylase ORF1 from Streptomyces verticillus

253rd National Meeting (2017)

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