Enzymatic Studies on the Biosynthesis of Atypical Angucycline Antibiotic Lugdunomycin in Streptomyces
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Lugdunomycin, a novel antibacterial natural product isolated from Streptomyces sp. QL37, is a highly rearranged angucycline-derived polyketide with strikingly complex structure. Its proposed biosynthetic pathway involves unprecedented chemistry, including complex redox and rearrangement reactions. My aim is to elucidate the early tailoring steps in lugdunomycin biosynthesis to gain more tools for engineering synthetic pathways that produce novel antibiotics.
Previous knockout studies indicated that LugOI is a C12-hydroxylase like its homolog PgaE from Streptomyces sp. PGA64. I show that LugOIIred ketoreductase coupled with PgaE, converts the common angucycline precursor UWM6 into tetrangomycin. The addition of LugM methylase into the reaction yields 8-O-methyltetrangomycin, a putative precursor for lugdunomycin. I also show that SDR-family enzyme LugG is a C7 ketoreductase acting upon 8-O-methyltetrangomycin and its C1 ketoreduced derivative produced by LugOIIred. Furthermore, I show that TacA from tac-cluster in Streptomyces sp. CB00072 is a promiscuous C12 ketoreductase
In this study, I produced and purified angucycline precursors using engineered Streptomyces strains, and enzymes encoded in the lug-cluster heterologously in E. coli. To determine the functions of the enzymes, I incubated the precursors with different combinations of proteins, and analyzed the reaction samples by HPLC. The structure elucidation of the products of my key reactions were done using LC-MS and NMR techniques.
Our future objective is to elucidate the mechanism of the proposed exotic ring-cleavage of 8-O-methyltetrangomycin, which is putatively catalyzed by LugOIII and LugOV. Ring-cleavage reactions require chemically challenging C-C bond cleavage. Insight into the mechanism of this reaction could help us design synthetic antibiotics with novel structures.