Natural products are organic compounds synthesized by bacteria or other organisms that offer them evolutionary advantages. An example of such is nocardioazine. Produced by the marine bacterium Nocardiopsis sp.CMB-M0232, nocardioazine has the ability to inhibit cancer drug transport proteins and in doing so reverse drug resistance in colon cancer cells (Raju, 2011). This could have great implications in colon cancer research, but Nocardiopsis takes over a week to culture and can only produce a few mg of nocardioazine per liter of culture. Our goal was to then genetically modify a more accessible organism such as E.coli with genes from Nocardiopsis sp., to determine how nocardiozine is enzymatically produced, with the hope of optimizing artificial nocardiozine production. Assuming nocardioazine’s precursor is Cyclo-L-trp-L-trp, we used pCDFDuet vector to insert genes into E.coli that we hypothesized would encode methyltransferase and racemase proteins required to modify this precursor into nocardioazine. Though gel electrophoresis and DNA sequencing we found that both racemase and methyltransferase DNA sequences were successfully ligated into pCDFDuet vectors and introduced into E.coli BL21(DE3) pET30 nozA expression hosts, producing cWW. However, the majority of proteins targeted for characterization were found in insoluble fractions of E.coli cell lysate, so future research will vary the conditions under which the expression hosts are grown to increase protein solubility. We also aim to introduce both methlytransferase and racemase genes into pCDFDuet vector simultaneously, as this opens up the possibility of exploring the functions of both corresponding enzymes in E.coli simultaneously.

Genetic engineering of Escherichia coli to produce nocardioazine natural products

2019 Spring National Meeting

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