Metabolic Engineering and Directed Evolution of Perillyl Alcohol Biosynthetic Pathway in Escherichia coli to Produce Compounds with Anti-cancer Activity Against Breast cancer cell lines

Terpenoids are a large diverse class of naturally occurring organic chemicals, which are used extensively for their aromatic and pharmaceutical qualities. One such terpenoid is perillyl alcohol that has potential anti-cancer activity. It is derived from plant extracts (i.e. mint, cherries, and celery seeds) where it is produced via the mevalonate pathway. Initial work focuses on engineering the bacterium, Escherichia coli to produce large quantities of terpenoids, including perillyl alcohol and its derivatives. The genes involved in the perillyl alcohol biosynthetic pathway include geranyl pyrophosphate synthase (GPPS), limonene synthase (LS), a cytochrome P450 alkane hydroxylase ( ahpG), a ferredoxin reductase (ahpH) and a ferredoxin (ahpI). When codon-optimized versions of these genes were successfully installed in E. coli, the effect of pathway expression on growth of E. coli was negligible. Expression of these genes did not produce perillyl alcohol, however, we were able to see a new peak that was determined to be cis-p-mentha-2,8, dien-1-ol using retention time data and mass spectral analysis. Chapter 2 also describes the effect of cis-p-mentha-2,8, dien-1-ol on MCF-7 and MDA-MB-231 breast cancer cell lines. This compound was able to significantly inhibit the proliferation of MCF-7 and MDA-MB-231 cells, and the IC50 was determined to be 2.5 muM for MCF-7 cells and 3muM for MDA-MB-231 cells. Chapter 3 describes the evolution of the biosynthetic pathway to obtain novel compounds with anti-cancer activity. We introduced random mutations in the limonene synthase gene via Kunkel mutagenesis and generated a library of mutants, each capable of producing a potential anti-cancer compound. The entire library was screened in pools of 15 clones each, and one pool of mutants showed antiproliferative activity against the estrogen receptor (ER)-negative MDA-MB-231 breast cancer cells. When the pool of clones was deconvoluted, no single clone with this activity was identified, it may be currently concluded that the clones of the pool have a combined effect on the cells. This work describes the construction and expression of a biosynthetic pathway in E. coli as well as evolution of the pathway to produce new compounds with antiproliferative activities against breast cancer cell lines.