Date of Award

12-1-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Yi-Qiang Cheng

Committee Members

Daad Saffarini, Graham Moran, Steven Forst, Gyaneshwar Prasad

Keywords

Anticancer, Cell Penetrating Peptide, HDAC Inhibitor, Heterologous Expression, L-Form Bacteria, PKS/NRPS

Abstract

Histone deacetylase (HDAC) inhibitors are becoming increasingly valuable therapeutic agents in treatment of several types of malignancies. FK228 is a depsipeptde anticancer compound produced by Chromobacterium violaceum no. 968 through a nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid assembly line. In the present study, reconstitution of the biosynthetic pathway responsible for the production of FK228 revealed cross-talk between modular PKS and fatty acid synthase. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We reported through heterologous expression of the FK228 biosynthetic pathway in E. coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl CoA acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the "AT-less" PKS modules on DepBC for polyketide chain elongation. Concurrently a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Importantly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward engineering of novel tumor-targeting bio-agents. The second part of the dissertation focused on the engineering of cell penetrating peptides (CPPs) with anticancer activity. A fusion protein combining the transmembrane activity of a bacterial CPP with a eukaryotic mitochondrial disrupting protein (MDP) was constructed to become a new CPP-MDP protein with anticancer activity. A new E. coli BL21(DE3)-based expression system that facilitates the secretion of CPP fusion proteins into the growth media for rapid and efficient protein purification was also created. This protein secretion system utilizes induced L-form bacterial spheroplasts to generate a yield of 32 mg/L of CPP-MDP directly from the media. Construction of an L-form based E. coli BL21 (DE3) protein secretion system and a modular CPP expression system allows future rapid CPP-fusion protein production with new fusion protein partners to develop new CPP-based anticancer agents.

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