METABOLIC ENGINEERING OF THE DEOXYXYLULOSE PHOSPHATE PATHWAY FOR ISOPRENOID PRODUCTION IN E.COLI

Abstract

Metabolic engineering offers a sustainable approach for the production of various compounds from inexpensive and renewable starting materials, including bulk chemicals, fine chemicals, drugs and biofuels. This thesis is focused on developing engineering tools on optimizing strains and global environments to maximize isoprenoid production and attempting to elucidate the underlying biochemical mechanisms. First, precursor was identified as a limiting factor and its availability was increased through engineering carbon uptake pathway. Second, an experimental design aided systematic pathway optimization method was developed to systematically optimize the expression of carbon uptake and isoprenoid pathway genes. Built on the genetically optimized strain, isoprenoid production was further improved by optimizing the medium and cultural conditions, resulting in significant improvement. Third, the efficient transporter screening and engineering method was established, resulting in the significant enhancement of cytotoxic isoprenoid production. Lastly, a semi-closed dual-flux platform was established to monitor the concentration of intracellular and extracellular metabolite intermediates. This platform and the observed correlation of the efflux and intracellular concentration pave the road to understand quantitatively the isoprenoid pathway in the future.