DESIGN AND DEVELOPMENT OF MOLECULAR TOOLS FOR THE COMBINATORIAL MULTIVARIANT-MODULAR APPROACH TO ENGINEERING METABOLIC PATHWAYS IN E.COLI

Abstract

METABOLIC ENGINEERING OF MICROORGANISMS IS A SUSTAINABLE AND RENEWABLE APPROACH TO PRODUCE A VARIETY OF HIGH-VALUED FINE CHEMICALS. CONSIDERING THE COMPLEX GENETIC NETWORKS AND BIOCHEMICAL INTERACTIONS IN THE HOST, A MULTIVARIATE-MODULAR APPROACH TO ENGINEERING THE BIOCHEMICAL PATHWAYS IS AN ESSENTIAL STEP IN THE IDENTIFICATION AND MODULATION OF CONDITIONS FOR THE OPTIMAL PRODUCTION OF THE DESIRED COMPOUNDS. THIS THESIS IS FOCUSED ON THE DEVELOPMENT OF NOVEL TECHNOLOGIES FOR THE COMBINATORIAL MULTIVARIATE-MODULAR APPROACH TO ENGINEERING METABOLIC PATHWAYS IN ESCHERICHIA COLI. THESE INCLUDE METHODS FOR 1) THE DIRECT AND HIGH-THROUGHPUT TRANSCRIPTIONAL MEASUREMENT; 2) THE HIGHLY EFFICIENT, LABOR- AND TIME-SAVING IN VITRO ASSEMBLY OF DNA; 3) THE SIMULTANEOUS AND SYSTEMATIC PERTURBATION OF MULTIPLE PATHWAY MODULES USING A GENETIC CONTROL SYSTEM; 4) STABLY MAINTAINING THE CO-EXISTENCE OF ANTIBIOTIC SELECTION FREE MULTI-PLASMIDS IN ENGINEERED HOSTS. THESE WELL-DESIGNED TOOLS WERE USED TO O