ENGINEERING CO2-FIXING ESCHERICHIA COLI FOR HEXANOYL COENZYME A BIOSYNTHESIS THROUGH HETEROLOGOUS GENE EXPRESSION, GENE SILENCING AND TRANSCRIPTIONAL BIOCHEMICAL REPRESSION

Sustainably produced oleochemicals from microbial lipid metabolism are an attractive alternative to traditional industrial production that is using expensive or non-renewable feedstocks. Though, microbial chassis as cell factories for production of oleochemicals at high titer for commercial exploitation require biochemical and genetic manipulation towards specific gene programming. As such, Escherichia coli cells are the laboratory workhorse with significant importance in biotechnological development and are the prime microbial candidate to drive synthesis of valuable compounds. The aim of this thesis is to demonstrate a novel metabolic system in Escherichia coli for controlled expression of specific genes encoding putative heterologous lipid-related enzymes and silencing native enzymes as wells as transcription factors to augment metabolic flow of specific pathways towards hexanoyl-CoA accumulation in the cell at commercial concentrations using a cheap feedstock. First, a CO2-fixing pathway was constructed in an Escherichia coli strain by introducing a donated expression cassette containing genes encoding two sequential cyanobacterial Calvin cycle enzymes; Rubisco (RBC)-encoding genes (rbcL-rbcX-rbcS), and phosphoribulokinase-encoding gene (PRK) and a cyanobacterial carbonic anhydrase (CA)-encoding genes (ccaA), mimicking cyanobacterial carbon concentrating mechanism. Second, a hexanoyl-CoA-producing pathway was incorporated by including an Arabidopsis thaliana acyl-lipid thioesterase 4-encoding gene (ALT4) and acyl-activating enzyme-encoding gene (AAE17). Next, an antisense RNA pathway was incorporated into the previously engineered strain to prevent the flow of the produced hexanoyl-CoA to β-oxidation and phospholipid synthesis. Three RNA-silencing sequences targeting Escherichia coli anaerobic acyl-CoA dehydrogenase-encoding gene (Ydio), aerobic acyl-CoA dehydrogenase-encoding gene (FadE) and fused 2-acylglycerophospho-ethanolamine acyltransferase/acyl-acyl carrier protein synthetase-encoding gene (aas) were incorporated. Finally, DNA-binding transcriptional dual regulator-encoding gene (FadR) targeting β-oxidation repression was incorporated. Markedly, the engineered Escherichia coli productivity of hexanoyl‑CoA was 100% increase over wildtype. This multi-gene transformation system is the first report in increasing hexanoyl-CoA through controlling simultaneously fatty acid biosynthesis and β-oxidation, utilizing simple RNA silencing and transcriptional repression technology in Escherichia coli cells.