Kefale, Teshager Bitew2024-04-242024-04-242024-04-11http://hdl.handle.net/10012/20485This dissertation advances metabolic engineering by optimizing the genetic and metabolic capabilities of Yarrowia lipolytica and Escherichia coli to enhance their applications in biotechnology. It focuses on improving Y. lipolytica's mannitol and amino acids production by varying fermentation temperatures and employing techniques like shake flask fermentation, HPLC, and NMR. Notably, mannitol production was enhanced through targeted modifications of FBP1 gene at elevated temperatures. RNAseq analyses highlighted shifts in metabolic pathways under thermal stress, markedly in lipid, sugar and amino acids metabolism. Additionally, a dual-gRNA CRISPR-Cas9 system was integrated within the pCRISPRYL2 plasmid, noticeably improving genetic editing precision by overcoming the constraints of the non-homologous end joining (NHEJ) pathway. Furthermore, the study pioneered a Cell-Free Metabolic Engineering (CFME) strategy to synthesize 5-Aminolevulinic Acid (5-ALA) utilizing optimized enzymatic reactions and operational conditions, presenting a scalable and eco-friendly alternative to conventional whole-cell systems. In parallel, engineered E. coli demonstrated robust heme production capabilities in both whole-cell and cell-free systems. Heme derivatives, including valuable pigments like biliverdin, Phycocyanobilin (PCB) and Phycoerythrobilin (PEB) were also produced at a 1L bioreactor scale utilizing E. coli engineered with unexplored enzymes. Overall, this work not only expands the scope of metabolic engineering but also sets a foundational work for future innovations in biomanufacturing.enMetabolic EngineeringYarrowia lipolyticaE. coliFermentation temperatureCRISPR-Cas9 SystemCell-Free Metabolic Engineering5-Aminolevulinic AcidRNAseqHemeBiliverdinPhycocyanobilinPhycoerythrobilinDoctoral Thesis