The Embden-Meyerhoff-Parnas (EMP) pathway, commonly known as glycolysis, represents the fundamental biochemical infrastructure for sugar catabolism in almost all organisms, as it provides key components for biosynthesis, energy metabolism, and global regulation. EMP-based metabolism synthesizes three-carbon (C3) metabolites prior to two-carbon (C2) metabolites, and must emit one CO2 in the synthesis of the C2 building block, acetyl-CoA, a precursor for many industrially important products. Thus, a key limitation for producing acetyl-CoA derived bioproducts is the intrinsic carbon loss in acetyl-CoA biosynthesis. Pyruvate decarboxylation releases the carboxyl group of pyruvate as carbon dioxide or formate to the environment. To overcome this limitation, we constructed and evolved an E. coli strain that relies on non-oxidative glycolysis (NOG) for carbon catabolism to support growth. In this talk, we will discuss how the fundamental metabolic pathways can be re-wired and how regulatory circuits can be altered through rational design, genome editing and evolution. (Lin, P. P. et al. (2018) Construction and evolution of an Escherichia coli strain relying on nonoxidative glycolysis for sugar catabolism. PNAS 115, 3538–3546).