Metabolic engineering and biocatalysis aim to produce new value-added products from biological routes through careful and deliberate genetic engineering in different microbial chassis with suitable metabolic repertoire. Hitherto, the endeavour has largely been confined to the production of new products from glucose and a few other well characterized substrates. But, as the bioeconomy gains in importance and the world seeks to explore new sources of carbon as feedstocks, a possibility exists in developing alternative C2 compounds as feedstocks in contrast to the current reliance on C6 (glucose and fructose), C5 (xylose) and C3 (glycerol) substrates. Two potential C2 alternative substrates explored in this work are ethanol and ethylene glycol. Ethanol is a biofuel whose biotechnological potential has not been fully explored. In the first part of the book, the potential of using the NADH generated from a reconstructed ethanol utilization pathway to power a biotransformation reaction is explored in Escherichia coli. Experimental results indicate that ethanol utilization delivers higher biotransformation efficiency compared to state-of-the-art glucose dehydrogenase system, which confirmed theoretical predictions of relative efficiency of the two systems. Next, the book switches attention to ethylene glycol which could be derived as breakdown products of lignocellulosic waste or from plastic waste. Here, the proposal is to demonstrate the potential of an NADH generating ethylene glycol utilization pathway that terminates at glycolate to support biocatalytic reactions in E. coli. Subsequently, the two-step ethylene glycol utilisation pathway would be extended to malate and 2-phosphoglycerate to help another biotechnology workhorse, Bacillus subtilis, to grow on ethylene glycol as the sole carbon source. Different connection points to central carbon metabolism would provide a natural experiment to decipher the relative efficiencies of activating tricarboxylic acid cycle and glycolysis in supporting cell growth, and illuminate insights into the relative efficiency and evolutionary importance of different branches of metabolism in B. subtilis. Finally, ethylene glycol utilization would be combined with product formation to help B. subtilis to synthesise green fluorescent protein using flux from the C2 alternative substrate. This system would provide a platform for examining the efficacy of different ethylene glycol utilization pathways and entry points in central carbon metabolism in synthesising amino acids and proteins. Overall, this book hopes to explore the biotechnological potential of two alternative C2 substrates (ethanol and ethylene glycol) in biocatalysis and metabolic engineering from the substrate utilisation perspective.