The production of sustainable fuels for transportation and energy represents an essential technology enabling the transition away from fossil fuel use. Over the past two decades several biological routes for the production of chemically identical molecules to those in fossil fuels (‘drop-in’ fuels) have been discovered and characterized. These have held promise as breakthrough technologies but have yet to reach commercial viability. Industrial scale bioprocess development for drop-in fuel production has been held back by high feedstock prices and inadequate process efficiencies. Currently, bioethanol and biodiesel are produced using crop-derived sugars and oils, so called ‘1st generation’ biofuels. Although their use positively impacts net CO2 emissions, their derivatization from food crops means they compete for arable land. To avoid this ‘food vs. fuel’ issue, alternative carbon sources for bioproduction must be explored. In this thesis the use of two non-sugar substrates, acetate and ethanol, were explored as carbon sources for hydrocarbon biosynthesis. Either carbon source enabled the production of these compounds at titres and product/substrate yields similar to that of glucose. The rate and efficiency of substrate conversion to product largely governs the viability of a bioprocess. These properties are in part defined by the enzymes that make up the relevant metabolic pathways. One of these pathways is the cyanobacterial alkane biosynthesis pathway discovered in 2010, consisting of acyl-ACP reductase (AAR) and aldehyde deformylating oxygenase (ADO). The terminal ADO enzyme is known for its low catalytic efficiency, and as a result many enzyme engineering efforts have targeted this enzyme. This thesis describes the design of growth-coupled approaches for engineering ADO. Growth-coupling involves linking enzyme activity to cell fitness and taking advantage of the principles of directed evolution to select for improved enzyme variants. Several growth-coupling approaches were assessed in this work through a combination of in silico flux balance analysis and experimental work, mostly focussing on the byproduct of ADO – formate. These efforts resulted in functional growth-coupling of ADO activity via the reductive glycine pathway. Another approach to growth-couple ADO activity was based on redox cofactor auxotrophy. An E. coli strain lacking all central metabolic pathways for NAD+ reduction was constructed, dubbed NADHaux, and its growth could be tightly linked to NADH generation by formate oxidation. Taken together, this thesis provides insights into the extremes of E. coli redox metabolism and presents hopeful results regarding the use of non-sugar carbon sources for bioproduction. In addition, it describes valuable novel methodologies for the evolutionary engineering of alkane biosynthesis.