Although biological systems hold great potential for the sustainable production of fuels and chemicals from one-carbon (C1) feedstocks (1), their C1 utilization reactions rely on specific acceptor molecules, complex metabolic pathways, and originate from difficult to engineer microorganisms, limiting applicability and implementation in the biotechnology industry (2–4). As a result, to date no non-native C1 utilizing organism has been engineered for growth or product synthesis from solely C1 substrates. In this thesis, we report that 2-hydroxyacyl-CoA lyase (HACL), an enzyme involved in the α-oxidation of long-chain fatty acids, catalyzes a novel C1 addition reaction resulting in the ligation of carbonyl-containing molecules with formyl-CoA to produce C1-elongated 2-hydroxyacyl-CoAs. We characterized the first prokaryotic variant of HACL and found that it can use a wide range of carbonyl substrates of different chain lengths, including both aldehydes and ketones, which when combined with enzymes comprising a de novo designed pathway, supported the conversion of C1 feedstocks to industrially relevant chemicals such as glycolate, ethylene glycol, ethanol, acetate, glycerate, and 2-hydroxyisobutyrate. Homology-guided mutagenesis allowed the identification of key residues influencing the in vivo activity of HACL and its ability to support C1 bioconversion. We implemented an HACL-based pathway in E. coli to utilize formaldehyde as the sole carbon substrate for glycolate production and demonstrated the potential of the pathway to support growth in a two-strain system, which was supported by genome scale modeling and flux balance analysis. The previously undescribed condensation reaction catalyzed by HACL is a direct and flexible means for C1 addition that can facilitate engineering of C1 bioconversion and synthetic methylotrophy/autotrophy in industrial organisms.