One-carbon (C1) compounds derived from waste gases such as carbon dioxide, carbon monoxide and methane start to be recognized as carbon feedstock in the field of metabolic engineering and industrial biotechnology. Numerous enzymes and pathways have been identified and engineered for efficient C1 assimilation into multi-carbon molecules and the list continues to increase at an unprecedented pace with advances in synthetic biology. This thesis aims to provide two synthetic pathways into the list, based on new-to-nature biochemistries, each having unique characteristics and advantages over the preexisting pathways. Specifically, there are four chapters in the thesis: first chapter provides a comprehensive review on C1-utilizing enzymes and metabolic pathways, both natural and synthetic, with comments on cross-platform capabilities and industrial applications and highlighting the pathway dependency to the host metabolism. Second chapter introduces synthetic C1 utilization pathways named Formyl-CoA Elongation (FORCE) pathways. FORCE pathways operate in an orthogonal manner to the host metabolism, exemplified by the abilities to generate products directly from C1 compounds in a growth-decoupled bioconversion in Escherichia coli. Also, FORCE pathways’ potential to be harnessed in a synthetic C1-trophy segregating C1 assimilation and native substrate utilization is demonstrated in a two-strain co-culture system. Third chapter discusses approaches to improve FORCE pathway flux by identifying and engineering more efficient variants of the key condensation enzyme, 2-hydroxyacyl-CoA synthase (HACS), which is also identified as a major rate limiting step in the pathways. A variant with more than 10-fold improvement in activity was discovered, which was applied in the pathway to demonstrate significantly improved product titer, rate, and yield. Fourth chapter explores the journey to engineer E. coli to utilize a non-native substrate methylsuccinate, a metabolic precursor from oxygen-independent methane activation via fumarate addition. Combination of rational pathway design and adaptive laboratory evolution is used to achieve a strain growing efficiently on methylsuccinate as sole carbon source, which could be used as a selection platform to screen for methane activation enzymes and ultimately as a chassis for synthetic methanotrophy.