Throughout the past several decades, the petroleum industry has remained a dominant player in the production of fuels, chemicals, and consumer products. However, rising global temperatures and declining fossil fuel reserves demand the need for sustainable processes that can compete with the existing crude oil-based economy. Employing microbes such as E. coli for the chemical production using renewable starch-derived feedstocks such as glucose has been identified as a preferred alternative. Although lignocellulosic feedstocks have been explored as an alternative to renewable sugars, these processes are yet to be successfully implemented in an industrial setting. Despite more recent research efforts to incorporate C1 feedstocks such as methane, methanol and carbon dioxide in microbial catalysis, development of efficient bacterial metabolic pathways using these feedstocks have proven to be challenging. Herein, acetylenecarboxylic acid (ACA) is proposed as a unique feedstock for microbial catalysis. ACA can be derived from dehydrodimerization of methane to acetylene and subsequent carboxylation of acetylene. Cg10062 was previously identified as an enzyme capable of hydrating ACA to form a mixture of malonate semialdehyde (MSA) and acetaldehyde. In this study, novel variant Cg10062(E114N)) that forms exclusively MSA from ACA was discovered using rational mutagenesis. Cg10062(E114N) was coupled with NADPH-dependent dehydrogenase YdfG to develop a unique biocatalytic route to building block chemical 3-hydroxypropionic acid (3-HP). In vitro synthesis of 3-HP from ACA was demonstrated using catalytic amounts of NADP(H) where cofactor regeneration was achieved using NADP+-dependent phosphite dehydrogenase PTDH.
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