Despite the potential of biotechnological processes for one-carbon (C1) bioconversion， efficient biocatalysts required for their implementation are yet to be developed. To address intrinsic limitations of native C1 biocatalysts， here we report that 2-hydroxyacyl CoA lyase (HACL)， an enzyme involved in mammalian α-oxidation， catalyzes the ligation of carbonyl-containing molecules of different chain lengths with formyl-coenzyme A (CoA) to produce C1-elongated 2-hydroxyacyl-CoAs. We discovered and characterized a prokaryotic variant of HACL and identified critical residues for this newfound activity， including those supporting the hypothesized thiamine pyrophosphate-dependent acyloin condensation mechan... More
Despite the potential of biotechnological processes for one-carbon (C1) bioconversion， efficient biocatalysts required for their implementation are yet to be developed. To address intrinsic limitations of native C1 biocatalysts， here we report that 2-hydroxyacyl CoA lyase (HACL)， an enzyme involved in mammalian α-oxidation， catalyzes the ligation of carbonyl-containing molecules of different chain lengths with formyl-coenzyme A (CoA) to produce C1-elongated 2-hydroxyacyl-CoAs. We discovered and characterized a prokaryotic variant of HACL and identified critical residues for this newfound activity， including those supporting the hypothesized thiamine pyrophosphate-dependent acyloin condensation mechanism. The use of formyl-CoA as a C1 donor provides kinetic advantages and enables C1 bioconversion to multi-carbon products， demonstrated here by engineering an Escherichia coli whole-cell biotransformation system for the synthesis of glycolate and 2-hydroxyisobutyrate from formaldehyde and formaldehyde plus acetone， respectively. Our work establishes a new approach for C1 bioconversion and the potential for HACL-based pathways to support synthetic methylotrophy.
