One-carbon (C1) compounds are emerging as cost-effective and potentially carbon-negative feedstocks for biomanufacturing, which require efficient, versatile metabolic platforms for the synthesis of value-added products. Synthetic formyl-CoA elongation (FORCE) pathways allow diverse product synthesis from C1 compounds via iterative C1 elongation, operating independently from the host metabolism with reduced engineering complexity and improved theoretical yields. However, a major bottleneck was identified as the suboptimal kinetics of the core C1-C1 condensation enzyme, 2-hydroxyacyl-CoA synthase (HACS), catalyzing the acyloin condensation reaction between formaldehyde and formyl-CoA. Here, we used a combinatorial approach of bioprospecting and rational protein engineering to identify multiple HACS variants with significantly improved activities toward C1 substrates. Sequence and structure alignment of the active variants elucidated the key regions for the catalytic function, which were targeted for mutagenesis, leading to improved catalytic efficiency. In parallel, a consecutive round of bioprospecting for homologs with high similarity with active variants revealed a highly active HACS variant exhibiting up to 7-fold improvement in catalytic efficiency (k(cat)/K-M) and 14-fold improvement in the FORCE pathway flux in vivo compared to the previous reports. Upon further optimization of the downstream pathway, the orthogonal C1-to-product bioconversion system showed a metabolic flux of up to 700 mu M glycolate OD-1 h(-1) (2.1 mmol gDCW(-1) h(-1)) and an industrially relevant glycolate titer, rate, and yield of 5.2 g L-1 (67.8 mM), 0.22 g L-1 h(-1), and 94% carbon yield, respectively.
