Identification of 2‑Hydroxyacyl-CoA Synthases with High Acyloin Condensation Activity for Orthogonal One-Carbon Bioconversion

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
(kcat/KM)
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 μM 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.