Abstract
One of the biggest challenges to realize a circular carbon economy is the synthesis of complex carbon compounds from one-carbon (C1) building blocks. Since the natural solution space of C1–C1 condensations is limited to highly complex enzymes, the development of more simple and robust biocatalysts may facilitate the engineering of C1 assimilation routes. Thiamine diphosphate-dependent enzymes harbor great potential for this task, due to their ability to create C–C bonds. Here, we employed structure-guided iterative saturation mutagenesis to convert oxalyl-CoA decarboxylase (OXC) from Methylobacterium extorquens into a glycolyl-CoA synthase (GCS) that allows for the direct condensation of the two C1 units formyl-CoA and formaldehyde. A quadruple variant MeOXC4 showed a 100 000-fold switch between OXC and GCS activities, a 200-fold increase in the GCS activity compared to the wild type, and formaldehyde affinity that is comparable to natural formaldehyde-converting enzymes. Notably, MeOCX4 outcompetes all other natural and engineered enzymes for C1–C1 condensations by more than 40-fold in catalytic efficiency and is highly soluble in Escherichia coli. In addition to the increased GCS activity, MeOXC4 showed up to 300-fold higher activity than the wild type toward a broad range of carbonyl acceptor substrates. When applied in vivo, MeOXC4 enables the production of glycolate from formaldehyde, overcoming the current bottleneck of C1–C1 condensation in whole-cell bioconversions and paving the way toward synthetic C1 assimilation routes in vivo.
Highlights
The synthesis of complex molecules from one-carbon (C1)compounds is key to a circular economy
No hydroxyacyl-CoA lyase (HACL) structure is available and homology models fail to accurately predict the structure of the C-terminal active site loop, which exhibits low sequence homology throughout the HACL/oxalyl-CoA decarboxylase (OXC) family (Figure S1). Another limitation of HACLs is their poor production in E. coli.[23,24]. To overcome these challenges with HACLs, we recently focused on repurposing OXCs, which naturally catalyze the decarboxylation of oxalyl-CoA, as formyl-CoA condensing enzymes.[24]
None of the newly introduced amino acids of MeOXC4 is found in any HACL homolog (Figure S1), indicating that through directed evolution, an alternative maximum in the glycolyl-CoA synthase (GCS) activity landscape was found
Summary
Compounds is key to a circular economy. C1 compounds, in particular formate and methanol, can be derived directly from. The four substitutions in MeOXC4 directly affected the catalytic activity, as well as the kon and koff rates of the different substrates, causing a specificity switch between native OXC (oxalyl-CoA decarboxylation) and GCS activity (formyl-CoA condensation with formaldehyde) of greater than 100 000-fold. Three-ordered water molecules (W1−3) are observed in a structure with a trapped α-carbanion/enamine intermediate, with W2 (in close contact to W1 and W3) proposed to protonate the Cα of the intermediate (Figure S2).[28] W1 is hydrogen-bonded to residues corresponding to Y134 and E135, while Y497 and S568 form hydrogen bonds to W3.28 In MeOXC4, this hydrogen bonding network to W1 and W3 is lost due to the E135G, Y497F, and S568G substitutions This change in the water network likely helps to promote GCS activity at the expense of the OXC reaction. Enhancing ACR activity will be key toward improving and further developing this C1 fixation pathway for biotechnological applications
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