Engineering a Highly Efficient Carboligase for Synthetic One-Carbon Metabolism

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 diphosphatedependent 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.

Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA

“…5 E ). Accordingly, the tyrosine and serine residues were replaced by phenylalanine and glycine, respectively, in the quadruple MeOXC mutant (Y497F and S568G) to improve the condensation reaction of formyl-CoA with formaldehyde, since the latter as well as the resulting glycolyl-CoA do not contain a carboxylic group anymore ( 10 ). Furthermore, the substrate Cα oxygen interacting glutamine residue found in HACL and other ThDP-dependent enzymes (Q113 and Q128 in RuHACL and AcHACL, respectively) is replaced by a conserved glutamic acid in WT OXC (E121 and E135 in OfOXC and MeOXC, respectively).…”

“…5 E ). The mutation of the glutamic acid to a glycine residue in MeOXC increased the rate of the synthase reaction, most likely because the tyrosine side chain is given more space for optimal interaction with the intermediate Cα-hydroxyl group ( 10 ). Unfortunately, substrate or covalently bound intermediate structures have not yet been solved for the optimized MeOXC mutant and only a structure in open conformation has been deposited (PDB ID: 7B2E ).…”

“…5 E ) clearly indicates that for transferring the HACL reaction to OXC, thus far, focus has been only laid on mutations to avoid unfavorable interaction during substrate accommodation. Consequently, the resulting MeOXC mutant is quite promiscuous, as carbonyl compounds of varying size (from formaldehyde to phenylacetaldehyde) serve as substrate ( 10 ). Furthermore, since the MeOXC mutations E135G, Y497F, and S568G do not provide the acid/base catalyst required for reverse steps 3 and 4 in the HACL catalytic cycle ( Fig.…”

“…1 ), it is tempting to transfer the HACL reaction to an OXC template. Accordingly, a quadruple mutant of the OXC from Methylorubrum extorquens (MeOXC) has recently been established for the condensation of formyl-CoA with formaldehyde by directed evolution ( 10 ). However, the analogy between OXC and HACL is limited.…”

A computational walk to the hidden peaks of protein performance

“…But can it be used to facilitate the engineering of new protein functions, for example, as recently experimentally achieved for the direct enzymatic catalysis of a C1–C1 condensation? ( 8 ) Also, exciting fundamental questions could be explored: is it possible to improve any protein for human uses? The example of the carbon fixing enzyme RuBisCO suggests that in some cases nature might have already found functional peak performance ( 9 , 10 ).…”