Assimilation of formic acid and CO2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways.

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2 can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO2 through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing the gcvTHP genes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO2 in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO2 The pyruvate-forming flux from FA and CO2 could be increased to 14.9% by knocking out gcvR, pflB, and serA, chromosomally expressing gcvTHP under trc, and overexpressing the reconstructed THF cycle, gcvTHP, and lpd genes in one vector. To reduce glucose usage required for energy and redox generation, the Candida boidinii formate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO2 consumption rates of 370.2, 145.6, and 14.9 mg⋅g dry cell weight (DCW)-1⋅h-1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO2 after glucose depletion, suggesting that combined use of the C1 assimilation pathway and C. boidinii Fdh will be useful for eventually developing a strain capable of utilizing FA and CO2 without an additional carbon source such as glucose.

Structural insight into a molecular mechanism of methenyltetrahydrofolate cyclohydrolase from Methylobacterium extorquens AM1.

Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.