Assimilation of formic acid and CO2by engineeredEscherichia coliequipped with reconstructed one-carbon assimilation pathways

Abstract

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2can be an attractive raw material for bio-based chemicals. Here, we report the development ofEscherichia colistrains assimilating FA and CO2through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. TheMethylobacterium extorquensformate-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 thegcvTHPgenes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO2in 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 CO2could be increased to 14.9% by knocking outgcvR,pflB, andserA, chromosomally expressinggcvTHPundertrc, and overexpressing the reconstructed THF cycle,gcvTHP, andlpdgenes in one vector. To reduce glucose usage required for energy and redox generation, theCandida boidiniiformate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO2consumption 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 CO2after glucose depletion, suggesting that combined use of the C1 assimilation pathway andC. boidiniiFdh will be useful for eventually developing a strain capable of utilizing FA and CO2without an additional carbon source such as glucose.