Assimilation of formic acid and CO ₂ by engineered <i>Escherichia coli</i> equipped with reconstructed one-carbon assimilation pathways

Abstract

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 <i>Escherichia coli</i> strains assimilating FA and CO₂ through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The <i>Methylobacterium extorquens</i> 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 (<i>gcvR</i>) and overexpressing the <i>gcvTHP</i> 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 <i>gcvR</i>, <i>pflB</i>, and <i>serA</i>, chromosomally expressing <i>gcvTHP</i> under <i>trc</i>, and overexpressing the reconstructed THF cycle, <i>gcvTHP</i>, and <i>lpd</i> genes in one vector. To reduce glucose usage required for energy and redox generation, the <i>Candida boidinii</i> 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 <i>C. boidinii</i> Fdh will be useful for eventually developing a strain capable of utilizing FA and CO₂ without an additional carbon source such as glucose.