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

Abstract

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

Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO2 Fixation

“…Unlike the CBB cycle, the rGly process has few overlaps with cellular central metabolism, reducing the influence of metabolic regulations. Bang and Lee (2018) introduced this pathway in E. coli to realize one-carbon assimilation in vivo by overexpressing enzymes related to the rGly pathways as well as NAD-dependent FDH for producing reducing power from formate. After glucose depletion, the engineered strain maintained a slight growth using only formate and CO 2 as substrates, confirming its feasibility for supplying energy and reducing power via NAD-dependent FDH oxidative activity.…”

Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate

“…By using NCD‐linked FDH* and ME*, we demonstrated successful reductive carboxylation of pyruvate driven by formate and NCD in vitro and in vivo. Recently, formate has been applied to support cell growth of E. coli and Saccharomyces cerevisiae , or to provide reducing power for autotrophic growth on CO 2 by engineered E. coli . With FDH* and other NCD‐linked enzymes, it may provide fascinating opportunities to selectively deliver reducing power, together with CO 2 if an NCD‐linked enzyme such as ME* is involved, for valuable metabolite production, which fits well with the idea of the formate bio‐economy .…”

Expression of Formate-Tetrahydrofolate Ligase Did Not Improve Growth but Interferes With Nitrogen and Carbon Metabolism of Synechocystis sp. PCC 6803

“…In plants and other oxygenic phototrophs, the CO 2 -releasing step via glycine cleavage in the photorespiratory pathway produces high amounts of methylene-THF, which is then used by SHMT to synthesize serine on the expense of a second glycine molecule. It has been discussed that an increased pool of methylene-THF due to efficient formate incorporation could turn photorespiration into less CO 2 -releasing or even CO 2 -fixing, when the glycine-decarboxylase reaction is reversed Recently, the formate-assimilation pathway including a reversed glycine decarboxylase flux was successively established in E. coli , proving the afore calculated kinetical feasibility and functionality of the designed CO 2 -fixing shunt ( Bar-Even et al, 2010 ; Yishai et al, 2017 ; Bang and Lee, 2018 ; Döring et al, 2018 ; Kim et al, 2020 ).…”