Growth of E. coli on formate and methanol via the reductive glycine pathway

Abstract

Engineering a biotechnological microorganism for growth on one-carbon (C1) intermediates, produced from the abiotic activation of CO2, is a key synthetic biology step towards the valorization of this greenhouse gas to commodity chemicals. Here, we redesign the central carbon metabolism of the model bacterium Escherichia coli for growth on C1 compounds using the reductive glycine pathway. Sequential genomic introduction of the four metabolic modules of the synthetic pathway resulted in a strain capable of growth on formate and CO2 with a doubling time of ~70 hours and growth yield of ~1.5 gCDW / molformate. Short-term evolution decreased doubling time to less than 8 hours and improved biomass yield to 2.3 gCDW / mol-formate. Growth on methanol and CO2 was achieved by the expression of methanol dehydrogenase in the evolved strain. Establishing synthetic formatotrophy and methylotrophy, as demonstrated here, paves the way for sustainable bioproduction rooted in CO2 and renewable energy.

Awakening a latent carbon fixation cycle inEscherichia coli

“…Formate is then assimilated via one of several variants of the reductive glycine pathway 18,19 (see Fig. 1B for an example), the activity of which was recently demonstrated in E. coli 20,21 . Alternatively, formate is assimilated via variants of the serine cycle, or, more precisely, the previously suggested serine-threonine cycle 22 (see Fig. 1C for an example).…”

In vivoselection for formate dehydrogenases with high efficiency and specificity towards NADP+

“…Using an NAD + -dependent FDH to provide reducing power requires high flux via the membrane-associated transhydrogenase to regenerate NAPDH 51,52 , at the cost of dissipating the proton motive force and reducing biomass yield. Expressing a NADP + -dependent FDH would enable direct production of NADPH (which can regenerate NADH via the soluble transhydrogenase 51 ), thus saving energy and increasing biomass yield.…”

Bioelectrochemical engineering analysis of formate-mediated microbial electrosynthesis

“…We consider the growth of two different model organisms: Cupriavidus necator, a well-studied chemolithoautotrophic organism in MES systems that is capable of growth on both formate and H2 as energy sources, and Escherichia coli, the biotechnology workhorse bacterium that has been recently engineered to support formatotrophic growth. [22]…”

“…By mole balance, the stoichiometric coefficients are for biomass with the composition 1 1.77 0.44 0.25 . [23] The reductive glycine pathway (rGlyP), recently engineered in both C. necator [21] and E. coli [22] and discovered in wild-type phosphite-oxidizing organisms, [36] is predicted to enable higher biomass yield using formate as a growth substrate than the Calvin cycle (Fig. 1B).…”

“…[3,5] Formate/ic acid stands out as an especially promising redox mediator because it is readily and specifically produced from CO2 [18][19][20] and multiple natural and engineered formatotrophic growth mechanisms exist in workhorse bacteria. [21][22][23][24][25] Initial scale-up, [26] component integration, [27] and media optimization [28] studies have been performed for MES systems, demonstrating the need for careful attention to process parameters including the gas/liquid mass transfer coefficient (kLa). However, progress towards scaled, optimized systems has been limited, in part due to the complex nature of coupled bioelectrochemical systems.…”