Replacing the Calvin cycle with the reductive glycine pathway inCupriavidus necator

Abstract

14Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial 15 feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant 16 hosts that can grow on formate, but it uses the inefficient Calvin cycle. Here, we redesign C. necator 17 metabolism for formate assimilation via the highly efficient synthetic reductive glycine pathway. First, we 18 demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore 19 the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By 20 integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the 21 synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism 22 and use short-term evolution to optimize pathway activity, doubling the growth yield on formate and 23 quadrupling the growth rate. This study thus paves the way towards an ideal microbial platform for realizing 24 the formate bioeconomy. 25 26 27 28 Microbial biosynthesis offers an environmentally friendly alternative to fossil-based production. However, the 31 limited availability and questionable sustainability of microbial feedstocks hamper the expansion of 32 biotechnological production and the establishment of a circular carbon economy. The common substrates for 33 microbial bioproduction are plant-based sugars, the utilization of which competes with food supply and 34 necessitates vast land use that negatively impacts the environment. Moreover, alternative feedstocks, such 35 as lignocellulosic biomass, suffer from crucial drawbacks, such as difficult and expensive processing 1 . A 36 fundamental limitation of all photosynthesis-based resources is the low energy conversion efficiency 37 associated with this process, typically below 1% 2,3 . 38Electromicrobial production has gained attention as an alternative route towards sustainable biotechnology 4,5 . 39 This strategy is based on the use of two key feedstocks: CO2-free electricitye.g. from solar, wind, hydro -40 the production of which is rapidly growing, and CO2, a virtually unlimited carbon source, captured either from 41 point sources or directly from air. Some microbes can grow by receiving electrons directly from a cathode; 42 however, low current densities limit the economic viability of this approach 6,7 . A more feasible option is the 43 electrochemical production of small reduced compounds 6 that are subsequently fed to microbes and then 44 converted into value-added chemicals. Among the possible mediator compounds, hydrogen, carbon 45 monoxide, and formate can be produced at high efficiency and rate 8 . Whereas hydrogen and carbon monoxide 46 are gases of low solubility, formate is completely miscible and can be readily introduced to microbial cells 47 without mass transfer limitations and without major safety concerns 9 . Hence, establishing a "formate bio-48 economy" has been proposed as a ro...

From CO2 to Bioplastic – Coupling the Electrochemical CO2 Reduction with a Microbial Product Generation by Drop‐in Electrolysis

“…To the author's opinion, two different approaches appear promising: on the one hand, a biotransformation in a bioreactor system, providing ideal fermentation conditions such as constant feed and automated pH regulation, might improve the substrate to product conversion. On the other hand, the rather inefficient Calvin cycle, on which the PHB production is based on might be replaced by a more efficient metabolic cycles such as the reductive glycine pathway, as it has been recently demonstrated by Claassens et al [38] Nevertheless, the molecular complexity of PHB as model polymer must be taken into account for the evaluation of the process. Comparing with literature, Krieg et al reached a FE of 0.92 % for the microbial electrosynthesis of the complex molecule α-Humulen from CO 2 .…”

Bioelectrochemical engineering analysis of formate-mediated microbial electrosynthesis

“…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).…”

“…2). [21,23,37] We used experimental parameters describing electrochemical reduction towards HCOOand H2 on a Sn electrode because of its ability to selectively produce formate [18,53] and we compared operation with two different gas feed compositions: , 2 = 0.5, corresponding to an equal mixture of CO2 and O2 in the gas feed stream; and , 2 = 0.833, corresponding to a CO2 partial pressure of 1 atm for our model. Current density towards CO2 reduction or H2 production increases exponentially as a function of voltage following Butler-Volmer kinetics ( Fig.…”

“…[24] We evaluated the potential for improved productivity, carbon utilization, and energy efficiency by formatotrophic growth using the rGlyP since this pathway has recently been engineered in C. necator and E. coli. [21,22] The maximum biomass productivity for E. coli is ~22% higher than for wild-type C. necator (i.e. C. necator using the Calvin cycle), reaching ~2.15 g L -1 hr -1 at ~2.3 V (Fig.…”

“…[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.…”