RubisCO selection using the vigorously aerobic and metabolically versatile bacterium Ralstonia eutropha

Abstract

Recapturing atmospheric CO2 is key to reducing global warming and increasing biological carbon availability. Ralstonia eutropha is a biotechnologically useful aerobic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) for CO2 utilization, suggesting that it may be a useful host to bioselect RubisCO molecules with improved CO2-capture capabilities. A host strain of R. eutropha was constructed for this purpose after deleting endogenous genes encoding two related RubisCOs. This strain could be complemented for CO2-dependent growth by introducing native or heterologous RubisCO genes. Mutagenesis and suppressor-selection identified amino-acid substitutions in a hydrophobic region that specifically influences RubisCO’s interaction with its substrates, particularly O2, which competes with CO2 at the active site. Unlike most RubisCOs, the R. eutropha enzyme has evolved to retain optimal CO2-fixation rates in a fast-growing host, despite the presence of high levels of competing O2. Yet its structure-function properties resemble those of several commonly found RubisCOs, including the higher-plant enzymes, allowing strategies to engineer analogous enzymes. Because R. eutropha can be cultured rapidly under harsh environmental conditions (e.g., with toxic industrial flue gas), in the presence of near saturation levels of oxygen, artificial selection and directed evolution studies in this organism could potentially impact efforts towards improving RubisCO-dependent biological CO2 utilization in aerobic environments.

Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle

“…In this study, we explore metabolic routes involved in phosphoglycolate salvage in Cupriavidus necator H16 (formerly known as Ralstonia eutropha H16 or Alcaligenes eutrophus H16), the best-studied chemolithoautotrophic microorganism that uses the Calvin cycle under aerobic conditions (but also under anaerobic conditions with nitrate as electron acceptor) ( 18 – 20 ). Unlike cyanobacteria, C. necator does not harbor a CO 2 concentrating mechanism (i.e., a carboxysome with appropriate inorganic carbon transporters), as evident from the relatively high CO 2 specificity of its Rubisco, which falls within the range reported for plants but is much higher than that found in cyanobacteria ( 21 – 23 ). Very little is known about phosphoglycolate salvage in C. necator .…”

Photorespiration pathways in a chemolithoautotroph

“…In this study, we explore metabolic routes involved in photorespiration of Cupriavidus necator H16 (formerly known as Ralstonia eutropha or Alcaligenes eutrophus), the best-studied chemolithoautotrophic microorganism that fixes CO 2 fixation via the Calvin cycle (12)(13)(14). Unlike cyanobacteria, C. necator does not harbor a CO 2 concentrating mechanism (i.e., a carboxysome with appropriate inorganic carbon transporters), as evident from the relatively high CO 2 specificity of its Rubisco, which falls within the range reported for plants but is much higher than that found in cyanobacteria (15)(16)(17). Very little is known about photorespiration in C. necator.…”

Applications of Protein Engineering and Directed Evolution in Plant Research

“…In a clever approach, the problem of false positives was combated by expressing a phosphoribulokinaseneomycin phosphotransferase fusion protein and including the additional selection pressure of antibiotic resistance (Wilson et al, 2018). In an approach similar to the one taken in E. coli, the soil bacterium Ralstonia eutropha has also been developed for in vivo screening of Rubisco variants (Satagopan and Tabita, 2016).…”