Engineering the Calvin-Benson-Bassham cycle and hydrogen utilization pathway of Ralstonia eutropha for improved...

CO₂ is fixed by all living organisms with an autotrophic metabolism, among which the Calvin-Benson-Bassham (CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO₂ fixation efficiency of cells is an important subject of biological research with significant application potential.

In this work, the autotrophic microbe Ralstonia eutropha (Cupriavidus necator) was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha, we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were functional during autotrophic growth of R. eutropha. SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system(A quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins) and RbcX(A chaperone in the folding of Rubisco), the culture OD₆₀₀ of engineered strain increased 89.2% after 72 h of autotrophic growth, although the difference was decreased at 96 h, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and lead to improved growth in comparison to the host specific enzyme. Meanwhile, expression of hydrogenases was optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD₆₀₀ to 93.4% at 72 h. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.7%.

To our best knowledge, this is the first report of successfully engineering the CBB pathway and hydrogenases of R. eutropha for improved activity, and is one of only a few cases where the efficiency of CO₂ assimilation pathway was improved. Our work demonstrates that R. eutropha is a useful platform for studying and engineering the CBB for applications.