The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism.

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.