Phototrophic growth of a Rubisco-deficient mesophilic purple nonsulfur bacterium harboring a Type III Rubisco from a hyperthermophilic archaeon

The hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 harbors a structurally novel, Type III Rubisco (Rbc_Tk). In terms of protein engineering of Rubiscos, the enzyme may provide an alternative target to the conventional Type I and Type II enzymes. With a future aim to improve the catalytic properties of Rbc_Tk, here we examined whether or not the enzyme could support growth of a mesophilic organism dependent on CO₂ fixation. Via double-crossover homologous recombination, we first deleted three Rubisco genes present on the chromosome of the photosynthetic mesophile Rhodopseudomonas palustris No. 7. The mutant strain (Δ3) could neither grow under photoautotrophic nor photoheterotrophic conditions. We introduced the rbc_Tk gene into strain Δ3 either on a plasmid, or by integrating the gene onto the chromosome. The two transformant strains harboring rbc_Tk displayed growth under photoautotrophic and photoheterotrophic conditions, both dependent on CO₂ fixation. Specific growth rates and Rubisco activity levels were compared under photoheterotrophic conditions among the two transformants and the wild-type strain. We observed that the levels of Rubisco activity in the respective cell-free extracts correlated well with the specific growth rates. Immunoprecipitation experiments revealed that Rubisco activity detected in the transformants was derived solely from Rbc_Tk. These results demonstrated that the Type III Rbc_Tk from a hyperthermophile could support CO₂ fixation in a mesophilic organism, and that the specific growth rate of the transformant can be used as a convenient parameter for selection of engineered proteins with improved Rubisco activity.