Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803†rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803Δrbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.