Metabolic engineering approach to elevate the carbon dioxide assimilation of cyanobacteria, which serves as a raw material for biofuel ethanol

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Cyanobacteria are fast-growing photolithototrophs that require radiant energy, using water as electron donor. The biomass of cyanobacteria that are rich in D-glucose may serve as a raw material for biofuel ethanol. Despite this fact, large quantities of CO_2, accumulated from the external medium through a CO_2 concentrating mechanism, are in inorganic form around the CO_2 fixing enzyme Rubisco. The present study was undertaken to elevate the conversion of inorganic carbon to biomaterials of cyanobacterial biomass. To elevate the carbon utilization of cyanobacteria, a metabolic engineering approach to activate a chemosynthesis of cyanobacteria was performed. The enzyme of our concern was carbamoyl-phosphate synthetase (CPSase) that catalyzes the formation of carbamoyl phosphate from CO_2 and NH_3, derived from glutamine and ATP. CPSase consists of a 40kDa glutaminase (OLN) subunit and a 120kDa synthetase (CPS) subunit. The carB gene encoding CPS subunit was cloned from cyanobacterium Synechocystis sp. strain PCC6803 using PCR with a cosmid cs0499 as the template. Escherichia coli BL21(DE3) was transformed with the plasmid containing carB gene from Synechocystis sp. strain PCC6803, however, it represented no activity of CPS subunit. Cloning of carB gene was repeated using E.coli DH5α as the PCR template. E.coli C600 that was transformed with the bacterial carB expressed CPS subunit. Specific activity of CPS subunit was 146 mmol/g.min. High production of CPS subunit was confirmed on SDS-PHAGE. Attempt to transform Synechococcus sp. strain PCC7942-Spc with carB gene was also made.