Our goal is to improve the catalytic efficiency and specificity of the Synechococcus PCC6301 ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) through directed evolution. This enzyme catalyzes the rate-limiting step in biological carbon fixation, and its activity probably limits the growth rates of plants and the size of the biosphere. We have developed a novel ultra-high throughput genetic selection for RuBisCO function by engineering the pentose phosphate pathway of wild-type Escherichia coli. Our selection system enables the simultaneous evaluation of millions of RuBisCO mutants, and is significantly more sensitive and rapid than previously reported systems. We have directed the evolution of RuBisCO hypermorphs in three rounds of iterated random mutagenesis and selection. E. coli expressing these RuBisCO variants incorporate radioactive CO2 over ten times more rapidly than the parental strain expressing the wild-type protein. These results demonstrate the utility of our novel genetic selection, and suggest the possibility of bioconverting carbon dioxide into high value energy or chemical products.

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