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PEDS Advance Access originally published online on January 19, 2006
Protein Engineering Design and Selection 2006 19(3):113-119; doi:10.1093/protein/gzj010
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli

Monal R. Parikh1, Dina N. Greene1, Kristen K. Woods2 and Ichiro Matsumura1,3

1Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University School of Medicine, Rollins Research Center, Room 4119, 1510 Clifton Road, Atlanta, GA 30322, USA 2Present address: United States Department of Agriculture, Plant Polymer Research, NCAUR, 1815 N. University Street Peoria, IL 61604, USA

3 To whom correspondence should be addressed. E-mail: imatsum{at}emory.edu

The Calvin Cycle is the primary conduit for the fixation of carbon dioxide into the biosphere; ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the rate-limiting fixation step. Our goal is to direct the evolution of RuBisCO variants with improved kinetic and biophysical properties. The Calvin Cycle was partially reconstructed in Escherichia coli; the engineered strain requires the Synechococcus PCC6301 RuBisCO for growth in minimal media supplemented with a pentose. We randomly mutated the gene encoding the large subunit of RuBisCO (rbcL), co-expressed the resulting library with the small subunit (rbcS) and the Synechococcus PCC7492 phosphoribulokinase (prkA), and selected hypermorphic variants. The RuBisCO variants that evolved during three rounds of random mutagenesis and selection were over-expressed, and exhibited 5-fold improvement in specific activity relative to the wild-type enzyme. These results demonstrate a new strategy for the artificial selection of RuBisCO and other non-native metabolic enzymes.

Keywords: carbon dioxide fixation/horizontal transfer/in vitro evolution/metabolic engineering/ribulose 1,5-bisphosphate carboxylase oxygenase

Received October 8, 2005; revised December 11, 2005; accepted December 16, 2005.

Edited by Don Hivert


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