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Author Notes:

To whom correspondence should be addressed. E-mail: imatsum@emory.edu

Present address: United States Department of Agriculture, Plant Polymer Research, NCAUR, 1815 N. University Street Peoria, IL 61604, USA

DG showed that rbcLS-pET30 is more reliable (genetically stable) than rbcLS-pMAL-2px. K.K.W. helped develop the genetic selection (Figure 1b) and CO2 incorporation assay. IM cloned the wild-type rbcL, rbcS and prkA genes and made the wild-type and mutant rbcL-6his-rbcS-pET30 constructs.

We thank Ms Lori Rowe and Mr Charles O’Brien for sequencing them. We are grateful to Dr Tabita for providing the polyclonal anti-RuBisCO antibodies (Figure 2a), Drs Robert Tabita and Robert Houtz for reading the manuscript and to Dr Spencer Whitney for his helpful comments.

Subjects:

Research Funding:

M.R.P. was supported by NSF BIO/MCB (#0109668); D.N.G. by NIH training grant (T32 GM008367); I.M. by NIH/NIAID (1 R21AI054602-01).

Keywords:

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

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

Tools:

Journal Title:

Protein Engineering, Design and Selection

Volume:

Volume 19, Number 3

Publisher:

, Pages 113-119

Type of Work:

Article | Post-print: After Peer Review

Abstract:

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.

Copyright information:

©The Author 2006. Published by Oxford University Press. All rights reserved.

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