期刊
PROTEIN ENGINEERING DESIGN & SELECTION
卷 19, 期 3, 页码 113-119出版社
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzj010
关键词
carbon dioxide fixation; horizontal transfer; in vitro evolution; metabolic engineering; ribulose 1; 5-bisphosphate carboxylase oxygenase
资金
- NIAID NIH HHS [R21 AI054602-02, 1 R21 AI054602-01, R21 AI054602] Funding Source: Medline
- NIGMS NIH HHS [T32 GM008367] Funding Source: Medline
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.
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