Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli.
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.
Parikh
Monal R
MR
Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University School of Medicine, Rollins Research Center, Room 4119, Atlanta, GA 30322, USA.
Greene
Dina N
DN
Woods
Kristen K
KK
Matsumura
Ichiro
I
D015153:Blotting, Western; D002245:Carbon Dioxide / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D014644:Genetic Variation; D007700:Kinetics; D009154:Mutation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D046940:Synechococcus / Q000201:enzymology / Q000254:growth & development
D004269:DNA, Bacterial; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase