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Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

Mueller-Cajar, Oliver; Whitney, Spencer

Description

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1, 5-bisphosphate) carboxylase/ oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions 1174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to...[Show more]

dc.contributor.authorMueller-Cajar, Oliver
dc.contributor.authorWhitney, Spencer
dc.date.accessioned2015-12-10T22:55:17Z
dc.identifier.issn0264-6021
dc.identifier.urihttp://hdl.handle.net/1885/60046
dc.description.abstractThe photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1, 5-bisphosphate) carboxylase/ oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions 1174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XLI-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield ε15-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.
dc.publisherPortland Press
dc.sourceBiochemical Journal
dc.subjectKeywords: Chaperonin; CO fixation; Directed evolution; Protein engineering; RbcX; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco); Escherichia coli; amino acid; chaperonin; glutamine; isoleucine; leucine; methionine; mutant protein; phenylalanine; protein Chaperonin; CO2 fixation; Directed evolution; Protein engineering; RbcX; Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)
dc.titleEvolving improved Synechococcus Rubisco functional expression in Escherichia coli
dc.typeJournal article
local.description.notesImported from ARIES
local.identifier.citationvolume414
dc.date.issued2008
local.identifier.absfor060409 - Molecular Evolution
local.identifier.absfor060107 - Enzymes
local.identifier.ariespublicationu9204316xPUB519
local.type.statusPublished Version
local.contributor.affiliationMueller-Cajar, Oliver, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationWhitney, Spencer, College of Medicine, Biology and Environment, ANU
local.description.embargo2037-12-31
local.bibliographicCitation.startpage205
local.bibliographicCitation.lastpage214
local.identifier.doi10.1042/BJ20080668
dc.date.updated2015-12-10T07:48:51Z
local.identifier.scopusID2-s2.0-50949090470
local.identifier.thomsonID000258799400005
CollectionsANU Research Publications

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