Increased expression of a bacterial phosphotriesterase in Escherichia coli through directed evolution.
We devised a growth-based strategy for screening phosphotriesterase mutant libraries for variants with enhanced activity towards organophosphates that generate dimethyl phosphate when hydrolysed. Phosphotriesterase mutants were screened for activity by growing transformed Escherichia coli on agar plates containing methyl paraoxon as a sole phosphorus source. E. coli is capable of growth under these conditions when coexpressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) and the glycerophosphodiester phosphodiesterase from Enterobacter aerogenes (GpdQ). The latter enzyme can hydrolyse the dimethyl phosphate produced by the phosphotriesterase to methyl phosphate, which can then be used by E. coli as a source of phosphate. Phosphotriesterase was expressed from the lac promoter at levels such that its activity was growth-rate limiting. Cultures of the largest colonies (1% of the transformants) were assayed for activity towards paraoxon spectrophotometrically in microtitre plates. This process produced E. coli variants with higher whole cell activity than wild-type, which was found to be a consequence of increased protein expression rather than any increase in enzymatic activity. The mutations present in these mutant enzymes with increased expression were exclusively in the coding region, suggesting the improvement occurs post-transcriptionally.
McLoughlin
Sean Yu
SY
Research School of Chemistry, Building 35 Science Road, Australian National University, Canberra ACT 0200, Australia.
Jackson
Colin
C
Liu
Jian-Wei
JW
Ollis
David
D
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006868:Hydrolysis; D009154:Mutation; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D010261:Paraoxon / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D009943:Organophosphorus Compounds; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase; D044345:Phosphoric Triester Hydrolases; D010261:Paraoxon