Overexpression in Escherichia coli of Soluble Aristolochene Synthase from Penicillium roqueforti

Abstract

Aristolochene synthase, a fungal cyclase which has been isolated from Aspergillus terreus and Penicillium roqueforti, catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon aristolochene. The aristolochene synthase gene ( Ari1) of P. roqueforti has previously been cloned and expressed at low levels as a protein A-aristolochene synthase fusion protein in Escherichia coli. We have now used the polymerase chain reaction to amplify the aristolochene synthase coding sequence using engineered primers which produced dsDNA carrying an EcoRI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon and a BamHI site adjacent to the TAA stop codon. The PCR product was digested with EcoRI and BamHI and inserted into the multiple cloning site of the expression vector pLM1 which carried the promoter and translational leader sequence from T7 gene 10 and the E. coli rrnBT 1T 2 tandem transcription terminator. Cloning of the resulting construct into E. coli XL1-Blue and sub-cloning into the expression host E. coli BL21(DE3) gave transformants which expressed aristolochene synthase at levels up to 40% of soluble protein when induced with isopropyl β-D-thiogalactoside. Purification of the recombinant protein by ammonium sulfate precipitation, ion-exchange chromatography on Q Sepharose, and affinity dye chromatography on Reactive Blue 4-agarose gave homogenous aristolochene synthase which had the expected N-terminal sequence, ATSTE, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady-state kinetic parameters when compared to native fungal protein.