Aristolochene synthase: Purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase

Aristolochene synthase catalyzes the cyclization of farnesyl diphosphate (6 ) to (+)-aristolochene (1), The Aspergillus terreus enzyme has been purifie d 75-fold to homogeneity in six steps. Based on the sequence of 3 internal peptides obtained by Lys-C digestion of the native protein, a set of degene rate PCR primers was used to amplify a 550-bp segment of cDNA corresponding to a portion of the aristolochene synthase transcript, A second round of P CR using specific primers was used to prepare a P-32-labeled 180-bp segment , which was used to screen an A. terreus cDNA library prepared using lambda ZapII, resulting in the identification and sequencing of the A. terreus ar istolochene synthase cDNA Aristolochene synthase was encoded by an open rea ding frame (ORF) of 960 bp, corresponding to a protein of 320 amino acids w ith a predicted M-D of 36,480. Comparison of the A. terreus ORF with the se quence of the previously described aristolochene synthase from Penicillium roqueforti revealed a 66% of identity at the nucleic acid level and a 70% i dentity at the deduced amino acid level between the aristolochene synthases from the two different fungal sources. PCR was used to insert the A. terre us aristolochene synthase gene into the T7lac expression vector pET11a. Clo ning of the resultant construct into Escherichia coli XL1-Blue and subcloni ng into the expression host E. coli BL21(DE3)/pLysS gave, after induction w ith IPTG, soluble aristolochene synthase as 5-10% of total protein. The rec ombinant aristolochene synthase, which was purified 13-fold to homogeneity, appeared to be identical in all respects with the native A. terreus enzyme , displaying essentially the same steady-state kinetic parameters, with a K -m of 15 nM and k(cat) 0.015 s(-1). Using PCR to amplify the aristolochene synthase gene (Aril) from A. terreus genomic DNA revealed the presence of 2 introns, identical in relative location but different in both sequence and length compared to the corresponding Aril gene of P. roqueforti. (C) 2000 Academic Press.