Production of isokaurenoic and kaurenoic acids in double transformed cells of Saccharomyces cerevisiae

摘要

We report the bifunctional activity of the native ent-kaurene oxidase from Montanoa tomentosa (MtKO) and its N-terminal modified version (LMtKO) for producing both isokaurenoic acid and kaurenoic acid in Saccharomyces cerevisiae. The K-m app of MtKO showed more affinity for ent-kaurene (80.5 mu M) than for isokaurene (96.4 mu M). Interestingly, LMtKO exhibited an increase of the affinity for isokaurene (79.6 mu M) but simultaneously showed an enhancement in the V-max for both substrates (32.6-38.9 mu mol(-1) mg(-1) h(-1)). Biotransformation assays using isokaurene and yeasts containing LMtKO, resulted in 70% more production of isokaurenoic acid, when compared with the yields from yeasts expressing MtKO. Likewise, biotransformation assays using geranylgeraniol and double transformed cells of S. cerevisiae containing an optimized version the ent-kaurene synthase from Phaeosphaeria sp. L487 (optKS) and the LMtKO, produced similar to 25% more kaurenoic acid than the yeasts containing optKS and MtKO. The isokaurenoic acid synthesized by transgenic yeasts was tested for its anti-acetylcholinesterase and antimicrobial properties. Isokaurenoic acid generated a non-competitive inhibition on acetylcholinesterase, decreasing the V-max from 0.0249 to 0.0104 mM min(-1) but not affecting the K-m (0.714 mM). The same diterpene showed antifungal activity against Fusarium oxysporum, Aspergillus niger and Phytophtora infestans with a minimum inhibitory concentration of 15.3, 18.3 and 19.2 mu g mL(-1), respectively.