Production of isokaurenoic and kaurenoic acids in double transformed cells of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> |
| |
Authors: | Iván Dávila-Olivares María Blanca Estela Lara-Vergara Yesenia Pacheco-Hernández Nemesio Villa-Ruano |
| |
Institution: | 1.Unidad Académica de Ciencias Biológicas,Universidad Autónoma de Zacatecas,Zacatecas,Mexico;2.Departamento de Ciencias Químico-Biológicas,Universidad de las Américas Puebla,Cholula,Mexico;3.Instituto Politécnico Nacional,Centro de Investigación en Biotecnología Aplicada,Tepetitla,Mexico;4.Universidad de la Sierra Sur,Miahuatlán de Porfirio Díaz,Mexico |
| |
Abstract: | 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 Km app of MtKO showed more affinity for ent-kaurene (80.5 µM) than for isokaurene (96.4 µM). Interestingly, LMtKO exhibited an increase of the affinity for isokaurene (79.6 µM) but simultaneously showed an enhancement in the Vmax for both substrates (32.6–38.9 μmol?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 ~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 Vmax from 0.0249 to 0.0104 mM min?1 but not affecting the Km (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 µg mL?1, respectively. |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|