Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae

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• 作者：Dayu Yu^a ; ^b ; ¹ ; Fuchao Xu^a ; ^b ; ¹ ; Jiachen Zi^b ; Siyuan Wang^b ; David Gage^b ; Jia Zeng^b ; Jixun Zhan^b ; ^{jixun.zhan@usu.edu}
• 关键词：Fungal cyclooligomer depsipeptide synthetases ; Beauvericins ; Bassianolide ; Ketoisovalerate reductase ; Heterologous expression ; Saccharomyces cerevisiae
• 刊名：Metabolic Engineering
• 出版年：2013
• 出版时间：July, 2013
• 年：2013
• 卷：18
• 期：Complete
• 页码：60-68
• 全文大小：1060 K

文摘

Two fungal cyclooligomer depsipeptide synthetases (CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC 7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.8¡À1.4 mg/l) and bassianolide (21.7¡À0.1 mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding d-hydroxyisovaleric acid (d-Hiv) and the corresponding l-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of d-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins. The total titer of beauvericin and its congeners (beauvericins A-C) was increased to 61.7¡À3.0 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC 7159. Supplement of l-Val at 10 mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.8¡À2.1 mg/l. Using this yeast system, we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules.