Department of Applied Chemistry and Biological Engineering, College of Chemical Engineering, Northeast Dianli University, Jilin 132012, China.
Metab Eng. 2013 Jul;18:60-8. doi: 10.1016/j.ymben.2013.04.001. Epub 2013 Apr 19.
Two fungal cyclooligomer depsipeptide synthetases(CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC7159, 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 ± 1.4 mg/l) and bassianolide (21.7± 0.1 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 ± 3.0 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC7159. 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 ± 2.1 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.
两种真菌环寡肽脱酰基酶合成酶（CODS），即来自白僵菌 ATCC7159 的 BbBEAS（352 kDa）和 BbBSLS（348 kDa），在酿酒酵母 BJ5464-NpgA 中进行了重组，分别导致相应的抗癌天然产物 beauvericins 和 bassianolide 的产生。工程酿酒酵母 BJ5464-NpgA 菌株中 beauvericins（33.8±1.4 mg/l）和 bassianolide（21.7±0.1 mg/l）的产量与天然产生菌白僵菌相当。喂养 D-羟基异戊酸（D-Hiv）和相应的 L-氨基酸前体可提高 beauvericins 和 bassianolide 的产量。然而，D-Hiv 的高价格限制了其在这些环寡肽的大规模生产中的应用。相反，我们将另一种来自白僵菌的酶，酮异戊酸还原酶（KIVR），工程改造到酿酒酵母 BJ5464-NpgA 中，以增强这种昂贵底物的原位合成。酵母中 BbBEAS 和 KIVR 的共表达导致 beauvericins 产量的显著提高。beauvericin 和其同系物（beauvericins A-C）的总产量增加到 61.7±3.0 mg/l，达到天然产生菌白僵菌 ATCC7159 的 2.6 倍。补充 10 mM 的 L-Val 可提高 KIVR 的底物酮异戊酸的供应，从而进一步将 beauvericins 的总产量提高到 105.8±2.1 mg/l。使用该酵母系统，我们对来自尖孢镰刀菌 NRRL 26139 的未知 CODS 进行了功能表征，将其鉴定为 beauvericin 合成酶，命名为 FvBEAS。我们的工作为真菌 CODS 的功能重建和工程改造提供了一种有用的方法，以有效生产这类抗癌分子。
