Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae

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±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 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±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.