title: Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Department of Applied Chemistry and Biological Engineering, College of Chemical Engineering, Northeast Dianli University, Jilin 132012, China.
authors.forename: Dayu
authors.initials: D
authors.lastname: Yu
authors.forename: Fuchao
authors.initials: F
authors.lastname: Xu
authors.forename: Jiachen
authors.initials: J
authors.lastname: Zi
authors.forename: Siyuan
authors.initials: S
authors.lastname: Wang
authors.forename: David
authors.initials: D
authors.lastname: Gage
authors.forename: Jia
authors.initials: J
authors.lastname: Zeng
authors.forename: Jixun
authors.initials: J
authors.lastname: Zhan
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism*; D052982:Beauveria / Q000201:enzymology* / Q000235:genetics; D047630:Depsipeptides / Q000096:biosynthesis* / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D047630:Depsipeptides; D005656:Fungal Proteins; C004456:beauvericin; D010453:Peptide Synthases