Supplementary file 1_Developing Emericellopsis sp. XJ1056 as a versatile fungal platform for high-yield biosynthesis of nonribosomal peptides: a case study on beauvericin.docx

IntroductionBeauvericin is a cyclodepsipeptide with insecticidal, antitumor, and antimicrobial activities, yet its application is limited by low yields. Filamentous fungi represent promising cell factories for complex natural products, but constraints in genetic manipulation and productivity hinder their widespread use.
MethodsIn this study, we developed a high-efficiency fungal cell factory for the de novo biosynthesis of beauvericin. The filamentous fungus Emericellopsis sp. XJ1056, which natively produces high levels of the peptidyl product antiamoebins (5.2 g/kg), was selected as the host due to its exceptional peptide synthetic capacity. A robust genetic toolkit was established, including the construction of a Δku70strain to enhance homologous recombination efficiency (from 9.4% to 50–73.68%). Using Δku70 as the chassis, we integrated the ∼10-kb beauvericin synthetase gene (bbBeas) via multiplexed homologous recombination, along with kivr (encoding 2-ketoisovalerate reductase) under the native helA promoter to supply the precursor D-hydroxyisovaleric acid (D-Hiv). Fermentation conditions were optimized, and strategies including split expression and codon optimization were evaluated.
ResultsThe engineered strain Δku70-bbBeas-kivr synthesized beauvericin without precursor feeding. Optimal production (663.40 mg/kg) was achieved using rice supplemented with wheat bran as the solid medium. While split expression did not further enhance yield, codon optimization (opbbBeas) significantly increased transcriptional efficiency, raising beauvericin production to 921.24 mg/kg dry weight—surpassing the previously reported maximum (98.56 mg/L) in Beauveria bassiana.
DiscussionThis study achieves the highest beauvericin yield reported to date and establishes a generalizable platform for the heterologous production of diverse nonribosomal peptides (NRPs), with broad implications for sustainable biomanufacturing and natural product discovery.