A dedicated vector for efficient library construction and high throughput screening in the hyphal fungus Chrysosporium lucknowense

A self-replicating vector was designed that enables the construction of complex libraries in the fungus Chiysosporium lucknowense. The circular vector is linearized in vivo and results in a transformation frequency up to 13,000 transformants/ug ofplasmid DNA. Upon prolonged cultivation of the transformants in liquid medium, the linearized vector finally integrates, mainly as a single copy, at the telomere associated region of the chromosomes. Screening for colonies with compact filamentous growth on agar plates resulted in a C. lucknowense strain with significantly reduced viscosity during fermentation. Further medium modification resulted in conditions that enable cultivation, replication, and screening of a large collection of fungal transformants in microtiter plates. This paper describes the successful integration of library construction and screening in an automated setup using different configurations for a hyphal fungal system.