Targeted Cloning of a Large Gene Cluster from Lecanicillium Genome by Cre/loxP Based Method.

Functionally related genes often form a large gene cluster on fungal genomes. To analyze, by heterologous expression system, overall pathway in which a series of related genes are involved, the whole gene cluster should be introduced intact into the host strain. However, the construction of a genomic library based on cosmid or bacterial artificial chromosome, and screening of a clone harboring the target region are time consuming and usually require additional cloning of missing regions. The available PCR-based methods are convenient, but are likely to cause unexpected errors during long-range PCR. Therefore, in this study we developed a method for targeted cloning of a large gene cluster based on Cre/loxP-mediated recombination. loxP sequences were integrated at both edges of the targeted region, and the region was excised and cloned as a circular fosmid by in vitro Cre recombination. To facilitate the Cre/loxP-based method, a competent host-vector system was developed, including a double auxotrophic Lecanicillium PTk3 (ΔpyrG trp1−ku80−) strain and two vectors for introducing the loxP sequences, pUTlox and pCCPlox. A targeted region longer than 45 kb in length was successfully cloned by the Cre/loxP-based method.