Characterization of an efficient gene cloning strategy for Aspergillus niger based on an autonomously replicating plasmid: cloning of the nicB gene of A. niger

Abstract

The development of an improved gene cloning strategy by complementation of mutant alleles in Aspergillus niger is described. The strategy is based on the use of a fungal autonomously replicating vector, pAB4-ARp1. This vector was constructed by the introduction of a previously described sequence involved in autonomous replication (AMA1), into a pyrG integrative vector, pAB4-1. With vector pAB4-ARp1, a 10-100-fold increase in transformation frequency was obtained, as compared to pAB4-1. Furthermore, the transformation frequency of a co-transformed plasmid is also increased using pAB4-ARp1. A. niger transformants containing pAB4-ARp1 are mitotically unstable. Co-transformed plasmids strictly co-segregated with the autonomously replicating vector, as a result of recombination between both vectors. The use of pAB4-ARp1 in gene cloning was demonstrated by the complementation of two linkage group-VII-specific A. niger mutants. Complementation of a lysF mutant was achieved by co-transformation of pAB4-ARp1 with total genomic A. niger DNA ('instant bank'). A nicB-deficient A. niger was complemented by co-transformation with pAB4-ARp1 and an A. niger cosmid library. The complementing DNA was re-isolated from a Nic+ transformant by transforming Escherichia coli with total genomic DNA of this transformant. Gene disruption and genetic analysis was carried out to prove that the previously unknown A. niger nicB gene had been cloned.