Transformation of Aspergillus oryzae using the A. niger pyrG gene

Abstract

A transformation system for Aspergillus oryzae based on the orotidine-5'-phosphate decarboxylase gene (pyrG) was developed. Transformation frequencies of up to 16 transformants per microgram of DNA were obtained with the vector pAB4-1, which carries the pyrG gene of A. niger. Southern blotting analysis showed that vector DNA sequences were integrated into the chromosomal DNA, in various copy numbers and presumably at different sites. Efficient cotransformation of an unselectable gene was also shown. Under the conditions used no transformants were obtained with the equivalent pyr4 gene of Neurospora crassa.

Adenine Auxotrophic Mutants ofAspergillus oryzae: Development of a Novel Transformation System with Triple Auxotrophic Hosts

“…Although single and double auxotrophic mutant strains have earlier been constructed for use as suitable transformation hosts, [3][4][5][6] the need to improve a heterologous protein production system and facilitate extensive molecular genetic analyses in A. oryzae has prompted this study.…”

“…In recent years several auxotrophic (argB, pyrG, niaD, and sC) [3][4][5][6] and dominant (amdS and ptrA) 7,8) selectable markers have been developed for transformation of A. oryzae. While most of these transformation techniques used a single marker gene, a double auxotrophic transformation system (niaD À , sC À ) was developed by Yamada et al 6) which enabled two genetic modifications in a strain.…”

Diverse data supports the transition of filamentous fungal model organisms into the post-genomics era

“…While the yield was fewer than one transformant per microgram of plasmid DNA, these transformants were stable for multiple transfers without selection. At the same time, transformation was accomplished by complementation of pyrG (Mattern et al 1987), met (Iimura et al 1987), and later niaD (Unkles et al 1989). Similarly, dominant selectable markers based upon carboxin resistance (Shima et al 2009) and recyclable markers (Maruyama & Kitamoto 2008) as well as strains for targeted transformation (Takahashi et al 2006) based upon the production of non-homologous-integration-deficient strains has facilitated use of A. oryzae .…”

3 Genetic Transfer Applied to Traditional Sake Brewing

“…Shortly thereafter, Gomi, Iimura and Hara (1987) and Mattern et al (1987) further developed the transformation systems using an argB gene encoding ornithine carbamoyltransferase (OCfase) of A. nidulans and a pyrG gene encoding orotidine-5'-phosphate decarboxylase of A. niger, respectively, as selectable markers. The recipient argB mutants can be easily distinguished among many arginine-auxotrophic mutants by their growth phenotype on a medium containing citrulline, although there are no available positive selection procedures for isolating the argB mutants.…”