The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation

Strategies for the transformation of filamentous fungi

“…This then enables the transformation system to be optimized, and then used to clone the homologous nitrate reductaseencoding gene. This method has been used with the industrially important A. niger (Unkles et al 1989a) and A. oryzae (Unkles et al 1989b). Similar systems employing selenate resistance to isolate mutants lacking ATP sulphurylase, followed by complementation with the sC gene of A. nidulans (Buxton et al 1989) and¯uoroacetate selection of acetyl CoA synthetase mutants (Gouka et al 1993), are available.…”

Efficient Expression of Mono- and Diacylglycerol Lipase Gene fromPenicillium camembertiiU-150 inAspergillus oryzaeunder the Control of Its Own Promoter

“…A, oryzae niaD mutant AO 1.1 was used as a transformation host throughout this study. Transformation of A, oryzae was done as described by Unkles et al 11) Plasmid pLGG300 and pLGR80, containing the P. camembertii mdlA gene and its introndeleted gene, respectively, were described previously, 8,10) Plasmid construction. Plasmid pNLGG2 and pNLGR2, containing the P. camembertii mdlA gene and its intronless gene, respectively, and the A. oryzae niaD selection marker gene, were constructed in the following way: a 2.0-kb HindIII fragment from pLGG300 or 1.9-kb HindIII fragment from pLGR80 was blunt-ended and ligated into the SmaI site of pUC19.…”

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 .…”