Date Published: December 14, 2011
Author(s): Emi Kunitake, Shuji Tani, Jun-ichi Sumitani, Takashi Kawaguchi.
Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus.
The imperfect fungus Aspergillus aculeatus no. F-50 [NBRC 108796], which was isolated from soil in our laboratory, forms black-pigmented asexual spores similar to those of Aspergillus niger. This A. aculeatus strain produces cellulases and hemicellulases that are applicable for synergistic pulp hydrolysis in combination with cellulases from Trichoderma reesei (Murao et al. 1979). Another feature of A. aculeatus is its ability to secrete endogenous proteins in high quantities; A. aculeatus expresses its own β-mannosidase at levels 9 times greater than those of A. oryzae, which is one of the most widely used hosts for protein production (Kanamasa et al. 2007). Therefore, we aimed to genetically modify A. aculeatus to create a high-quality host for the production of autologous cellulases and hemicellulases, and thereby facilitate the production of effective enzymes for the saccharification of unutilized cellulosic biomass and its subsequent bioconversion. To achieve this goal, a method to increase the amount of secreted enzymes is necessary. Although it is important to understand the molecular mechanisms underlying the effective secretion of endogenous enzymes and the associated gene regulation mechanisms, these mechanisms remain unclear (Ooi et al. 1999; Takada et al. 1998 and 2002). Thus, there is an increasing need to establish methods for functional genetic analyses in A. aculeatus.
The results presented here demonstrate that the developed AMT method is applicable for high-throughput insertional mutagenesis in A. aculeatus. This method was developed by optimizing parameters that affect the AMT frequencies such as AS concentration, the ratio of A. tumefaciens cells to A. aculeatus cells, co-cultivation conditions, and starting materials (Michielse et al. 2008). Using the AMT method optimized for A. aculeatus wild-type, 30 transformants per 104 conidia were formed, on average, when using solid IM for co-cultivation. The transformation frequency on solid IM was relatively higher than that for other fungi, e.g., 150-300 transformants per 106 recipients in C. lagenarium, 200 transformants per 106 recipients in A. awamori, 5 transformants per 107 recipients in A. niger, and 50 transformants per 105 recipients in N. crassa (de Groot et al. 1998; Tsuji et al. 2003). A. tumefaciens C58C1 and a binary vector, pBIG2RHPH2, seem to be appropriate for the AMT of A. aculeatus because the transformation frequency is influenced by differences in the binary vector or bacterial strain used (Mullins et al. 2001).
The authors declare that they have no competing interests.