Transformation of IntactAspergillus nigerby Electroporation

Abstract

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per micrograms of integrative vector and 100 colonies per micrograms of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.

Strategies for the transformation of filamentous fungi

“…The ®rst procedure involves the pre-treatment of conidial preparations with a cell wall weakening agent (Chakraborty et al 1991;Ozeki et al 1994). An alternative method is to use electroporation of protoplasts (Kothe and Free 1996).…”

“…A method for electroporation of germinated conidia of A. nidulans, which circumvents the requirements for protoplast formation, has been described (Sa Ânchez and Aguirre 1996). Electroporation systems have been developed for a large number of ®lamentous fungi including N. crassa, Penicillium urticae, Leptosphaeria maculans, A. oryzae (Chakraborty et al 1991), Scedosporium proli®cans (Ruiz-Dõ Âez and Martõ Ânez-Sua Ârez 1999), A. nidulans (Sa Ânchez and Aguirre 1996), A. niger (Ozeki et al 1994), Wangiella dermatitidis, A. fumigatus (Kwon-Chung et al 1998) etc. Actually, electroporation is the most used method for transforming ®lamentous fungi.…”

Genetic and Genomic Manipulations in Aspergillus niger

“…Recently, this method has been adapted for high-throughput platform with transformation and regeneration conducted in microtiterplate (Gielesen and van den Berg 2013 ). Transformation by electroporation of either protoplasts (Ward et al 1989 ) or germinating spores yields fewer transformants, 10-500 per μg DNA for integrative and episomal vectors, respectively (Ozeki et al 1994 ). Agrobacterium-mediated transformation has also been attempted, but it is rather ineffi cient with up to fi ve transformants per μg of DNA (de Groot et al 1998 ;Michielse et al 2005 ).…”

Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance

“…At the end of the experiments, the transformed conidia were plated on PDA medium, incubated at 40³C for 12^16 h, and then, the same volume of a PDA over-layer containing 200 Wg ml 3I of hygromycin was added. The EL procedure was carried out according to the method of Ozeki et al [8], using a Gene Pulser Apparatus (Bio-Rad) and electroporation disposable cuvettes (Bio-Rad, USA) with an inter-electrode distance of 0.2 cm. The electrical parameters were set up after experimental optimization.…”

“…Alternative methods have been developed to eliminate this step. Recent examples of whole-cell transformation include the electroporation of either germinating conidia treated with L-glucuronidase [8] or mycelial fragments [9] and the permeabilization of germinated conidia by lithium acetate treatment [10]. All such methods gave reproducible results, thus making transformation more reliable.…”