Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus.
The development of an efficient fungal expression system for recombinant proteins requires an improved transformation system for the host organism. We report a facile, efficient and highly reproducible electroporation-mediated transformation system for Thermomyces lanuginosus with a transformation efficiency of 1.27 x 10(3) transformants/microg DNA. Conidia of T. lanuginosus were stably transformed to hygromycin B resistance using the pBC-hygro plasmid construct. Optimal electroporation conditions for maximum transformation of 10(8) conidia ml(-1) in 1.2 M sorbitol buffer (15 mM DTT, 5% DMSO) were a field strength of 5.5 kV/cm for 10 ms and a DNA concentration of 0.5 microg microl(-1). Transformants were recovered in prewarmed potato dextrose broth supplemented with 1.2 M sorbitol for 1-2 h at 50 degrees C. The presence of the hygromycin B phosphotransferase (hph) gene and non-integrative transformation was confirmed by PCR, Southern hybridization analysis and plasmid recovery. Transformants exhibited altered phenotype with reduced pigmentation and transformants were found to be mitotically stable after 15 sequential transfers on nonselective media without selective pressure.
Gangavaram
Lakshmi Prasanna
LP
Department of Biotechnology and Food Technology, Durban University of Technology, Durban 4000, South Africa.
Mchunu
Nokuthula
N
Ramakrishnan
Priya
P
Singh
Suren
S
Permaul
Kugen
K