Protoplast transformation of Trichoderma reesei RUT C30

Brain-Isasi, Stephanie; Lienqueo, Maria E.

Keywords: heterologous expression, protoplast, fungal transformation


Single Cell Protein (SCP) is an alternative and sustainable protein source derived from microbial biomass. It is flexible to nutritional requirements because of wide microbial biodiversity. In order to provide an efficient SCP production, microbial cells should be able to ferment low-cost carbon sources. Microbial cells must be safe and able to grow in a high rate under fermentation conditions. Trichoderma reesei RUT C30 is a cellulolytic terrestrial fungi with a high protein content (up to 40 % dry weight). The aim of this work is to overexpress other cellulases in Trichoderma reesei, along with process optimization for biomass production using macroalgal waste derived from Ulva green tides. TO carry out this work, transformation of T. reesei RUT C30 with integration cassettes comprising cellobiohydrolase I gene. We initially assessed the electroporation procedure described by Linger et al. (2015). After four attempts, we assessed other electroporation protocols (Schuster et al., 2012; and US 2010/0304468 A1). Given that we were not able to transform T. reesei by means of electroporation, we leaned towards protoplast-mediated transformation (as described by Ballance et al., 1983 and Pentillä et al., 1987), although the main drawback of this method is its low efficiency. The average yield is between 3 × 108 and 1 × 109 protoplasts (starting from five 10-day-old plates) and the average viability of PEG-treated protoplasts is around 0.5 % to 1 % (Gruber et al. 1990a y 1990b). Results: We obtained two transformed strains comprising pTrEno-cbdI cassette. These results were also verified by sequence analysis.

Más información

Fecha de publicación: 2019
Año de Inicio/Término: 27-29 November 2019
Idioma: English
Financiamiento/Sponsor: Centre for Biotechnology and Bioengineering