Cellulase Production in Trichoderma Strains

Trichoderma reesei QM9414 (ATCC 26921), which is a general cellulase producer, and RUT-C30 (ATCC 56765), which is another cellulase high-producing strain that is less sensitive to glucose repression, were utilized as the control strains for the comparison of cellulase production. The strain SN1 is a hypercellulolytic variant isolated from our laboratory (Song et al., 2016 (link)) and used as the initial host for strain improvement. Escherichia coli DH5a (TransGen, Beijing, China) was used for vector construction and propagation. The pMD18-T cloning vector (Takara, Otsu, Japan) was purchased for TA cloning. The pTHB vector was used for bgl1 overexpression; this vector contained a T. reesei bgl1 expression cassette under the control of a modified four-copy cbh1 promoter, as described by Zhang et al. (2010) (link). The plasmid pAB4-1 contained the Aspergillus niger pyrG gene as a selection marker to encode orotidine-5′-phosphate decarboxylase (Hartl and Seiboth, 2005 (link)). All strains were grown and maintained on a potato dextrose agar plate (PDA) containing 20.0 g/L D-glucose and 20.0 g/L agar for 5–7 days at 30°C. The conidia were harvested, and 10⁶ conidia were inoculated in a 500 mL flask containing 150 mL minimal medium (MM; Penttilä et al., 1987 (link)). MM with 300 μg/mL hygromycin B and 1.5 mg/mL 5′-FOA (Sigma, USA) was applied as the selective medium to screen the uracil auxotroph transformants (Singh et al., 2015 (link)). An esculin plate containing 3 g/L of esculin, 10 g/L of sodium carboxymethy cellulose (CMC–Na), 0.5 g/L of ferric citrate, and 20 g/L agar was utilized to screen the strains showing β-glucosidase (BGL) activity. A CMC plate containing 10 g/L of CMC–Na, 1 g of yeast extract, and 20 g/L agar was utilized to screen the strains showing cellulase (EG) activity.