Polycistronic cellulase gene expression in Pichia pastoris

Cellulases are among the most important groups of industrial enzymes that are widely consumed in biofuel production, pulp and paper, textile, and detergent industries. The methylotrophic yeast Pichia pastoris was used for heterologous expression of a thermophilic cellulase collection. P. pastoris cells were transformed by the codon-optimized polycistronic EBG construct. This construct included egxA gene (from Ampullaria crossean, with endo- and exoglucanase activities), cglT gene (from Thermoanaerobacter brockii, with β-glucosidase activity), and zsgreen (a fluorescent marker). Gene expression was examined at mRNA level using RT-PCR technique. The results indicated successful transcription of all transgenes. CglT and ZsGreen recombinant proteins were respectively detected by enzymatic assay and fluorescent microscope, while endo- and exoglucanase activities were not determined by enzymatic assays. The highest β-glucosidase activity was measured at 65 ºC and pH 5.5. CglT is a good candidate for completing cellulase collections with low β-glucosidase activity. These cellulase sets could be used in biofuel production because of the high glucose tolerance property of CglT.