Constitutive expression of codon optimized Trichoderma reesei TrCel5A in Pichia pastoris using GAP promoter

Abstract

To address the deficient activity of TrCel5A in naturally secreted cellulase preparation, this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris. A recombinant TrCel5A was screened out after gene optimization, synthesis, and expression. The biochemical and enzymatic properties of the new recombinant were characterized. As a result, optimization of shake-flask fermentation of the recombinant was obtained at 28 °C, 2% inoculum volume, an initial pH of 6.0, as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L, respectively. Under the above-optimized conditions, the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation. To further enhance enzyme production, pilot-scale cultivation was evaluated using 5-L bioreactors. Using high-cell-density fermentation, the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L. In addition, the kinetic factors, including K _m and V _max values for TrCel5A, were detected to be 5.1 mg/mL and 265.9 μmol/(min^.mg), respectively. Thus, TrCel5A was effectively expressed in P.pastoris under the GAP promoter, and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.

Funding

national key research and development of China,(2019YFE0114600); National Natural Science Foundation of China,(21776114)