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.

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation

“…The correctly expression plasmids were linearized with Sal I endonuclease, and the DNA was purified using a PCR product recovery kit and transformed into P. pastoris X33 competent cells [30,31]. After quickly adding 1.0 mL of pre-chilled 1.0 M sorbitol, the cells were incubated at 30 °C, 220 r/min for 2 h before being inoculated in yeast extract peptone dextrose (YPD) plates containing 0.1% zeocin antibiotic, and randomly selecting single colonies for shake flask fermentation re-screening.…”

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

“…The slow methanol-utilizing strain KM71H exhibited the highest yield of endoglucanasethat is, 550 mg L −1 (25-fold higher than wild-type)with higher optimum temperature (80 °C). 27 Codon-optimized T. reesei endoglucanase 1 (egl1) gene under P. pastoris expression system increased egl1 expression as well as enzyme activity up to 1.24 fold. 68 Codon optimized xynA gene from Penicillium citrinum was expressed under pGAP and pAOX1 promoter in P. pastoris.…”

“…A codon‐optimized Acidothermus cellulolyticus endoglucanase synthetic gene was expressed under the pAOX1 promoter of P. pastoris (KM71H). The slow methanol‐utilizing strain KM71H exhibited the highest yield of endoglucanase – that is, 550 mg L −1 (25‐fold higher than wild‐type) – with higher optimum temperature (80 °C) 27 . Codon‐optimized T. reesei endoglucanase 1 (egl1) gene under P. pastoris expression system increased egl1 expression as well as enzyme activity up to 1.24 fold 68 .…”