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Constitutive expression of codon optimized Trichoderma reesei TrCel5A in Pichia pastorisusing GAP promoter
- Source :
- Systems Microbiology and Biomanufacturing; July 2022, Vol. 2 Issue: 3 p498-506, 9p
- Publication Year :
- 2022
-
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 Kmand Vmaxvalues for TrCel5A, were detected to be 5.1 mg/mL and 265.9 μmol/(min.mg), respectively. Thus, TrCel5A was effectively expressed in P.pastorisunder the GAP promoter, and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.
Details
- Language :
- English
- ISSN :
- 26627655 and 26627663
- Volume :
- 2
- Issue :
- 3
- Database :
- Supplemental Index
- Journal :
- Systems Microbiology and Biomanufacturing
- Publication Type :
- Periodical
- Accession number :
- ejs58795072
- Full Text :
- https://doi.org/10.1007/s43393-021-00071-7