Overexpression of cellulolytic enzymes in Pichia pastoris and its applications in ethanol fermentation of rice straw

Abstract

In recent years, the importance of economically viable biorefineries from lignocellulosic biomass is increasing and become a high research priority. However, cellulolytic organisms produce multiple enzymes that are difficult to purify and adapt for biorefining process configurations, notably concerning temperature and pH optima. Therefore, there is still a need to produce fungal cellulases in heterologous systems allowing easy transformation and purification in order to confer cellulolytic capabilities to other non-cellulolytic microorganisms, and develop biocatalysts posing some characteristics over the traditional catalyst through rational or directed evolution strategies. In this context, a glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae was favorably expressed under the control of either constitutive or inducible promoter in Pichia pastoris. Recombinant ExgA had an apparent of molecular weight about 40 kDa having 96% amino acid sequence homology with A. oryzae ExgA. The resulted showed enzymatic activity was highest at 2 U/ml after 42 h for inducible expression, and tolerated to glucose inhibition with Ki, KM and Vmax were 365 mM, 0.56 mM, 10042 μmol min-1 mg of protein-1, respectively. Moreover, the sequences encoding endoglucanase II (eglII) and cellobiohydrolase II (cbhII) from the fungus Trichoderma reesei QM9414 were also successfully cloned and expressed in Yarrowia lipolytica and P. pastoris expression system to point out the possibility to use Y. lipolytica as alternative cellulolytic yeast. Extracellular endoglucanase and cellobiohydrolase activity was maximized in Y. lipolytica Po1d strain using constitutive promoter and preoproLip2 secretion signal. The endoglucanase activity was less than seven-times when compared to recombinant P. pastoris induced by 3.0% (v/v) methanol, whereas, the expression level of cellobiohydrolase from Y. lipolytica was higher than in P. pastoris. The specific activity of both proteins was greater than their homologs produced by P. pastoris, and glycosylation level had little effect on their enzymatic activity and properties. After two rounds of directed evolution via error-prone PCR and site-saturation mutagenesis, variants T257N and T257D were the best thermostable EGII mutants. The thermostability of EGII mutants was improved which half of its activity was lost at 70 °C within 120 min. These results demonstrated that Y. lipolytica is potentially an excellent and attractive system for heterologous expression and high-throughput screening.