Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter

Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.

Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system

ABSTRACT The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Cloning, characterization and directed evolution of a xylosidase from Aspergillus niger

β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant.