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Minerva Biotecnologica 2014 March;26(1):23-30


lingua: Inglese

Molecular cloning of endoglucanase gene of Trichoderma Reesei 5A into Saccharomyces cerevisiae

Pannu R. 1, Kumar D. 2, Rani R. 1, Chaudhary K. 1

1 Department of Biotechnology and Molecular Biology CCS Haryana Agricultural University Hisar-125004 Haryana, India; 2 Department of Biotechnology Deenbandhu Chhotu Ram University of Science and Technology, Murthal Sonepat Haryana, India


Aim: In this study an attempt has been made to clone endoglucanase gene from Trichoderma reesei 5A into Saccharomyces cerevisiae for direct conversion cellulosic biomass into ethanol.
Methods: In the present study seven Trichoderma reesei strains were used for characterization of cellulase production and the most efficient strain, Trichoderma reesei 5A has been used as a source of endoglucanase gene. This gene was transformed into S. cerevisiae. The recombinant clones of S. cerevisiae were screened on YEP medium supplemented with 1% phosphocellulose and 100 mg/mL ampicillin and further tested for ethanol production using rice straw as substrate.
Results: It was observed that cellulase activity of T. reesei strains varies from 0.73 to 3.11 IU and T. reesei 5A shown maximum cellulase activity after 96 h of growth perod at 37 °C. Total genomic DNA was isolated from T. reesei 5A and then it was partially digested with Sau 3A and ligated to vector YEpFLAG-1, linearized with BamH1. The construct was used to transform Escherichia coli and recombinant clone(s) were screened on Reese medium supplemented with carboxymethyl cellulose and ampicillin. The E. coli recombinant clones were further confirmed by gene specific amplification using PCR. S. cerevisiae was transformed with the recombinant plasmids YEpFLAG-1-cel-exo and YEpFLAG-1-cel-endo isolated from E. coli transformants. Carboxymethyl cellulase (CMCase) activity was observed in three yeast transformants Tr-2, Tr-4 and Tr-6 as 0.50, 0.70 and 0.80 IU, respectively. no FPase (exoglucanse) activity was observed in any of the yeast transformants.
Conclusion: Direct conversion of cellulose to ethanol by the yeast transformants confirms that our cloning was successful and these clones can be further utilized for the production of cellulase.

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