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Polycistronic cellulase gene expression in Pichia pastoris

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Abstract

Cellulases are among the most important groups of industrial enzymes that are widely consumed in biofuel production, pulp and paper, textile, and detergent industries. The methylotrophic yeast Pichia pastoris was used for heterologous expression of a thermophilic cellulase collection. P. pastoris cells were transformed by the codon-optimized polycistronic EBG construct. This construct included egxA gene (from Ampullaria crossean, with endo- and exoglucanase activities), cglT gene (from Thermoanaerobacter brockii, with β-glucosidase activity), and zsgreen (a fluorescent marker). Gene expression was examined at mRNA level using RT-PCR technique. The results indicated successful transcription of all transgenes. CglT and ZsGreen recombinant proteins were respectively detected by enzymatic assay and fluorescent microscope, while endo- and exoglucanase activities were not determined by enzymatic assays. The highest β-glucosidase activity was measured at 65 ºC and pH 5.5. CglT is a good candidate for completing cellulase collections with low β-glucosidase activity. These cellulase sets could be used in biofuel production because of the high glucose tolerance property of CglT.

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Acknowledgements

We would like to appreciate Dr. Moein Farshchian for his assistance in gene construct design.

Funding

This work was supported by the Biotechnology Development Council (grant number, 100485) and Ferdowsi University of Mashhad (grant number, 29524).

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Authors and Affiliations

  1. Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

    Athar Sadat Javanmard, Maryam M. Matin & Ahmad Reza Bahrami

  2. Department of Biology, Faculty of Science, Yasouj University, Yasuj, Iran

    Athar Sadat Javanmard

  3. Novel Diagnostics and Therapeutics Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

    Maryam M. Matin

  4. Industrial Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

    Ahmad Reza Bahrami

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Conceptualization: Bahrami, Matin, Javanmard

Methodology: Javanmard

Data analysis: Javanmard

Writing—original draft preparation: Javanmard

Writing—review, and editing: Bahrami, Matin

Supervision: Bahrami, Matin

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Correspondence to Ahmad Reza Bahrami.

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Javanmard, A.S., Matin, M. & Bahrami, A.R. Polycistronic cellulase gene expression in Pichia pastoris. Biomass Conv. Bioref. 13, 7151–7163 (2023). https://doi.org/10.1007/s13399-021-01765-7

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