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Identification of the Talaromyces cellulolyticus Gene Encoding an Extracellular Enzyme with β-galactosidase Activity and Testing it as a Reporter for Gene Expression Assays

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Abstract

The filamentous fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is currently being intensively studied as a promising industrial producer of a number of secreted cellulolytic enzymes. In this study, the T. cellulolyticus gene lacA, which encodes a protein orthologous to the fungal extracellular β-galactosidases of family 35, was identified. The substitution of the lacA upstream region with a constitutive promoter demonstrated that the product of this gene is effectively secreted and possesses β-galactosidase activity. The optimal pH and temperature values for the hydrolysis of o-nitrophenyl-β-D-galactopyranoside by this enzyme were determined to be pH 4.5–5.5 and 50 °C, respectively. The negligible production of β-galactosidase activity by strains expressing lacA under native regulation raises the possibility of using lacA as a reporter gene. To test this hypothesis, the native promoter of lacA was replaced with the strong inducible promoter of the T. cellulolyticus cellobiohydrolase I gene. The cultivation of the resulting strain in various media showed that the β-galactosidase activity depends on cultivation conditions similar to the cellobiohydrolase activity. Thus, the suitability of lacA as a reporter for evaluating promoters with a wide range of expression profiles was demonstrated.

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Acknowledgements

We would like to thank Dr. Natalia P. Zakataeva (Ajinomoto-Genetika Research Institute, 1st Dorozhny proezd, 1-1, Moscow 117545, Russia) for critical reading of the manuscript and for fruitful discussions.

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  1. Ajinomoto-Genetika Research Institute, 1st Dorozhny proezd, 1-1, Moscow, 117545, Russia

    Alexandra P. Orleneva, Vsevolod A. Serebrianyi, Ekaterina A. Kutukova & Tatiana A. Yampolskaya

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Orleneva, A.P., Serebrianyi, V.A., Kutukova, E.A. et al. Identification of the Talaromyces cellulolyticus Gene Encoding an Extracellular Enzyme with β-galactosidase Activity and Testing it as a Reporter for Gene Expression Assays. Mol Biotechnol 64, 637–649 (2022). https://doi.org/10.1007/s12033-022-00453-9

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