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Applied and Environmental Microbiology, July 2002, p. 3550-3559, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3550-3559.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production

Francisco J. Moralejo,¹ Rosa Elena Cardoza,¹ Santiago Gutierrez,^1,2 Marta Lombraña,¹ Francisco Fierro,^1,2 and Juan F. Martín^1,2*

Instituto de Biotecnología de León INBIOTEC, Parque Científico de León, 24006 León,¹ Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain²

Received 27 November 2001/ Accepted 19 April 2002

Aspergillopepsin B was identified in culture broths of Aspergillus awamori by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The pepB gene encoding aspergillopepsin B of A. awamori was cloned and characterized. It is located in chromosome IV of A. awamori, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of Aspergillus niger var. macrosporus. The pepB gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the pepB gene in the antisense orientation downstream from the gpdA promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the pepB gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the pepB gene was deleted by double crossover. Two of the selected transformants lacked the endogenous pepB gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in A. awamori.

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* Corresponding author. Mailing address: Instituto de Biotecnología de León INBIOTEC, Parque Científico de León, Avda. del Real, no. 1, 24006 León, Spain. Phone: 34-987-210308. Fax: 34-987-210388. E-mail: degjmm{at}unileon.es.

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Applied and Environmental Microbiology, July 2002, p. 3550-3559, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3550-3559.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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