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.
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.
Moralejo
Francisco J
FJ
Instituto de Biotecnología de León INBIOTEC, Parque Científico de León, Aveñida del Real no. 1, 24006 León, Spain.
Cardoza
Rosa Elena
RE
Gutierrez
Santiago
S
Lombraña
Marta
M
Fierro
Francisco
F
Martín
Juan F
JF
D001230:Aspergillus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002876:Chromosomes, Bacterial; D003470:Culture Media; D015536:Down-Regulation; D010450:Endopeptidases / Q000378:metabolism*; D018628:Gene Dosage; D020868:Gene Silencing / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D010940:Plant Proteins / Q000378:metabolism*; D016372:RNA, Antisense / Q000096:biosynthesis / Q000494:pharmacology*; D013549:Sweetening Agents; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003470:Culture Media; C095962:PepB protein, Staphylococcus epidermidis; D010940:Plant Proteins; D016372:RNA, Antisense; D013549:Sweetening Agents; C003427:thaumatin protein, plant; D010450:Endopeptidases