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Kinetics of mRNA and protein synthesis of genes controlled by the 1,4- β-endoxylanase A promoter in controlled fermentations of Aspergillus awamori

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Author: Gouka, R.J. · Stam, H. · Fellinger, A.J. · Muijsenberg, R.J.G.T. · Wijngaard, A.J. van de · Punt, P.J. · Musters, W. · Hondel, C.A.M.J.J. van den
Type:article
Date:1996
Institution: TNO Voeding
Source:Applied and Environmental Microbiology, 10, 62, 3646-3649
Identifier: 233485
Keywords: Nutrition · Triacylglycerol lipase · Cell culture · Enzyme activity · Enzyme analysis · Gene expression · Gene expression regulation · Glucose metabolism · Promoter region · Protein synthesis · Rna analysis · Aspergillus · Endo-1,4-beta Xylanases · Fermentation · Gene Expression Regulation, Fungal · Glucose · Glucuronidase · Kinetics · Lipase · Promoter Regions (Genetics) · Protein Biosynthesis · Recombinant Fusion Proteins · RNA, Messenger · Xylose · Xylosidases · Aspergillus awamori · Escherichia coli · Thermomyces lanuginosus

Abstract

In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-β-endoxylanase A (EXLA)-overproducing strain. Directly after the D-xylose pulse, exlA mRNA was synthesized, and it reached a constant maximal level after 45 to 60 min. This level was maintained as long as D-xylose was present. The kinetics of mRNA synthesis of the genes encoding Thermomyces lanuginosa lipase (lplA) and Escherichia coli β- glucuronidase (uidA), which were also under the control of the exlA promoter, were similar to those observed for exlA mRNA. The repression of exlA expression was analyzed by pulsing D-glucose to a D-xylose-limited continuous culture. Immediately after the glucose pulse, the exlA mRNA level declined rapidly, with a half-life of approximately 20 to 30 min, and it reached a minimal level after 60 to 90 min. The time span between mRNA synthesis and the secretion of proteins was determined for EXLA and lipase. In both cases, mRNA became visible after approximately 7.5 min. After 1 h, both proteins became detectable in the medium but the rate of secretion of EXLA was faster than that of lipase.