A pyruvate decarboxylase gene from Aspergillus parasiticus

FEMS Microbiology Letters 117 (1994) 207-210 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 05884

A pyruvate decarboxylase gene from Aspergillus parasiticus Vicente Sanchis 1, Inmaculada Vinas 1, Ian N. Roberts, David J. Jeenes, Adrian J. Watson and David B. Archer * Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK (Received 24 December 1993; revision received and accepted 15 January 1994)

Abstract: A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase. Key words: Aspergillus parasiticus; Ethanol; Pyruvate decarboxylase

Introduction

Several species of filamentous fungi produce ethanol in submerged culture despite having an obligate growth requirement for oxygen [1]. This is a result of the onset of anoxic conditions which can occur in shake flask cultures that contain high levels of fungal mass [1,2]. Production of ethanol presumably occurs from pyruvic acid through the combined activities of pyruvate decarboxylase, which converts pyruvic acid to acetaldehyde, and alcohol dehydrogenase which converts acetaldehyde to ethanol. Although alco-

hol dehydrogenase genes have been isolated from filamentous fungi including species of Aspergillus [3,4] no pyruvate decarboxylase (PDC)-encoding gene has been described in fungi other than yeasts. In Saccharomyces cerevisiae, for example, three PDC genes have been described [5-7]. In this paper we describe the isolation and sequence analysis of pdcA from A. parasiticus. We also demonstrate that, while ethanol can be a growth substrate under some conditions, it can also be produced and accumulate in shake flask cultures of A. parasiticus, A. niger and A. nidulans.

Materials and Methods * Corresponding author. Tel.: (0603) 255 256; Fax: (0603) 507 723. x Present address: UdL IRTA, Centre R&D Lleida, Food Technology Unit, Rovira Roure 177, 25006 Lleida, Spain.

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Genomic DNA was extracted from A. parasiticus strain SU-1 (ATCC 56775) [8] and restricted by EcoRI. A fraction (4-10 kb) obtained from

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sucrose gradients [9] was used to prepare a library in Lambda ZAP I1 according to instructions (Stratagene). Oligonucleotide primers for DNA amplification by polymerase chain reaction (PCR) were synthesized on an applied Biosystems 394 CTTCTGTCATATATAAAGAAGAAASATGTATACAGTATTGAGGAGAGCAGCGCAAGTAAC l GTAAAATCTAGTTCACTCCGACAAAAAAACC

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