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Applied and Environmental Microbiology, October 2005, p. 5743-5751, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5743-5751.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Metabolic Engineering of the Purine Pathway for Riboflavin Production in Ashbya gossypii

Alberto Jiménez,¹ María A. Santos,¹ Markus Pompejus,² and José L. Revuelta^1*

Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain,¹ BASF-Aktiengesellschaft, GVF/C-A30, 67056 Ludwigshafen, Germany²

Received 18 April 2005/ Accepted 25 May 2005

Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid³¹⁰, lysine³³³, and alanine⁴¹⁷) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter).

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* Corresponding author. Mailing address: Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain. Phone: 34 923 294671. Fax: 34 923 224876. E-mail: revuelta{at}usal.es.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, October 2005, p. 5743-5751, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5743-5751.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Mateos, L., Jimenez, A., Revuelta, J. L., Santos, M. A. (2006). Purine Biosynthesis, Riboflavin Production, and Trophic-Phase Span Are Controlled by a Myb-Related Transcription Factor in the Fungus Ashbya gossypii.. Appl. Environ. Microbiol. 72: 5052-5060 [Abstract] [Full Text]