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J. Biol. Chem., Vol. 283, Issue 37, 25186-25199, September 12, 2008

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Antibiotic Overproduction in Streptomyces coelicolor A3(2) Mediated by Phosphofructokinase Deletion^*

Irina Borodina¹², Jeroen Siebring¹, Jie Zhang, Colin P. Smith^¶, Geertje van Keulen^||, Lubbert Dijkhuizen, and Jens Nielsen³

From the Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, 2800 Kongens Lyngby, Denmark, the Department of Microbiology, Groningen Biotechnology and Biomolecular Sciences Institute, University of Groningen, P. O. Box 14, 9750 AA, Haren, The Netherlands, ^¶Functional Genomics Laboratory, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom, and ^||Biological Sciences, School of the Environment and Society, Swansea University, Swansea SA2 8PP, United Kingdom

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by ¹³C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Received for publication, April 23, 2008 , and in revised form, June 16, 2008.

^* This work was sponsored in part by European Union 6FP ActinoGEN Project (Contract LSHM-CT-2004-005224). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6.

¹ Both authors contributed equally to this work.

² Supported by Ph.D. scholarship from Technical University of Denmark.

³ To whom correspondence should be addressed. Tel.: 45-45252696; Fax: 45-45884148; E-mail: jn{at}biocentrum.dtu.dk.

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