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Applied and Environmental Microbiology, June 2005, p. 3255-3268, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3255-3268.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rational Design of a Corynebacterium glutamicum Pantothenate Production Strain and Its Characterization by Metabolic Flux Analysis and Genome-Wide Transcriptional Profiling

Andrea T. Hüser,¹ Christophe Chassagnole,^2,3, Nic D. Lindley,² Muriel Merkamm,⁴ Armel Guyonvarch,⁴ Veronika Eliáková,⁵ Miroslav Pátek,⁵ Jörn Kalinowski,^6,7 Iris Brune,¹ Alfred Pühler,¹ and Andreas Tauch^7*

Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany,¹ Laboratoire Biotechnologie-Bioprocédés, UMR INSA/CNRS N°5504 & UMR INSA/INRA 792, Centre de Bioingénierie Gilbert Durand, Institut National de Sciences Appliquées, 135 Avenue de Rangueil, F-31077 Toulouse Cedex 4, France,² CRITT Bio-Industries, 135 Avenue de Rangueil, F-31077 Toulouse Cedex 4, France,³ Institut de Génétique et Microbiologie, Université Paris-Sud, Centre d'Orsay, F-91405 Orsay Cedex, France,⁴ Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeská 1083, CZ-14220 Praha 4, Czech Republic,⁵ Institut für Innovationstransfer an der Universität Bielefeld GmbH, Geschäftsbereich BioTech, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany,⁶ Institut für Genomforschung, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany⁷

Received 19 October 2004/ Accepted 18 December 2004

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

Present address: Physiomics plc, Magdalen Centre, Oxford Science Park, Oxford OX4 4GA, United Kingdom.

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