Downloaded from www.microbiologyresearch.org by IP: 23.22.50.124 On: Sun, 29 May 2016 15:29:33 Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions Masayuki Inui, Masako Suda, Shohei Okino, Hiroshi Nonaka, La ´ szlo ´ G. Puska ´ s, Alain A. Verte ` s and Hideaki Yukawa Correspondence Hideaki Yukawa mmg-lab@rite.or.jp Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizugawa, Kyoto, 619-0292, Japan Received 9 January 2007 Revised 4 March 2007 Accepted 10 April 2007 A transcriptional profiling of the metabolism of Corynebacterium glutamicum under oxygen deprivation conditions is reported. It was observed that the glucose consumption rate per cell when C. glutamicum cells were incubated under oxygen deprivation conditions was higher than that achieved by cells incubated under aerobic growth conditions. Furthermore, DNA microarray and quantitative RT-PCR analyses revealed that the genes of several key enzymes of the glycolytic and organic acid production pathways, including gapA, pgk, tpi, ppc, ldhA and mdh, were significantly upregulated under oxygen deprivation conditions. The corresponding enzymic activities consistently correlated with the regulation patterns of the genetic expression observed at the transcriptional level. Studies of lacZ fusions with the gapA, ldhA and mdh genes indicated not only that these genes are strongly induced at the onset of the stationary phase under aerobic growth conditions, but also that high expression levels are maintained under oxygen deprivation conditions. These results indicate that the genetic expression of several key metabolic enzymes in C. glutamicum cells incubated under oxygen deprivation conditions is chiefly regulated at the transcriptional level. The physiological consequence of the observed increased transcription under oxygen deprivation conditions is an increased rate of carbon source consumption, which is accompanied by a concomitant increase in organic acid production. INTRODUCTION Corynebacterium glutamicum is widely used as a cost- effective bioconverter for the industrial production of numerous metabolites, including amino acids and organic acids (Eggeling & Sahm, 1999; Hermann, 2003; Inui et al., 1999a; Kelle et al., 2005; Kumagai, 2000; Verte `s et al., 2005), as well as heterologous proteins (Billman-Jacobe et al., 1995; Salim et al., 1997). We previously observed that when aerobically grown C. glutamicum R (Yukawa et al., 2007) is packed to a high cell density under oxygen deprivation conditions, despite the cessation of cellular growth, the cells remain able to excrete in significant amounts several metabolites, including lactate and succi- nate (Inui et al., 2004b). There are two main advantages of using these oxygen deprivation conditions for the produc- tion of organic acids in corynebacteria. First, energy is primarily used for compound production, since microbial growth is essentially halted. This results in high yields and low by-product formation. Second, the absence of growth enables the use of micro-organisms at high density. This leads to high volumetric productivity. The metabolism of C. glutamicum R for organic acid production has previously been investigated by measuring organic acid production, glucose consumption and intra- cellular NAD + /NADH ratios in the wild-type and various mutant strains in which the genes coding for key metabolic enzymes have been inactivated (Inui et al., 2004b). For L- lactate production, glucose is metabolized to pyruvate via Abbreviations: DCW, dry cell weight; DO, dissolved oxygen; FUM, fumarase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; MQO, malate: quinone oxidoreductase; OAA, oxaloacetate; ORP, oxidation–reduction potential; PC, pyruvate carboxylase; PEPC, phosphoenolpyruvate carboxylase; PGK, phosphoglycerate kinase; SDH, succinate dehydro- genase; TCA cycle, tricarboxylic acid cycle; TPI, triosephosphate isomerase. The GenBank/EMBL/DDBJ accession nos for the complete C. glutamicum R genome DNA and native episome sequences are AP009044 and AP009045, respectively. The array data discussed in this publication have been deposited in GenomeNet EXPRESSION (http://www.genome.jp/kegg/expression/) and are accessible through accession number ex0001754. Tables showing oligonucleotides used in this study, expression data of genes observed to be up- or down-regulated under oxygen deprivation conditions, and the COGs functional annotation of genes and the numbers of genes showing increased or decreased transcriptional levels during oxygen deprived reactions, are available as supplementary data with the online version of this paper. Microbiology (2007), 153, 2491–2504 DOI 10.1099/mic.0.2006/005587-0 2006/005587 G 2007 SGM Printed in Great Britain 2491