Journal of Biotechnology 167 (2013) 309–315 Contents lists available at ScienceDirect Journal of Biotechnology jo u r n al homep age: www.elsevier.com/locate/jbiotec Hydrogen peroxide stress provokes a metabolic reprogramming in Pseudomonas fluorescens: Enhanced production of pyruvate Adam Bignucolo, Varun P. Appanna, Sean C. Thomas, Christopher Auger, Sungwon Han, Abdelwahab Omri, Vasu D. Appanna ∗ Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario P3E 2C6, Canada a r t i c l e i n f o Article history: Received 10 April 2013 Received in revised form 28 June 2013 Accepted 2 July 2013 Available online 16 July 2013 Keywords: Pyruvate production Metabolic reconfiguration Phosphoenolpyruvate synthase NADPH-generating enzymes Bioconversion a b s t r a c t Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production of pyruvate under the challenge of hydrogen peroxide (H 2 O 2 ). Although this stress led to a sharp reduction in the activities of numerous tricarboxylic acid (TCA) cycle enzymes, there was a marked increase in the activities of catalase and various NADPH-generating enzymes to counter the oxidative burden. The upre- gulation of phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) coupled with the reduction of pyruvate dehydrogenase (PDH) in the H 2 O 2 -challenged cells appear to be important contributors to the elevated levels of pyruvate found in these bacteria. Increased pyruvate synthesis was evident in the presence of a variety of carbon sources including d-glucose. Intact cells rapidly consumed d-glucose with the concomitant formation of this monocarboxylic acid. At least a 12-fold increase in pyruvate produc- tion within 1 h was observed in the stressed cells. These findings may be exploited in the development of technologies aimed at the conversion of carbohydrates into pyruvate. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Microorganisms are crucial effectors of numerous biotechnolo- gical processes that are commercially utilized to produce a variety of value-added products. These include the production of ethanol through the use of anaerobic respiration in yeast, and the gen- eration of lactic acid in dairy products, a process mediated by the bacterium Lactobacillus plantarum (Fu and Mathews, 1999). Complex biomolecules such as surfactants and proteases may also be manufactured by microbial systems. Indeed, various Pseu- domonas species are known to produce rhamnolipids, while Bacillus species commercially produce proteases which are widely uti- lized as detergents (Hadj-Ali et al., 2007). In an effort to render these biotechnological processes effective, the microbes often need to undergo genetic modifications that are tailored for these spe- cific duties. These manipulations may include the alteration of a microbe’s genetic makeup by deleting genes, introducing new ones Abbreviations: 6PGDH, 6-phosphogluconate dehydrogenase; BN-PAGE, blue native polyacrylamide gel electrophoresis; BSA, bovine serum albumin; CFE, cell free extract; ETC, electron transport chain; FUM, fumarase; GDH, glutamate dehydroge- nase; G6PDH, glucose-6-phosphate dehydrogenase; HPLC, high performance liquid chromatography; ICDH, isocitrate dehydrogenase; KGDH, alpha-ketoglutarate dehydrogenase; PDH, pyruvate dehydrogenase; PEPS, phosphoenolpyruvate syn- thase; PK, pyruvate kinase; TCA, tricarboxylic acid. ∗ Corresponding author. Tel.: +1 705 675 1151x2112; fax: +1 705 675 4844. E-mail address: vappanna@laurentian.ca (V.D. Appanna). from other organisms, or over-expressing the desired genetic infor- mation (Daniell et al., 2002; Masukawa et al., 2012). Recombinant technology is often utilized to create bacteria with unique commer- cial properties. The production of human insulin by Escherichia coli, where the gene for this protein is over-expressed, is an example of such a technique (Goeddel et al., 1979; Min et al., 2011). The enhancement of a given trait by natural means is also a common method utilized to tailor microbes to perform certain des- ignated tasks. Modulation of environmental factors can also help stimulate bacteria to produce specific by-products. These effectors can either be physical or chemical in nature (Akbari et al., 2012; Huang et al., 2010). A common example of this is phosphate star- vation, which is known to trigger the synthesis of phosphatase (Del Pozo et al., 1999). Adaptation to metal-polluted environments tends to favor microbial consortia that are very effective in biore- mediation processes. Indeed numerous microbial systems isolated from extreme surroundings are utilized in various industrial pro- cesses. The goal of these modifications is to help facilitate the production and secretion of a specific product (Aislabie et al., 2006; FitzPatrick et al., 2010). In this study, we have utilized oxidative stress in the form of hydrogen peroxide (H 2 O 2 ) on the nutrient versatile soil microbe Pseudomonas fluorescens in an effort to remodel its metabolic networks, with the aim of generating pyruvate from simple car- bohydrates. Pyruvate is an important ingredient in the synthesis of food additives, pharmaceuticals, nutraceuticals, weight control supplements, and amino acids such as tyrosine and tryptophan 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.07.002