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Altered Acetylation and Succinylation Profiles in Corynebacterium Glutamicum in Response to Conditions Inducing Glutamate Overpr

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Altered Acetylation and Succinylation Profiles in Corynebacterium Glutamicum in Response to Conditions Inducing Glutamate Overpr ORIGINAL RESEARCH Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction Yuta Mizuno1,2, Megumi Nagano-Shoji1,2, Shosei Kubo1,3, Yumi Kawamura4, Ayako Yoshida1, Hisashi Kawasaki3, Makoto Nishiyama1, Minoru Yoshida4 & Saori Kosono1,4 1Biotechnology Research Center, The University of Tokyo, Tokyo, Japan 2Kyowa Hakko Bio Co., Ltd., Tokyo, Japan 3Department of Environmental Materials Science, Tokyo Denki University, Tokyo, Japan 4RIKEN Center for Sustainable Resource Science, Saitama, Japan Keywords Abstract 2-oxoglutarate dehydrogenase complex, corynebacterium glutamicum, label-free The bacterium Corynebacterium glutamicum is utilized during industrial fermen- semi-quantitative proteomic analysis, tation to produce amino acids such as l-­glutamate. During l-­glutamate fer- L-glutamate overproduction, lysine mentation, C. glutamicum changes the flux of central carbon metabolism to acetylation, lysine succinylation. favor l-­glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two Correspondence major lysine acyl modifications were significantly altered upon glutamate over- Saori Kosono, Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, production in C. glutamicum; acetylation decreased, whereas succinylation Bunkyo-ku, Tokyo 113-8657, Japan. ­increased. A label-­free semi-­quantitative proteomic analysis identified 604 acety- Tel: +81 3 5841 3069; Fax: +81 3 5841 8030; lated proteins with 1328 unique acetylation sites and 288 succinylated proteins E-mail: [email protected] with 651 unique succinylation sites. Acetylation and succinylation targeted ­enzymes in central carbon metabolic pathways that are directly related to glu- Funding Information tamate production, including the 2-­oxoglutarate dehydrogenase complex This work was partially supported by JSPS (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping KAKENHI (grant no. 21580105 and 24580133). Some of the authors, including revealed that several critical lysine residues in the ODHC components were the corresponding author, are members of a susceptible to acetylation and succinylation. Furthermore, induction of glutamate laboratory, which is financed by an production was associated with changes in the extent of acetylation and endowment from Kyowa Hakko Kirin Co, Ltd. ­succinylation of lysine, suggesting that these modifications may affect the activity to Biotechnology Research Center, the of enzymes involved in glutamate production. Deletion of phosphotransacetylase University of Tokyo. The authors are not paid decreased the extent of protein acetylation in nonproducing condition, suggest- directly from the company, nor have shares ing that acetyl phosphate-­dependent acetylation is active in C. glutamicum. of the company. The company does not have any role in study design, data collection and However, no effect was observed on the profiles of acetylation and succinylation analysis, decision to publish, or preparation of in glutamate-­producing condition upon disruption of acetyl phosphate metabo- the article. lism or deacetylase homologs. It was considered likely that the reduced acetyla- tion in glutamate-­producing condition may reflect metabolic states where the Received: 29 July 2015; Revised: 23 October flux through acid-producing­ pathways is very low, and substrates for acetylation 2015; Accepted: 3 November 2015 do not ­accumulate in the cell. Succinylation would occur more easily than acetylation in such conditions where the substrates for both acetylation and MicrobiologyOpen 2016; 5(1): 152–173 succinylation are limited. This is the first study investigating the acetylome and doi: 10.1002/mbo3.320 succinylome of C. glutamicum, and it provides new insight into the roles of acyl modifications in C. glutamicum biology. Y. M. and M. N. S. contributed equality to this work. 152 © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Y. Mizuno et al. Acetylome and Succinylome of C. glutamicum Increasing evidence indicates that acyl modifications Introduction play a role in controlling metabolic enzymes (Wang et al. Nε- lysine acetylation is one of the most common post- 2010; Guan and Xiong 2011; Choudhary et al. 2014; translational modifications in both eukaryotes and prokary- Hirschey and Zhao 2015). Lysine acyl modifications may otes (Hu et al. 2010; Jones and O’Connor 2011; Kim provide an elegant mechanism to coordinate metabolic and Yang 2011; Choudhary et al. 2014; Huang et al. processes by utilizing metabolic intermediates such as 2014; Hentchel and Escalante-Semerena 2015). Recent acyl- CoA and nicotinamide adenine dinucleotide (NAD+) proteomic studies in diverse bacterial species have identi- as sensors (Wellen and Thompson 2012). Recently, enzyme fied hundreds of lysine- acetylated proteins that function modification has emerged as important mechanism to in various cellular processes (Yu et al. 2008; AbouElfetouh control metabolic enzymes and flux (Chubukov et al. et al. 2014; Castano- Cerezo et al. 2014; Kuhn et al. 2014; 2013). Thus, we speculate that lysine acyl modifications Liu et al. 2014; Hentchel and Escalante-Semerena 2015; may underlie metabolic flux change during glutamate Kosono et al. 2015; Schilling et al. 2015). New acyl- overproduction in C. glutamicum. To explore this pos- modifications of lysine residues, such as propionylation sibility, we performed a label-free semi- quantitative pro- (Chen et al. 2007; Garrity et al. 2007; Cheng et al. 2009; teomic analysis of lysine acetylation and succinylation Zhang et al. 2009; Okanishi et al. 2014), butyrylation substrates in glutamate-producing and nonproducing C. (Chen et al. 2007; Zhang et al. 2009), succinylation (Zhang glutamicum. We found that lysine acetylation and suc- et al. 2010; Xie et al. 2012; Colak et al. 2013; Weinert cinylation targeted most enzymes in central carbon meta- et al. 2013b; Hirschey and Zhao 2015; Kosono et al. 2015; bolic pathways that are directly linked to glutamate Pan et al. 2015; Yang et al. 2015), malonylation (Peng production, and furthermore that the extent of modification et al. 2011; Xie et al. 2012; Hirschey and Zhao 2015), changed in response to glutamate overproduction. To our crotonylation (Tan et al. 2011), 2-hydroxyisobutyrylation knowledge, this is the first to report the acetylation and (Dai et al. 2014), and glutarylation (Tan et al. 2014; succinylation profiles of proteins in C. glutamicum. Hirschey and Zhao 2015) have been recently discovered, and they often target the same lysine residues as acetyla- Materials and Methods tion. Of these acyl modifications, succinylation, as well as acetylation, are considered to frequently occur in both Bacterial strains and culture conditions eukaryotes and prokaryotes (Weinert et al. 2013b; Kosono et al. 2015). C. glutamicum ATCC13869 (laboratory stock) and Corynebacterium glutamicum is an aerobic, gram- positive ATCC13032 (JCM 1318, obtained from the Japan bacterium that grows on a variety of sugars, organic acids, Collection of Microorganisms, RIKEN-BRC) were used and alcohols, and it is utilized for the industrial produc- as the wild type strains. In- frame deletion mutants of tion of amino acids, particularly l- glutamate and l- lysine lysine deacetylase (KDAC) homologs (NCgl0078 and (Eggeling and Bott 2005). During glutamate fermentation, NCgl0616), acetate kinase (ackA, NCgl2656), phospho- the depletion of biotin, the addition of a detergent such transacetylase (pta, NCgl2657), isocitrate lyase (aceA, as Tween 40, or the addition of lactam antibiotics such NCgl2248), and malate synthase (glcB, NCgl2247) were as penicillin triggers glutamate overproduction. These trig- constructed by a two-step homologous recombination gers open mechano- sensitive channels to excrete glutamate procedure. Upstream and downstream regions (approxi- from the cell (Nakamura et al. 2007; Hashimoto et al. mately 1.5 kb each for the NCgl0078 or NCgl0616 dele- 2010, 2012) and simultaneously change the flux of central tion, and approximately 0.5 kb each for the other deletions) carbon metabolism to favor glutamate production (Shimizu of the target gene were amplified by PCR using oligo- et al. 2003; Shirai et al. 2007). In glutamate overproduc- nucleotide pairs corresponding to f1/r1 and f2/r2 primers, tion, the decrease in 2- oxoglutarate dehydrogenase complex respectively. The resulting products served as a template (ODHC) activity, which is positioned at the branch point for overlap- extension PCR using f1/r2 primers. The where the citrate cycle and glutamate biosynthesis pathways resulting PCR products were cloned into pK18mobsacB diverge, is a well- characterized phenomenon (Kawahara (Schafer et al. 1994). C. glutamicum was electroporated et al. 1997; Shimizu et al. 2003). OdhI (encoded by with the plasmid construct (1.25 kV mm−1, 25 μF, 200 Ω) NCgl1385) is known to negatively regulate the ODHC by and screened for kanamycin resistance to identify trans- binding to the E1o component (encoded by NCgl1084), formants that had undergone the initial recombination depending on its phosphorylation status (Niebisch et al. event. Transformants were grown in CM2B agar plates 2006; Schultz et al.
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