FEMS Microbiology Letters, 366, 2019, fnz187 https://doi.org/10.1093/femsle/fnz187 Advance Access Publication Date: 30 August 2019 Research Letter R E S E A R C H L E T T E R – Physiology & Biochemistry Contrasting effects of isocitrate dehydrogenase deletion on fuxes through enzymes of central metabolism in Escherichia coli Mansi El-Mansi 1,2, * ,† 1 Elizade University, Ilara Mokin, Ondo State, Nigeria and 2 University of Africa (UAT), Bayelsa State, Nigeria ∗ Corresponding author: Faculty of Science, University of Africa (UAT), Bayelsa State, Nigeria.Tel: +447470460114; E-mail: Bioed.uk@gmail.com and Mansi.elmansi@uat.edu.ng † Present address: Faculty of Science, University of Africa (UAT), Bayelsa State, Nigeria. One sentence summary: Loss of isocitrate dehydrogenase activity led to carbon fux redistribution among enzymes of central metabolism and increased phosphotransacetylase and acetate kinase activities. Editor: Robert Gunsalus ABSTRACT Flux analysis is central to understanding cellular metabolism and successful manipulation of metabolic fuxes in microbial cell-factories. Isocitrate dehydrogenase (ICDH) deletion conferred contrasting effects on fuxes through substrate-level phosphorylation (SLP) reactions. While signifcantly increasing fux through pyruvate kinase, it diminishes fux through succinyl CoA synthetase and upregulates phosphotransacetylase (PTA) and acetate kinase (AK). In addition to acetate, the ICDH-less strain excretes pyruvate, citrate and isocitrate. While effux to acetate excretion by the Escherichia coli parental strain and its ICDH-less derivative is a refection of high throughput of glycolytic intermediates, excretion of pyruvate is a refection of high throughput via pyruvate kinase. On the other hand, citrate and isocitrate excretion is a refection of truncating the Krebs cycle at the level of ICDH. Furthermore, another striking fnding is the inability of the ICDH-less cultures to utilize acetate as a source of carbon despite the availability of an adequate supply of extracellular glutamate (for biosynthesis) and elevated levels of AK and PTA (for acetate uptake). This striking observation is now explicable in the light of the newly proposed hypothesis that the expression of the ace operon enzymes is controlled in response to a minimum threshold signal (ATP), which could not be achieved in the ICDH-less strain. Keywords: Escherichia coli; fux analysis; acetate excretion; isocitrate dehydrogenase; pyruvate kinase; central metabolism INTRODUCTION The fow of glycolytic intermediates into central metabolism and out to intermediary metabolism for biosynthesis is rigor- ously regulated at the level of transcription (Baba et al. 2006; Brown, Jones-Mortimer and Kornberg 1977; Seshasayee et al. 2006; Gutierrez-R´ ıos et al. 2007; Klumpp, Zhhonnge and Hwa 2011) and posttranslational modifcations, e.g. reversible phos- phorylation (Cozzone and El-Mansi 2005) and reversible acety- lation (Hu, Lima and Wolfe 2010; Wang et al. 2010), with allosteric controls (feedback inhibition and feed-forward activa- tion) (Link, Kochanowski and Sauer 2013) protecting the pools of key metabolites through fne-tuning of the activities of irre- versible enzymes and isoenzymes (Fig. 1). Such a sophisticated net of regulatory control mechanisms is coordinated in such a way that ensures the unidirectional fow of glycolytic intermedi- ates into central metabolism (Ramseier et al. 1995; Kochanowski et al. 2013; Shimizu 2013) and the successful partition of carbon fux at metabolic junctions (Fischer and Sauer 2003; Cozzone and El-Mansi 2005; El-Mansi et al. 2015). Flux through Received: 29 April 2019; Accepted: 29 August 2019 C  The Author(s) 2019. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 1 Downloaded from https://academic.oup.com/femsle/article/366/15/fnz187/5556941 by guest on 14 March 2023