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      Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of d-glucose and l-arabinose

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          Abstract

          Background

          l-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to d-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as d-glucose, leading to a preferential utilization of d-glucose over pentoses. In order to simultaneously utilize both d-glucose and l-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis.

          Results

          Corynebacterium glutamicum ATCC 31831 utilized d-glucose and l-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized d-glucose over l-arabinose at a high concentration (15 g/L each), although l-arabinose and d-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for d-glucose and l-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of l-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of d-glucose and l-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized d-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of l-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of d-glucose and l-arabinose.

          Conclusions

          Metabolome analysis identified potential bottlenecks in d-glucose and l-arabinose metabolism and was then applied to the following rational metabolic engineering. Manipulation of only two genes enabled simultaneous utilization of d-glucose and l-arabinose at the same rate in metabolically engineered C. glutamicum. This is the first report of rational metabolic design and engineering for simultaneous hexose and pentose utilization without inactivating the phosphotransferase system.

          Electronic supplementary material

          The online version of this article (10.1186/s12934-018-0927-6) contains supplementary material, which is available to authorized users.

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          Most cited references55

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          Features of promising technologies for pretreatment of lignocellulosic biomass.

          N. Mosier (2005)
          Cellulosic plant material represents an as-of-yet untapped source of fermentable sugars for significant industrial use. Many physio-chemical structural and compositional factors hinder the enzymatic digestibility of cellulose present in lignocellulosic biomass. The goal of any pretreatment technology is to alter or remove structural and compositional impediments to hydrolysis in order to improve the rate of enzyme hydrolysis and increase yields of fermentable sugars from cellulose or hemicellulose. These methods cause physical and/or chemical changes in the plant biomass in order to achieve this result. Experimental investigation of physical changes and chemical reactions that occur during pretreatment is required for the development of effective and mechanistic models that can be used for the rational design of pretreatment processes. Furthermore, pretreatment processing conditions must be tailored to the specific chemical and structural composition of the various, and variable, sources of lignocellulosic biomass. This paper reviews process parameters and their fundamental modes of action for promising pretreatment methods.
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            Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites.

            Quantification of metabolites is of pivotal relevance in biology, where it complements more established omics techniques such as transcriptomics and proteomics. Here, we present a 25 min ion-pairing ultrahigh performance liquid chromatography-tandem mass spectrometry method that was developed for comprehensive coverage of central metabolism (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) and closely related biosynthetic reactions. We demonstrate quantification of 138 compounds, including carboxylic acids, amino acids, sugar phosphates, nucleotides, and functionalized aromatics. Biologically relevant isomers such as sugar phosphates are individually quantified by combining chromatographic separation and fragmentation. The obtained sensitivity and robustness enabled the detection of more than half all tested compounds in each of eight diverse biological samples of 0.5-50 mg dry cell weight. We recommend this method for routine and yet comprehensive quantification of primary metabolites in a wide variety of biological matrices.
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              Bio-based production of chemicals, materials and fuels -Corynebacterium glutamicum as versatile cell factory.

              Since their discovery almost 60 years ago, Corynebacterium glutamicum and related subspecies are writing a remarkable success story in industrial biotechnology. Today, these gram-positive soil bacteria, traditionally well-known as excellent producers of L-amino acids are becoming flexible, efficient production platforms for various chemicals, materials and fuels. This development is intensively driven by systems metabolic engineering concepts integrating systems biology and synthetic biology into strain engineering. Copyright © 2011 Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                kawaguchi_h@port.kobe-u.ac.jp
                kumikoro0829@gmail.com
                k-hara@u-shizuoka-ken.ac.jp
                hasunuma@port.kobe-u.ac.jp
                ochiaki@port.kobe-u.ac.jp
                +81-78-803-6192 , akondo@kobe-u.ac.jp
                Journal
                Microb Cell Fact
                Microb. Cell Fact
                Microbial Cell Factories
                BioMed Central (London )
                1475-2859
                17 May 2018
                17 May 2018
                2018
                : 17
                : 76
                Affiliations
                [1 ]ISNI 0000 0001 1092 3077, GRID grid.31432.37, Graduate School of Science, Technology and Innovation, , Kobe University, ; 1-1 Rokkodai, Nada, Kobe, 657-8501 Japan
                [2 ]ISNI 0000 0000 9209 9298, GRID grid.469280.1, Department of Environmental and Life Sciences, School of Food and Nutritional Sciences, , University of Shizuoka, ; 52-1 Yada, Suruga, Shizuoka, 422-8526 Japan
                [3 ]ISNI 0000 0001 1092 3077, GRID grid.31432.37, Department of Chemical Science and Engineering, Graduate School of Engineering, , Kobe University, ; 1-1 Rokkodai, Nada, Kobe, 657-8501 Japan
                [4 ]ISNI 0000000094465255, GRID grid.7597.c, Biomass Engineering Research Division, , RIKEN, ; 1-7-22 Suehiro, Turumi, Yokohama, Kanagawa 230-0045 Japan
                Author information
                http://orcid.org/0000-0002-2740-0384
                Article
                927
                10.1186/s12934-018-0927-6
                5956887
                29773073
                b1def24d-9b23-421b-9e43-9bd79d58bebf
                © The Author(s) 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 4 March 2018
                : 11 May 2018
                Funding
                Funded by: Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Inter-disciplinary Research Areas (Innovative Bioproduction, Kobe) from the Ministry of Education, Culture, Sports, Science and Technology, Japan
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Biotechnology
                corynebacterium glutamicum,simultaneous utilization,l-arabinose,metabolic engineering,metabolome analysis

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