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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

A0136 / / / A0960 / A0339 / A1715

Kawaguchi, Hideo
Yoshihara, Kumiko
Hara, Kiyotaka Y.
Hasunuma, Tomohisa
Ogino, Chiaki
Kondo, Akihiko

川口, 秀夫 / / / 蓮沼, 誠久 / 荻野, 千秋 / 近藤, 昭彦

17

76

BioMed Central

2018-05-17

© The Author(s) 2018. This 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.

2018-06-06

14752859

eng

1000050463873 / / / 1000020529606 / 1000000313693 / 1000040205547

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.

Corynebacterium glutamicum / Simultaneous utilization / L-Arabinose / Metabolic engineering / Metabolome analysis

https://kuid-rm-web.ofc.kobe-u.ac.jp/profile/ja.28b260930fa04130520e17560c007669.html / / / https://kuid-rm-web.ofc.kobe-u.ac.jp/profile/ja.73b63639d47d0a4b520e17560c007669.html / https://kuid-rm-web.ofc.kobe-u.ac.jp/profile/ja.cb7c84b4e252c367520e17560c007669.html / https://kuid-rm-web.ofc.kobe-u.ac.jp/profile/ja.a324eb4a1b052e53520e17560c007669.html

2018-06-06