title: Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel 13C-metabolite fingerprinting.
abstract: For rapid analysis of microbial metabolisms, (13)C-fingerprinting employs a set of tracers to generate unique labeling patterns in key amino acids that can highlight active pathways. In contrast to rigorous (13)C-metabolic flux analysis ((13)C-MFA), this method aims to provide metabolic insights without expensive flux measurements. Using (13)C-fingerprinting, we investigated the metabolic pathways in Rhodococcus opacus PD630, a promising biocatalyst for the conversion of lignocellulosic feedstocks into value-added chemicals. Specifically, seven metabolic insights were gathered as follows: (1) glucose metabolism mainly via the Entner-Doudoroff (ED) pathway; (2) lack of glucose catabolite repression during phenol co-utilization; (3) simultaneous operation of gluconeogenesis and the ED pathway for the co-metabolism of glucose and phenol; (4) an active glyoxylate shunt in acetate-fed culture; (5) high flux through anaplerotic pathways (e.g., malic enzyme and phosphoenolpyruvate carboxylase); (6) presence of alternative glycine synthesis pathway via glycine dehydrogenase; and (7) utilization of preferred exogenous amino acids (e.g., phenylalanine). Additionally, a (13)C-fingerprinting kit was developed for studying the central metabolism of non-model microbial species. This low-cost kit can be used to characterize microbial metabolisms and facilitate the design-build-test-learn cycle during the development of microbial cell factories.
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri, 63130.
authors.forename: Whitney D
authors.initials: WD
authors.lastname: Hollinshead
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri, 63130.
authors.forename: William R
authors.initials: WR
authors.lastname: Henson
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri, 63130.
authors.forename: Mary
authors.initials: M
authors.lastname: Abernathy
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri, 63130. tsmoon@wustl.edu.
authors.forename: Tae Seok
authors.initials: TS
authors.lastname: Moon
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri, 63130. yinjie.tang@seas.wustl.edu.
authors.forename: Yinjie J
authors.initials: YJ
authors.lastname: Tang
mesh_terms: D002247:Carbon Isotopes / Q000032:analysis*; D053858:Metabolic Networks and Pathways; D055432:Metabolomics / Q000379:methods*; D012240:Rhodococcus / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors
chemical_list: D002247:Carbon Isotopes
keywords: 13C-MFA; Entner-Doudoroff (ED) pathway; catabolite repression; gluconeogenesis; phenol