Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel 13 C‐metabolite fingerprinting

Hollinshead, Whitney D.; Henson, William R.; Abernathy, Mary; Moon, Tae Seok; Tang, Yinjie J.

doi:10.1002/bit.25702

Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel ¹³ C‐metabolite fingerprinting

Journal Article · Fri Sep 04 04:00:00 EDT 2015 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.25702· OSTI ID:1401150

Hollinshead, Whitney D. ^[1]; Henson, William R. ^[1]; Abernathy, Mary ^[1]; Moon, Tae Seok ^[1]; Tang, Yinjie J. ^[1]

Department of Energy, Environmental and Chemical Engineering Washington University in St. Louis St. Louis Missouri 63130

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. Biotechnol. Bioeng. 2016;113: 91–100. © 2015 Wiley Periodicals, Inc.

View Accepted Manuscript (Publisher)

Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0012705

OSTI ID:: 1401150

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 1 Vol. 113; ISSN 0006-3592

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United States

Language:: English

References (56)

Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis Wiechert, Wolfgang; Siefke, Claudia; de Graaf, Albert A. Biotechnology and Bioengineering, Vol. 55, Issue 1 https://doi.org/10.1002/(SICI)1097-0290(19970705)55:1<118::AID-BIT13>3.0.CO;2-I	journal	July 1997
Characterization of gas-liquid mass transfer phenomena in microtiter plates: Gas-Liquid Mass Transfer in Microtiter Plates Hermann, Robert; Lehmann, Mathias; Büchs, Jochen Biotechnology and Bioengineering, Vol. 81, Issue 2 https://doi.org/10.1002/bit.10456	journal	November 2002
In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineeredEscherichia coli Hong, Soon Ho; Park, Si Jae; Moon, Soo Yun Biotechnology and Bioengineering, Vol. 83, Issue 7 https://doi.org/10.1002/bit.10733	journal	July 2003
New tools for mass isotopomer data evaluation in13C flux analysis: Mass isotope correction, data consistency checking, and precursor relationships Wahl, S. Aljoscha; Dauner, Michael; Wiechert, Wolfgang Biotechnology and Bioengineering, Vol. 85, Issue 3 https://doi.org/10.1002/bit.10909	journal	January 2004
Glucose metabolism in the antibiotic producing actinomyceteNonomuraea sp. ATCC 39727 Gunnarsson, Nina; Bruheim, Per; Nielsen, Jens Biotechnology and Bioengineering, Vol. 88, Issue 5 https://doi.org/10.1002/bit.20279	journal	January 2004
Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630 Alvarez, Hector M.; Mayer, Frank; Fabritius, Dirk Archives of Microbiology, Vol. 165, Issue 6 https://doi.org/10.1007/s002030050341	journal	June 1996
Analysis of catRABC operon for catechol degradation from phenol-degrading Rhodococcus erythropolis Veselý, M.; Knoppová, M.; Nešvera, J. Applied Microbiology and Biotechnology, Vol. 76, Issue 1 https://doi.org/10.1007/s00253-007-0997-6	journal	May 2007
Induction and carbon catabolite repression of phenol degradation genes in Rhodococcus erythropolis and Rhodococcus jostii Szőköl, Juraj; Rucká, Lenka; Šimčíková, Michaela Applied Microbiology and Biotechnology, Vol. 98, Issue 19 https://doi.org/10.1007/s00253-014-5881-6	journal	June 2014
Triacylglycerol accumulation and oxidative stress in Rhodococcus species: differential effects of pro-oxidants on lipid metabolism Urbano, Susana Bequer; Di Capua, Cecilia; Cortez, Néstor Extremophiles, Vol. 18, Issue 2 https://doi.org/10.1007/s00792-013-0623-8	journal	January 2014
Methods and advances in metabolic flux analysis: a mini-review Antoniewicz, Maciek R. Journal of Industrial Microbiology & Biotechnology, Vol. 42, Issue 3 https://doi.org/10.1007/s10295-015-1585-x	journal	January 2015
Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis Tang, Yinjie J.; Shui, Wenqing; Myers, Samuel Biotechnology Letters, Vol. 31, Issue 8 https://doi.org/10.1007/s10529-009-9991-7	journal	April 2009
Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method Zhao, Jiao; Shimizu, Kazuyuki Journal of Biotechnology, Vol. 101, Issue 2 https://doi.org/10.1016/S0168-1656(02)00316-4	journal	March 2003
Bioconversion of indene to cis (1S,2R) indandiol and trans (1R,2R) indandiol by Rhodococcus species Chartrain, Michel; Jackey, Barbara; Taylor, Colleen Journal of Fermentation and Bioengineering, Vol. 86, Issue 6 https://doi.org/10.1016/S0922-338X(99)80005-1	journal	January 1998
Cultivation of lipid-producing bacteria with lignocellulosic biomass: Effects of inhibitory compounds of lignocellulosic hydrolysates Wang, Baixin; Rezenom, Yohannes H.; Cho, Kun-Ching Bioresource Technology, Vol. 161 https://doi.org/10.1016/j.biortech.2014.02.133	journal	June 2014
Effects of inhibitory compounds in lignocellulosic hydrolysates on Mortierella isabellina growth and carbon utilization Ruan, Zhenhua; Hollinshead, Whitney; Isaguirre, Christine Bioresource Technology, Vol. 183 https://doi.org/10.1016/j.biortech.2015.02.026	journal	May 2015
A roadmap for interpreting 13 C metabolite labeling patterns from cells Buescher, Joerg M.; Antoniewicz, Maciek R.; Boros, Laszlo G. Current Opinion in Biotechnology, Vol. 34 https://doi.org/10.1016/j.copbio.2015.02.003	journal	August 2015
Mapping photoautotrophic metabolism with isotopically nonstationary 13C flux analysis Young, Jamey D.; Shastri, Avantika A.; Stephanopoulos, Gregory Metabolic Engineering, Vol. 13, Issue 6 https://doi.org/10.1016/j.ymben.2011.08.002	journal	November 2011
Parallel labeling experiments and metabolic flux analysis: Past, present and future methodologies Crown, Scott B.; Antoniewicz, Maciek R. Metabolic Engineering, Vol. 16 https://doi.org/10.1016/j.ymben.2012.11.010	journal	March 2013
Publishing 13C metabolic flux analysis studies: A review and future perspectives Crown, Scott B.; Antoniewicz, Maciek R. Metabolic Engineering, Vol. 20 https://doi.org/10.1016/j.ymben.2013.08.005	journal	November 2013
A novel platform for automated high-throughput fluxome profiling of metabolic variants Heux, Stéphanie; Poinot, Juliette; Massou, Stéphane Metabolic Engineering, Vol. 25 https://doi.org/10.1016/j.ymben.2014.06.001	journal	September 2014
Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis Au, Jennifer; Choi, Jungik; Jones, Shawn W. Metabolic Engineering, Vol. 26 https://doi.org/10.1016/j.ymben.2014.08.002	journal	November 2014
Integrated 13 C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli Crown, Scott B.; Long, Christopher P.; Antoniewicz, Maciek R. Metabolic Engineering, Vol. 28 https://doi.org/10.1016/j.ymben.2015.01.001	journal	March 2015
Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration Ng, Chiam Yu; Farasat, Iman; Maranas, Costas D. Metabolic Engineering, Vol. 29 https://doi.org/10.1016/j.ymben.2015.03.001	journal	May 2015
Engineering l-arabinose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production Kurosawa, Kazuhiko; Plassmeier, Jens; Kalinowski, Jörn Metabolic Engineering, Vol. 30 https://doi.org/10.1016/j.ymben.2015.04.006	journal	July 2015
Quantifying Biomass Composition by Gas Chromatography/Mass Spectrometry Long, Christopher P.; Antoniewicz, Maciek R. Analytical Chemistry, Vol. 86, Issue 19 https://doi.org/10.1021/ac502734e	journal	September 2014
Transcriptional regulation is insufficient to explain substrate‐induced flux changes in Bacillus subtilis Chubukov, Victor; Uhr, Markus; Le Chat, Ludovic Molecular Systems Biology, Vol. 9, Issue 1 https://doi.org/10.1038/msb.2013.66	journal	January 2013
Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism Fischer, Eliane; Sauer, Uwe Nature Genetics, Vol. 37, Issue 6 https://doi.org/10.1038/ng1555	journal	May 2005
Tuberculosis — metabolism and respiration in the absence of growth Boshoff, Helena I. M.; Barry, Clifton E. Nature Reviews Microbiology, Vol. 3, Issue 1 https://doi.org/10.1038/nrmicro1065	journal	January 2005
Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Alper, Hal; Stephanopoulos, Gregory Nature Reviews Microbiology, Vol. 7, Issue 10 https://doi.org/10.1038/nrmicro2186	journal	October 2009
The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse McLeod, M. P.; Warren, R. L.; Hsiao, W. W. L. Proceedings of the National Academy of Sciences, Vol. 103, Issue 42, p. 15582-15587 https://doi.org/10.1073/pnas.0607048103	journal	October 2006
Glycolytic strategy as a tradeoff between energy yield and protein cost Flamholz, A.; Noor, E.; Bar-Even, A. Proceedings of the National Academy of Sciences, Vol. 110, Issue 24 https://doi.org/10.1073/pnas.1215283110	journal	April 2013
CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics Zhang, Zhengdong; Shen, Tie; Rui, Bin Nucleic Acids Research, Vol. 43, Issue D1 https://doi.org/10.1093/nar/gku1137	journal	November 2014
De novo design of heat-repressible RNA thermosensors in E. coli Hoynes-O'Connor, Allison; Hinman, Kristina; Kirchner, Lukas Nucleic Acids Research, Vol. 43, Issue 12 https://doi.org/10.1093/nar/gkv499	journal	May 2015
Recent advances in mapping environmental microbial metabolisms through ¹³ C isotopic fingerprints Tang, Joseph Kuo-Hsiang; You, Le; Blankenship, Robert E. Journal of The Royal Society Interface, Vol. 9, Issue 76 https://doi.org/10.1098/rsif.2012.0396	journal	June 2012
A Rhodococcus species that thrives on medium saturated with liquid benzene Paje, M. L. F.; Neilan, B. A.; Couperwhite, l. Microbiology, Vol. 143, Issue 9 https://doi.org/10.1099/00221287-143-9-2975	journal	September 1997
Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation Zhang, Y.; Adams, I. P.; Ratledge, C. Microbiology, Vol. 153, Issue 7 https://doi.org/10.1099/mic.0.2006/002683-0	journal	July 2007
The Entner-Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress : Perturbing the upper metabolism of Chavarría, Max; Nikel, Pablo I.; Pérez-Pantoja, Danilo Environmental Microbiology, Vol. 15, Issue 6 https://doi.org/10.1111/1462-2920.12069	journal	January 2013
Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord, Norway: isolation, diversity and biological activity Bredholdt, Harald; Galatenko, Olga A.; Engelhardt, Kerstin Environmental Microbiology, Vol. 9, Issue 11 https://doi.org/10.1111/j.1462-2920.2007.01387.x	journal	November 2007
Differences of heterotrophic 13CO2 assimilation by Pseudomonas knackmussii strain B13 and Rhodococcus opacus 1CP and potential impact on biomarker stable isotope probing: Differences of heterotrophic 13CO2 assimilation Feisthauer, Stefan; Wick, Lukas Y.; Kästner, Matthias Environmental Microbiology, Vol. 10, Issue 6 https://doi.org/10.1111/j.1462-2920.2008.01573.x	journal	March 2008
Prevalence of alkane monooxygenase genes in Arctic and Antarctic hydrocarbon-contaminated and pristine soils1 Whyte, L. G.; Schultz, A.; Beilen, J. B. FEMS Microbiology Ecology, Vol. 41, Issue 2 https://doi.org/10.1111/j.1574-6941.2002.tb00975.x	journal	August 2002
Metabolic flux analysis for a ppc mutant Escherichia coli based on ¹³ C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements Peng, Lifeng; Arauzo-Bravo, Marcos J.; Shimizu, Kazuyuki FEMS Microbiology Letters, Vol. 235, Issue 1 https://doi.org/10.1111/j.1574-6968.2004.tb09562.x	journal	June 2004
Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1 Goncalves, E. R.; Hara, H.; Miyazawa, D. Applied and Environmental Microbiology, Vol. 72, Issue 9 https://doi.org/10.1128/AEM.00947-06	journal	September 2006
Global Response to Desiccation Stress in the Soil Actinomycete Rhodococcus jostii RHA1 LeBlanc, J. C.; Goncalves, E. R.; Mohn, W. W. Applied and Environmental Microbiology, Vol. 74, Issue 9 https://doi.org/10.1128/AEM.02711-07	journal	March 2008
Large-Scale ¹³ C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose Klingner, Arne; Bartsch, Annekathrin; Dogs, Marco Applied and Environmental Microbiology, Vol. 81, Issue 7 https://doi.org/10.1128/AEM.03157-14	journal	January 2015
Selective Utilization of Exogenous Amino Acids by Dehalococcoides ethenogenes Strain 195 and Its Effects on Growth and Dechlorination Activity Zhuang, Wei-Qin; Yi, Shan; Feng, Xueyang Applied and Environmental Microbiology, Vol. 77, Issue 21 https://doi.org/10.1128/AEM.05676-11	journal	September 2011
Metabolic Network Analysis of Streptomyces tenebrarius, a Streptomyces Species with an Active Entner-Doudoroff Pathway Borodina, I.; Scholler, C.; Eliasson, A. Applied and Environmental Microbiology, Vol. 71, Issue 5 https://doi.org/10.1128/AEM.71.5.2294-2302.2005	journal	May 2005
ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase Giffin, M. M.; Modesti, L.; Raab, R. W. Journal of Bacteriology, Vol. 194, Issue 5 https://doi.org/10.1128/JB.05914-11	journal	December 2011
Correlation between Protein and mRNA Abundance in Yeast Gygi, Steven P.; Rochon, Yvan; Franza, B. Robert Molecular and Cellular Biology, Vol. 19, Issue 3 https://doi.org/10.1128/MCB.19.3.1720	journal	March 1999
Utilization of Halogenated Benzenes, Phenols, and Benzoates by Rhodococcus opacus GM-14. Zaitsev, G. M.; Uotila, J. S.; Tsitko, I. V. Applied and environmental microbiology, Vol. 61, Issue 12 https://doi.org/10.1128/aem.61.12.4191-4201.1995	journal	January 1995
The β-Ketoadipate Pathway And The Biology Of Self-Identity Harwood, Caroline S.; Parales, Rebecca E. Annual Review of Microbiology, Vol. 50, Issue 1, p. 553-590 https://doi.org/10.1146/annurev.micro.50.1.553	journal	October 1996
Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis Varman, Arul M.; He, Lian; You, Le Microbial Cell Factories, Vol. 13, Issue 1 https://doi.org/10.1186/1475-2859-13-42	journal	January 2014
Actinomycetes as host cells for production of recombinant proteins Nakashima, Nobutaka; Mitani, Yasuo; Tamura, Tomohiro Microbial Cell Factories, Vol. 4, Issue 1 https://doi.org/10.1186/1475-2859-4-7	journal	March 2005
Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli Zhang, Yan; Lin, Zhenquan; Liu, Qiaojie Microbial Cell Factories, Vol. 13, Issue 1 https://doi.org/10.1186/s12934-014-0172-6	journal	December 2014
A Genome-Scale Metabolic Reconstruction of Mycoplasma genitalium, iPS189 Suthers, Patrick F.; Dasika, Madhukar S.; Kumar, Vinay Satish PLoS Computational Biology, Vol. 5, Issue 2 https://doi.org/10.1371/journal.pcbi.1000285	journal	February 2009
Comparative and Functional Genomics of Rhodococcus opacus PD630 for Biofuels Development Holder, Jason W.; Ulrich, Jil C.; DeBono, Anthony C. PLoS Genetics, Vol. 7, Issue 9 https://doi.org/10.1371/journal.pgen.1002219	journal	September 2011
Carbohydrate Metabolism and Carbon Fixation in Roseobacter denitrificans OCh114 Tang, Kuo-Hsiang; Feng, Xueyang; Tang, Yinjie J. PLoS ONE, Vol. 4, Issue 10 https://doi.org/10.1371/journal.pone.0007233	journal	October 2009

Similar Records

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630

Journal Article · Tue Jul 02 00:00:00 EDT 2019 · Metabolic Engineering · OSTI ID:1572523

Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630

Journal Article · Mon Feb 01 23:00:00 EST 2016 · Nucleic Acids Research · OSTI ID:1255221

Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel 13 C‐metabolite fingerprinting

Citation Formats

References (56)

Similar Records

Related Subjects

Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel ¹³ C‐metabolite fingerprinting