Health Gesundheit Engineering of Corynebacterium glutamicum towards utilization of methanol as carbon and energy source Sabrina Witthoff 78 Corynebacterium glutamicum Methanol utilization by utilization by Methanol Member of the Helmholtz Association Member of the Sabrina Witthoff Gesundheit / Health Gesundheit / Health Band/ Volume 78 Band/ Volume 78 ISBN 978-3-95806-029-6 ISBN 978-3-95806-029-6 Schriften des Forschungszentrums Jülich Reihe Gesundheit / Health Band / Volume 78 Forschungszentrum Jülich GmbH Institute of Bio- and Geosciences Biotechnology (IBG-1) Engineering of Corynebacterium glutamicum towards utilization of methanol as carbon and energy source Sabrina Witthoff Schriften des Forschungszentrums Jülich Reihe Gesundheit / Health Band / Volume 78 ISSN 1866-1785 ISBN 978-3-95806-029-6 Bibliographic information published by the Deutsche Nationalbibliothek. The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de. Publisher and Forschungszentrum Jülich GmbH Distributor: Zentralbibliothek 52425 Jülich Tel: +49 2461 61-5368 Fax: +49 2461 61-6103 Email: [email protected] www.fz-juelich.de/zb Cover Design: Grafische Medien, Forschungszentrum Jülich GmbH Printer: Grafische Medien, Forschungszentrum Jülich GmbH Copyright: Forschungszentrum Jülich 2015 Schriften des Forschungszentrums Jülich Reihe Gesundheit / Health, Band / Volume 78 D 61 (Diss. Düsseldorf, Univ., 2014) ISSN 1866-1785 ISBN 978-3-95806-029-6 The complete volume is freely available on the Internet on the Jülicher Open Access Server (JuSER) at www.fz-juelich.de/zb/openaccess. Neither this book nor any part of it may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. CONTENT I Content 1 Summary.............................................................................................................................................. 1 1.1 Summary English.......................................................................................................................... 1 1.2 Summary German......................................................................................................................... 3 2 Introduction........................................................................................................................................ 5 2.1 Methylotrophic metabolism........................................................................................................ 6 2.1.1 Oxidation of methylated substrates....................................................................................... 8 2.1.2 Oxidation of formaldehyde to CO2........................................................................................ 9 2.1.3 Assimilation of formaldehyde, CO2 and/or methylene-H4F................................................. 13 2.2 Corynebacterium glutamicum as potential platform organism for methanol-based fermentation processes.............................................................................................................. 17 2.3 Aims of this thesis....................................................................................................................... 19 3 Results................................................................................................................................................ 21 3.1 Corynebacterium glutamicum harbours a molybdenum cofactor- dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate.............. 23 3.2 C1 metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide................................................................................ 35 3.3 Metabolic engineering of Corynebacterium glutamicum for the metabolization of methanol ................................................................................................................................. 45 4 Discussion......................................................................................................................................... 63 4.1 Identification and characterization of the endogenous pathway for oxidation of methanol to CO2.......................................................................................................................... 65 4.1.1 Oxidation of methanol by C. glutamicum wild type............................................................... 65 4.1.2 Oxidation of formaldehyde by C. glutamicum wild type........................................................ 67 4.1.3 Oxidation of formate by C. glutamicum wild type.................................................................. 69 4.2 Engineering of C. glutamicum towards methanol utilization............................................... 71 4.2.1 Engineering of C. glutamicum towards an increased methanol oxidation rate....................... 72 4.2.2 Engineering of C. glutamicum wild type towards assimilation of formaldehyde................... 73 II CONTENT 4.2.3 Impact of the absence of the dissimilatory pathway for formaldehyde oxidation on the assimilation of methanol-derived carbon.................................................................... 76 4.3 Conclusion and Perspective...................................................................................................... 77 5 References......................................................................................................................................... 79 6 Appendix........................................................................................................................................... 89 6.1 Supplementary material to “Formate dehydrogenase from Corynebacterium glutamicum.................................................................................................................................... 89 6.2 Supplementary material to “Endogenous methanol oxidation in Corynebacterium glutamicum.................................................................................................................................... 96 6.3 Supplementary material to “Engineering of Corynebacterium glutamicum towards methanol utilization................................................................................................................. 107 6.4 Supplementary references....................................................................................................... 113 ABBREVIATIONS III Abbreviations Abbreviations for Système International d'Unités (SI units) of measurement, chemical symbols for the elements and common abbreviations according to international standards, as for example listed in the author guidelines of the “Applied and Environmental Microbiology” journal are not listed in this section. ATCC American type culture collection FDH formate dehydrogenase AT aminotransferase FGH S-Formyl-glutathione hydrolase BHI(S) brain heart infusion (+ Sorbitol) FMH S-Formyl-MSH hydrolase BGSC Bacillus Genetic Stock Center FTIR Fourier transform infrared spectroscopy CBB Calvin-Benson-Bassham fw forward CD conserved domain F6P fructose-6-phosphate CDW cell dry weight GAP glyceraldehyde-3-phosphate CH cyclohydrolase GC gas chromatography DAK dihydroxyacetone kinase GFA GSH-formaldehyde-activating enzyme DAS dihydroxyacetone synthase GK glycerate kinase DH dehydrogenase GSH glutathione DHase dehydratase GST glutathione S-transferase DHa dehalogenase G6P glucose-6-phosphate DHA(P) dihydroxyacetone (phosphate) G6PDH glucose-6-phosphate dehydrogenase DSMZ Deutsche Sammlung für HPLC high-performance liquid Mikroorganismen & Zellkulturen chromatography DMS dimethylsufide HPR hydroxypyruvate reductase DTT dithiothreitol HPS 3-hexulose-6-phosphate synthase EMCP ethylmalonyl-CoA pathway H4F tetrahydrofolate et al. et alii H4MPT tetrahydromethanopterin E4P erythrose-4-phosphate H6P hexulose-6-phosphate FADH formaldehyde dehydrogenase IPTG isopropyl-thio-β-D-galactopyranoside FAE formaldehyde activating enzyme KanR kanamycin resistance FBA FBP aldolase KDPG(A) 2-keto-3-deoxy-6-phosphogluconate aldolase FBP fructose-1,6-bisphosphate LB Luria-Bertani broth FBPase fructose-1,6-bisphosphatase MADH methylamine dehydrogenase IV ABBREVIATIONS MAOX methylamine oxidase Ru5P ribulose-5-phosphate MDH methanol dehydrogenase SHMT serine hydroxymethyl transferase MeOX methane oxidase SBP sedoheptulose-1,7-bisphosphate MNO methanol:N,N′-dimethyl-4- SBPase sedoheptulose bisphosphatase nitrosoaniline oxidoreductases MOX methanol oxidase S7P sedoheptulose-7-phosphate MSA methanesulfonic acid TAL transaldolase MSH mycothiol TetR tetracycline resistance NMPG N-methylglutamate pathway TKT transketolase NMN nicotinamide mononucleotide TPI triosephosphate isomerase OD600 optical density at 600 nm XuMP xylulose monophosphate OAA oxaloacetate X5P xylulose-5-phosphate PEP phoshoenolpyruvate Xox/Xred unknown cofactor (oxidized/reduced) PEPC PEP carboxylase ZTG Zelltrockengewicht PGI phosphoglucoisomerase PHI 6-phospho-3-hexuloisomerase PFK phosphofructokinase PPP pentose phosphate pathway PRI phosphoriboisomerase PQQ pyrroloquinoline quinone 23PG 2-,3-phosphoglycerate 6PG 6-phosphogluconate 6PGDH 6-phosphogluconate dehydrogenase 6PGDHase 6-phosphogluconate dehydratase 6PGL 6-phosphoglucolactone 6PGLase 6-phosphoglucolactonase