Computational analysis and manipulation of the metabolic network of drug producing microorganisms INAUGURALDISSERTATION zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Albert-Ludwigs-Universität Freiburg im Breisgau Vorgelegt von Dennis Klementz aus Wiesbaden-Dotzheim Freiburg 2017 Dekan: Prof. Dr. Manfred Jung Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber Referent: Prof. Dr. Stefan Günther Korreferent: Prof. Dr. Andreas Bechthold Drittprüfer: Prof. Dr. Oliver Einsle Datum der Promotion: 26.02.2018 “What we observe is not nature itself, but nature exposed to our method of questioning.” Werner Heisenberg Index Index 1 Abstract ......................................................................................................................... 1 2 Introduction ................................................................................................................... 3 2.1 Streptomycetes, an important source of natural drugs .................................................... 3 2.2 Nucleocidin production in Streptomyces calvus ............................................................... 6 2.3 Griseorhodin A, a telomerase inhibitor from a marine Streptomyces strain .................... 9 2.4 Genome-scale metabolic modeling and Flux Balance Analysis ....................................... 11 2.5 ‘Omics’ data and genome-scale metabolic models ......................................................... 14 2.6 Enhanced secondary metabolite production and genome-scale metabolic models ...... 16 3 Materials ....................................................................................................................... 19 3.1 Chemicals ......................................................................................................................... 19 3.2 Equipment ....................................................................................................................... 21 3.3 Consumables .................................................................................................................... 23 3.4 Strains .............................................................................................................................. 24 3.5 Programs & Services ........................................................................................................ 25 3.6 Cultivation media ............................................................................................................. 26 3.6.1 TSB (Tryptone Soy Broth) ......................................................................................... 26 3.6.2 MS (Mannitol Soy) .................................................................................................... 26 3.6.3 SG+ (Soytone Glucose) .............................................................................................. 26 3.6.4 Minor Element Solution ........................................................................................... 27 3.6.5 Buffer ........................................................................................................................ 27 3.6.6 Minimal medium ...................................................................................................... 27 Index 4 Methods ....................................................................................................................... 28 4.1 StreptomeDB ................................................................................................................... 28 4.1.1 Data collection .......................................................................................................... 28 4.1.2 Genomes, taxonomy and gene clusters ................................................................... 28 4.1.3 Generation of the phylogenetic tree ....................................................................... 28 4.2 AdpA in Streptomyces asterosporus ............................................................................... 30 4.2.1 Detection of transposons ......................................................................................... 30 4.2.2 Detection of adpA binding sites ............................................................................... 30 4.2.3 Prediction of adpA regulon ...................................................................................... 30 4.2.4 Potential disrupted genes ........................................................................................ 30 4.2.5 G+C content and skew.............................................................................................. 31 4.2.6 Dotplot ...................................................................................................................... 31 4.3 Genome-scale metabolic modeling ................................................................................. 32 4.3.1 Model reconstruction ............................................................................................... 32 4.3.2 Analysis of essential elements ................................................................................. 32 4.3.3 Correlation plot and area under the curve (AUC) .................................................... 32 4.3.4 Knockout candidate identification ........................................................................... 33 4.3.5 Integration of transcriptomic data ........................................................................... 34 4.3.6 Generation of functional subsets ............................................................................. 35 4.3.7 Minimal medium prediction and validation ............................................................. 35 4.4 Strain cultivation .............................................................................................................. 36 4.4.1 Preculture ................................................................................................................. 36 4.4.2 Permanent culture ................................................................................................... 36 4.4.3 Agar cultures ............................................................................................................ 36 Index 4.4.4 Liquid culture in Erlenmeyer flasks .......................................................................... 36 4.4.5 Dry weight measurement ......................................................................................... 36 4.4.6 Sample preparation for HPLC-MS ............................................................................ 37 4.4.7 HPLC measurement .................................................................................................. 37 5 Results .......................................................................................................................... 38 5.1 StreptomeDB update ....................................................................................................... 38 5.2 Data Overview ................................................................................................................. 38 5.3 Phylogenetic tree ............................................................................................................. 43 5.3.1 NMR and MS prediction ........................................................................................... 45 5.3.2 Scaffold browser ....................................................................................................... 47 5.4 bldA function in Streptomyces calvus ............................................................................. 49 5.4.1 Metabolic model ...................................................................................................... 49 5.4.2 Gene expression ....................................................................................................... 50 5.4.3 Protein production ................................................................................................... 61 5.5 Role of adpA function in Streptomyces asterosporus ..................................................... 67 5.5.1 Genome Overview .................................................................................................... 67 5.5.2 Putative disrupted genes ......................................................................................... 70 5.5.3 Proteomic analysis ................................................................................................... 73 5.6 Enhancement of Griseorhodin A production in Streptomyces avermitilis SUKA22 ........ 75 5.6.1 Metabolic model ...................................................................................................... 75 5.6.2 Minimal-medium validation experiment ................................................................. 76 5.6.3 Knockout candidate identification ........................................................................... 79 5.6.4 Overview of all knockout candidates ..................................................................... 112 5.7 Experimental results ...................................................................................................... 114 Index 6 Discussion & Conclusion .............................................................................................. 118 6.1 StreptomeDB ................................................................................................................. 118 6.2 The effect of bldA in Streptomyces calvus....................................................................