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Systems Metabolic Engineering of Basfia Succiniciproducens For Systems metabolic engineering of Corynebacterium glutamicum for production of L-lysine and ectoine Dissertation zur Erlangung des Grades des Doktors der Ingenieurwissenschaften der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes von Gideon Gießelmann Saarbrücken 2019 Tag des Kolloquiums: 16.7.2019 Dekan: Prof. Guido Kickelbick Berichterstatter 1: Prof. Christoph Wittmann Berichterstatter 2: Prof. Gert-Wieland Kohring Vorsitz: Prof. Bruce Morgan Akad. Mitarbeiter: Dr. Björn Becker Publications Partial results of this work have been published previously. This was authorized by the Institute of Systems Biotechnology, represented by Prof. Dr. Christoph Wittmann. Peer-reviewed articles: Gießelmann G, D. Dietrich, L. Jungmann, M. Kohlstedt, E. Jung Jeon, S. Sun Yim, F. Sommer, D. Zimmer, T. Mühlhaus, M. Schroda, K. Jun Jeong, J. Becker, and C. Wittmann, 2019. Metabolic engineering of Corynebacterium glutamicum for high-level ectoine production – design, combinatorial assembly and implementation of a transcriptionally balanced heterologous ectoine pathway. Biotechnology Journal, under revision Becker J, G. Gießelmann, SL. Hoffmann, C. Wittmann, 2016: Corynebacterium glutamicum for sustainable bioproduction: from metabolic physiology to systems metabolic engineering. In: Zhao H., Zeng AP. (eds) Synthetic Biology – Metabolic Engineering. Advances in Biochemical Engineering/Biotechnology, vol 162. Springer, Cham The following peer-reviewed articles were published during this work, but are not part of the dissertation: Rohles CM, L. Gläser, M. Kohlstedt, G. Gießelmann, S. Pearson, A. del Campo, J. Becker, C. Wittmann, 2018. A bio-based route to the carbon-5 chemical glutaric acid and to bionylon-6,5 using metabolically engineered Corynebacterium glutamicum, Green Chemistry 20:4662-4674. Vassilev I, G. Gießelmann, SK. Schwechheimer, C. Wittmann, B Virdis, JO. Krömer: 2018, Anodic Electro‐Fermentation: Anaerobic production of L-lysine by recombinant Corynebacterium glutamicum. Biotechnol Bioeng, 115:1499– 1508. Rohles CM, G. Gießelmann, M. Kohlstedt, C. Wittmann, J. Becker, 2016: Systems metabolic engineering of Corynebacterium glutamicum for the production of the carbon-5 platform chemicals 5-aminovalerate and glutarate. Microb Cell Fact, 15:154. Conference contributions Gießelmann G, R. Schäfer, A. Banz, SL. Hoffmann, S. Schiefelbein, J. Becker, and C. Wittmann 2018: Systems and synthetic biology of lysine producing Corynebacterium glutamicum at high temperature, PhD day Faculty NT, Saarland University, Saarbrücken, Germany. Gießelmann G, R. Schäfer, A. Banz, SL. Hoffmann, S. Schiefelbein, J. Becker, and C. Wittmann, 2018: Systems and synthetic biology of L-lysine producing Corynebacterium glutamicum at high temperature, GASB German Association for Synthetic Biology Annual Conference, Berlin, Germany. Gießelmann G, S. Heiermann, C. Mattes, J. Becker and C. Wittmann, 2016: Metabolic engineering of Corynebacterium glutamicum for biosynthesis of health-care products from renewables, PhD day Faculty NT, Saarland University, Saarbrücken, Germany. Gießelmann G, R. Schäfer, N. Buschke, SL. Hoffmann, J. Becker and C. Wittmann, 2016: Systems metabolic engineering of Corynebacterium glutamicum for bioproduction from xylose, Association for General and Applied Microbiology, VAAM Annual Conference, Jena, Germany. Danksagung I Table of content Summary .................................................................................................. V Zusammenfassung ................................................................................. VI 1. Introduction ........................................................................................ 1 1.1 General introduction ...................................................................................... 1 1.2 Main objectives .............................................................................................. 3 2. Theoretical Background ................................................................... 4 2.1 Corynebacterium glutamicum as industrial cell factory .................................. 4 2.2 Systems and synthetic biology of C. glutamicum........................................... 6 2.3 Metabolic engineering of C. glutamicum ...................................................... 12 2.4 L-Lysine – world leading feed amino acid .................................................... 15 Industrial manufacturing and application ............................................... 15 Strain engineering for L-lysine production ............................................. 19 2.5 Ectoine – high-value amino acid for health and well-being .......................... 23 3. Materials and Methods.................................................................... 31 3.1 Bacterial strains and plasmids ..................................................................... 31 3.2 Genetic engineering .................................................................................... 34 3.3 Media and cultivation ................................................................................... 38 Shake flask cultivation .......................................................................... 38 II Parallel screening in mini-bioreactors ................................................... 39 Production in lab-scale bioreactors ....................................................... 39 3.4 Analytical Methods ...................................................................................... 40 Quantification of cell concentration ....................................................... 40 Quantification of sugars and organic acids ........................................... 40 Quantification of amino acids and ectoine............................................. 40 Quantification of intracellular amino acids ............................................. 40 Determination of enzyme activities ....................................................... 41 RNA sequencing ................................................................................... 41 4. Results and Discussion .................................................................. 43 4.1 Temperature impact on the metabolism of C. glutamicum LYS-12 .............. 43 Enzyme capacity in the central carbon metabolism .............................. 43 Establishment of protocols for transcriptome analysis .......................... 46 Transcriptome at different temperatures ............................................... 51 Physiological response to malic enzyme deletion in C. glutamicum LYS-12 .............................................................................................................. 58 Overexpression of lysGE for increased L-lysine yield ........................... 59 C. glutamicum LYS-12 reveals new target and production possibilities at high temperature ............................................................................................... 64 4.2 Metabolically engineered C. glutamicum for high-level ectoine production . 65 Increase of ectoine pathway flux through transcriptional balancing ...... 65 III Modulated ectABC expression via synthetic pathway design ............... 67 Optimal flux relies on specific combination of genetic control elements in the ectoine operon ............................................................................................. 71 Benchmarking of the best producer C. glutamicum ectABCopt .............. 75 Impact of transcriptional balancing on ectoine production performance 77 Driving industrial ectoine production at low salinity ............................... 77 Metabolic engineering of ectoine export ............................................... 79 C. glutamicum as promising production host for ectoine ....................... 82 5. Conclusion and Outlook ................................................................. 84 6. Appendix .......................................................................................... 86 7. References ..................................................................................... 108 IV Summary The soil bacterium Corynebacterium glutamicum has gained tremendous industrial interest, for the biotechnological production of L-lysine and L-lysine derived products. This work assessed the cellular function of the L-lysine producer C. glutamicum LYS-12 at high temperature. The interpretation of transcriptome and proteome- data together with previously generated fluxome data, provided a detailed picture of the regulatory adaption of the cells to 38°C and suggested potential targets for metabolic engineering. In line, the duplication of the lysGE gene cluster increased the L-lysine yield by 7% to 460 mmol mol-1. Additionally, the synthesis of the valuable compatible solute ectoine was optimized in C. glutamicum via transcriptional balancing of the heterologous ectoine cluster. The shuffling of synthetic promoter and spacer elements yielded a library of the terminal ectoine pathway. Strongly increased production was enabled by regulatory elements of medium strength, where the increased expression of the gene ectB, as compared to ectA and ectC, appeared crucial. The most advanced ectoine producer C. glutamicum ectABCopt achieved 65 g L-1 ectoine in a fed-batch process. In addition, transcriptome profiling of ectoine producing C. glutamicum revealed a yet unknown export mechanism for ectoine, an interesting target for further metabolic engineering. V Zusammenfassung Das Gram-positive Bodenbakterium Corynebacterium
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