Robot-assisted phenotyping of genome-reduced Corynebacterium glutamicum strain libraries to draft a chassis organism
Unthan, Simon; Wiechert, Wolfgang (Thesis advisor); Büchs, Jochen (Thesis advisor)
Jülich / Forschungszentrum Jülich GmbH, Zentralbibliothek (2016) [Dissertation / PhD Thesis]
Page(s): 1 Online-Ressource (122 Seiten) : Illustrationen, Diagramme
In this work, concepts were developed and applied to guide the construction of a Corynebacterium glutamicum chassis organism for synthetic biology approaches. The aim was to delete irrelevant genes from the wild type strain in order to obtain a chassis growing on defined CGXII medium with D-glucose with unaltered biological fitness, which was defined by the maximum specific growth rate and biomass yield.Initially, workflows were developed on a robotic Mini Pilot Plant (MPP), for example, to harvest cell-free cultivation supernatants from BioLector cultivations in response to individually defined triggers. Subsequently, assays for amino acids and D-glucose were established in 384-well plate scale in order to quantify these metabolites in cell-free culture supernatants in fully automated workflows .During initial reference experiments, protocatechuic acid was identified as a hidden co-substrate in the well-known defined CGXII medium. The additional TCA feed via acetyl-CoA and succinyl-CoA, which are derived from protocatechuic acid, elevates the growth rate by about 50 % in highly diluted cultures .The first step toward a chassis was the deletion of prophage elements contributing to about 6.7 % of the C. glutamicum genome. The respective strain MB001 showed unaltered biological fitness and an increased heterologous protein expression, caused by the removal of a restriction-modification system in prophage CGP3 .As a next step, 36 strains with deletion of non-essential gene clusters were tested thoroughly and 26 clusters were found irrelevant for the biological fitness of C. glutamicum and offered the potential to reduce the genome by about 22 % . Some clusters were also deleted in the L-lysine model producer DM1933 and the derived strain GRLP45 showed an 51 % increased L-lysine titer applying the automated MPP methods, what was finally confirmed in lab-scale bioreactors .During the final combinatorial deletion of irrelevant gene clusters, some interdependencies were observed resulting in a decreased of biological fitness of the respective strains. One of those strains was characterized in-depth and revealed the general interplay of ribosome capacity and maximum growth rate of C. glutamicum.In the end, two pre-chassis, namely W127 and W121, were obtained that displayed a total genome reduction of 8.8 % and 12.8 %, respectively. Both strains fulfilled the target criteria of unaltered biological fitness on defined CGXII medium in BioLector cultivations. Finally, the in-depth analysis of both pre-chassis in bioreactors revealed a morphological divergence of W121 which could be narrowed down to a single cluster deletion. However, W127 did not show any drawback compared to the wild type when tested under stress conditions and on different cultivation scales. In fact, this strain even grew faster on some C-sources, making it a good basis for synthetic biology approaches.