Towards a Chassis Organism for Synthetic Biology

Simon Unthan1, Meike Baumgart1, Marius Herbst2, Gerd Seibold3, Christian Rückert4, Volker F. Wendisch2, Wolfgang Wiechert1, Stephan Noack1

1Institute of Bio- and Geosciences, IBG-1:Biotechnology, Forschungszentrum Jülich, D-52428 Jülich, Germany

2Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, D-33615 Bielefeld,

Germany

3Institute of Biochemistry, University of Cologne, D-50674 Cologne, Germany

4Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, D-33615

Bielefeld, Germany

Synthetic biology is expected to introduce engineering principles to the field of life sciences. A prerequisite to enable the rational assembly of biological devices is not only a library of well-characterized genetic fragments (BioBricks) but also a robust structural basis (chassis organism). Such a chassis should display minimal biological complexity to ensure a predictable behavior and can either be constructed in a bottom-up or top-down strategy. The latter is followed in our joined project in which we construct a chassis organism by step-wise reduction of the natural genome of Corynebacterium glutamicum. This soil bacterium is a promising target organisms for a genome-reduction project, since deep insights into its physiology have already been gained in various systems biology approaches in the last decade [1, 2, 3].
As a starting point we deleted three prophages (CGP1-3) which make up almost 6 % of the wild type genome of C. glutamicum. The prophage-cured strain (Î?CGP123) showed no impaired phenotype with respect to growth rates or biomass yields during cultivation under various conditions. Interestingly, the loss of the prophages and their restriction- modification system resulted in a significantly increased plasmid stability and transformation efficiency [4].
Based on Î?CGP123 with improved genetic stability, we evaluated further genome-wide targets for subsequent deletion steps. In cooperation with project partners, we classified more than 50 % of the wild type genes as non-essential and identified several gene clusters for simultaneous deletion. These clusters were deleted one at a time in
Î?CGP123 to establish a strain library for subsequent phenotypic characterization and
evaluation of each genome reduction step.
To meet the demand of high-throughput cultivations we conducted the growth experiments in a novel Mini-Pilot-Plant (MPP) by embedding a BioLector-system in a robotic environment to automate complete workflows for upstream development [5]. To gain a deeper process understanding, cultivation samples are harvested and centrifuged automatically to provide supernatants for subsequent quantitative analysis with various fully automated assays in MTP scale.
As a result, the majority of gene cluster deletions did not impair the phenotype with respect to growth rate or biomass yield. These findings strongly support our prior classification of non-essential genes and thus prove the high potential to significantly reduce the wild type genome of C. glutamicum. In ongoing work we are combining multiple promising deletions steps to finalize our construction of a less complex and robust chassis organism for synthetic biology.

[1] van Ooyen J, Noack S, Bott M, Reth A, Eggeling L (2012) Improved L-lysine production with Corynebacterium glutamicum and systemic insight into citrate synthase flux and activity. Biotechnol Bioeng 109(8):2070-81

[2] Voges R, Noack S (2012) Quantification of proteome dynamics in Corynebacterium glutamicum by 15N-

labeling and selected reaction monitoring. J Proteomics 75(9):2660-9

[3] Unthan S., Grünberger A., van Ooyen J., Gätgens J., Heinrich J, Paczia N, Wiechert W., Kohlheyer D., Noack S. (2014) Beyond growth rate 0.6: What drives Corynebacterium glutamicum to higher growth rates in defined medium? Biotechnology & Bioengineering 111(2):359-371

[4] Baumgart M., Unthan S., Rückert C., Sivalingam J., Grünberger A., Kalinowski J., Bott M., Noack S., Frunzke J. (2013) Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology, Applied and environmental microbiology. 79(19):6006-6015

[5] Rohe P., Venkanna D., Kleine B., Freudl R., Oldiges M. (2012) An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform. Microbial cell factories 11(1):144