(1108-A) Transformation of the Gram-positive platform organism Corynebacterium glutamicum in a fully automated laboratory environment

Abstract: Corynebacterium glutamicum is a Gram-positive platform organism that is widely used for the industrial scale production of small molecules. We aim to expand the product spectrum of C. glutamicum towards more complex molecules, such as plant secondary metabolites or heterologous peptides, but the number of genetic modifications required to obtain optimized producer strains cannot be explored with manual labor alone. To alleviate this limitation, we present a robotic platform termed AutoBiotech, which encompasses fourteen different devices to perform most unit operations of molecular biology workflows. This allows the AutoBiotech platform to generate up to 96 microbial strains automatically in a single, µL scale workflow. Specifically, plasmids are assembled, purified, and directly used for transformations into C. glutamicum, where genome modifications can take place. The benefit of this workflow is the elimination of both Escherichia coli as a host for plasmid amplification and the consequent plasmid preparation. Besides generating plasmids, the transformation itself is challenging with Gram-positive organisms due to their strong cell wall. We evaluated automated electroporation and conjugation as transformation techniques for C. glutamicum with regard to method robustness and efficiency. Results showed that the transformation of C. glutamicum via electroporation could consistently be performed with a success rate of 98% and an efficiency of 1 - 10 CFU/fmol DNA. This was verified for plasmid sizes between 5.8 and 10.3 kbp when employing 20 - 40 fmol DNA. Contrary, conjugation resulted in lower experiment reproducibility and success rates as low as 13%. Furthermore, the results suggested a diminishing conjugation efficiency with increasing production of heterologous proteins. Therefore, electroporation was determined as the method of choice in automated environments. In conclusion, the purification of plasmid DNA directly after assembly represents a major advance for the strain-construction from Gram-positive organisms due to savings in costs and time. Moreover, the complete automation of this process is a solid foundation for automated genetic engineering with plasmid-based techniques such as CRISPR/Cas. Finally, the findings above showcase the adaptability of the AutoBiotech platform to implement novel workflows, implying a rising versatility towards further organisms and technologies.