Establishment and application of accelerated bioprocess development for filamentous organisms

  • Entwicklung und Anwendung von beschleunigter Bioprozessentwicklung für filamentöse Organismen

Koepff, Joachim; Oldiges, Marco (Thesis advisor); Blank, Lars M. (Thesis advisor)

Aachen : RWTH Aachen University (2020, 2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Filamentous organisms of the genus Streptomyces are key players in industrial production processes. Particularly their ability to synthezise an extensive bandwidth of bio-active compounds such as antibiotics and their efficiency in (heterologous) protein production have put these species into focus of researchers and biopharmaceutical companies, already for decades. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. To tackle this issue, an integrated phenotyping pipeline for Streptomyces lividans was developed and validated on the basis of shaken, parallelized 1000 µL microtiter-plate (MTP) cultivation. Starting from a standardized pre-culture procedure, highly reproducible results ((CV) ≤ 3.2 %) were obtainedfor biological independent batches. Even in comparison to a lab-scale bioreactor system with 1000-fold larger working volume, high comparable cultivation behavior was observed. This conformity could be further confirmed using state-of-the-art transcriptomics and proteomics. Remarkably, the optical biomass-related scattered light intensity signal revealed to be of great value, as it enabled to reliable deduce relevant parameters such as maximum specific growth rate and batch duration. Furthermore, tailored solutions for additional atline signals, such as robot-assisted cell-dry-weight and automated morphology analysis were successfully developed and validated. Hereby, the frame conditions of µL-scale cultivation and the special needs of the filamentous biological system were always considered.The developed accelerated cultivation workflow was subsequently employed and tested towards its applicability for further filamentous organisms, where it revealed excellent results for S. fradiae. Even for the filamentous fungi T. reesei useful information could be obtained, even though to a limited extend. Additionally, two S. lividans deletion strain libraries were phenotyped towards growth and production related parameters to investigate the function of relevant σ-factors and proteases in this filamentous Streptomycete.Embedded within the presented cultivation workflow, the integrated MTP-based phenotyping approach seems to be a suitable screening tool for filamentous and industrial relevant organisms like Streptomyces. The results of this thesis can thereby contribute to widen the field of application for such organisms and may narrow the technological gap towards their non-filamentous competitors.