Automated process development for enhanced pDNA production in Vibrio natriegens

Motivation and Introduction Acknowledgements/References Outputs and Vision Concept and Results – process development Automated process development for enhanced pDNA production in Vibrio natriegens Rosa Haßfurthera, M. Nicolas Cruz Bournazoua,b, Peter Neubauera,c , Annina Kemmera aTechnische Universität Berlin, Chair of Bioprocess Engineering, Ackerstraße 76, 13355 Berlin, Germany bDataHow AG, Vladimir-Prelog-Weg 1, 8093 Zurich, SwitzerlandcEnpresso GmbH, Frohnauer Str. 59, 13467 Berlin, Germany aTechnische Universität Berlin, Chair of Bioprocess Engineering, Ackerstraße 76, 13355 Berlin, Germany bDataHow AG, Vladimir-Prelog-Weg 1, 8093 Zurich, Switzerland cEnpresso GmbH, Frohnauer Str. 59, 13467 Berlin, Germany Evaluating V. natriegens Fitness for Industry Recent reviews highlight V. natriegens as a promising chassis for biomanufacturing due to its exceptionally fast growth and high biosynthetic capacity [1–3]. Its potential as pDNA production host may surpass Escherichia coli, which is currently dominant. Given the growing demand for pDNA as a precursor for mRNA and DNA vaccines, exploring V. natriegens as an alternative host with shorter process times and potentially higher yields is of strategic relevance. Its genetic and physiological similarity to E. coli allows the application of many established molecular biology and cultivation protocols for V. natriegens. Automated Process Development To harness the potential of V. natriegens as a pDNA production host, systematic optimization of cultivation conditions is required. Manual approaches are inefficient given the high number of possible combinations of process conditions. We therefore apply a high-throughput strategy using a robotic platform and parallelized mini bioreactor (MBR) system [4]. This setup accelerates process development by rapidly identifying conditions that maximize growth and productivity. Combining automation with parallelization reduces experimental bias and increases reproducibility, providing reliable data for process design. These capabilities make it possible to explore a much wider experimental space than feasible with conventional methods [5]. •Successful V. natriegens high throughput fed-batch cultivations. •High throughput product analysis via qPCR → Possible extension with multiplex analysis. •Initial results suggest that Vibrio is suitable for industrial processes. •Outlook: Demonstrating industrially relevant performance. •Apache Airflow (workflow management system) will be implemented as digital layer between the bioprocess system and its instruments. Experimental design The organism: V. natriegens fastest reported doubling time with 9.8 minutes; Gram-negative, curved rod-shaped; halophile; Metabolism: Facultative anaerobe, versatile in substrate use We gratefully acknowledge the financial support provided by HORIZON-WIDERA-2023-ACCESS-02 for the Dig4Bio project (project-ID 101159993). Further we acknowledge the support and contributions from our colleagues and members of the KIWI-Biolab that made this research possible. [1] Lima, Matthew, Charandatta Muddana, Zhengyang Xiao, Anindita Bandyopadhyay, Pramod P. Wangikar, Himadri B. Pakrasi, and Yinjie J. Tang. 2024. “The New Chassis in the Flask: Advances in Vibrio Natriegens Biotechnology Research.” Biotechnology Advances 77 [2] Thoma, Felix, and Bastian Blombach. 2021. “Metabolic Engineering of Vibrio Natriegens.” Edited by Diethard Mattanovich and Pablo Ivan Nikel. Essays in Biochemistry 65 (2): 381–92. https://doi.org/10.1042/EBC20200135. [3] Weinstock, Matthew T, Eric D Hesek, Christopher M Wilson, and Daniel G Gibson. 2016. “Vibrio Natriegens as a Fast-Growing Host for Molecular Biology.” Nature Methods 13 (10): 849–51. https://doi.org/10.1038/nmeth.3970. [4] Haby, B., Hans, S., Anane, E., Sawatzki, A., Krausch, N., Neubauer, P., & Cruz Bournazou, M. N. (2019). Integrated Robotic Mini Bioreactor Platform for Automated, Parallel Microbial Cultivation With Online Data Handling and Process Control. SLAS TECHNOLOGY: Translating Life Sciences Innovation, 24(6), 569–582. https://doi.org/10.1177/2472630319860775 [5] Prazeres, D. M.F, Silva-Santos, A. R., Gernaey, K. V., Gargalo, C. L., Hassfurther, R., & Kemmer, A. (2025). Incorporating digitalization in the conceptual design, research and development of plasmid biomanufacturing. [6] Kemmer, A., Cai, L., Cruz Bournazou, M. N., & Neubauer, P. (2023). High-Throughput Expression of Inclusion Bodies on an Automated Platform. In Inclusion Bodies—Methods and Protocols (1st ed.). Springer Protocols; Humana New York, NY. https://doi.org/10.1007/978-1-0716-2930-7 Results •Maximum cell dry weight (CDW): 40 g L-1 •Specific growth rate (µmax): 1.0 – 1.34 h⁻¹ •Biomass yield (Yx/s): 0.5 g g⁻¹ •Plasmid productivity:10 – 15 mg pDNA per g biomass •End of Fermentation: plasmid extraction All liquid additions, pH and temperature regulation, sampling, and related operations are managed through a central database, which distributes the information to support efficient data analysis, evaluation, and process optimization through integrated modelling strategies. Visit lecture or poster presentations from other members of Dig4Bio at: •L. Möller‘s talk on V. natriegens potential for plasmid productionTue 12.35 in Auditorium III •P1.001 - L. Gundersen – Poster on growth models for V. natriegens •Discussion on AI in Education led by F. Caccavale – Tue 10:35 in room 3.B •P4.049 - A. Santos – Poster on DNA-origami nanostructures •P2.016 - R. Hassfurther – Poster on Digitalization in Educationc qPCR (BioRad CFX 96) for pDNA analysis pDNA samples on the standard curve of the qPCR V. Natriegens microscopy image Tecan with integrated 2mag MBR platform Hamilton for atline analysis Roche - Cedex Bio HT analyzer All data, such as setpoints for the experiments or measurements, is stored in a central database. This database is then accessed to retrieve data for process control (adapted from [6]). Tested conditions in 24 MBR •Three different media types: •2xYT media with added salts •MSM (+) with S0 = 10 g L-1 •Thiele media S0 = 10 g L-1 •For every media – µset of 0.2 and 0.4 h⁻¹ •One condition to test pH of 7.5 •Shift to continuous feed after 3 hours •Glucose feed with salts and trace elements The product A 2967 bp high-copy vector carrying Amp resistance and an IPTG-inducible eGFP gene under T7 control. Experimental Setup[7] The mini-bioreactor experiments (~10 mL working volume) were carried out using a high-throughput cultivation system capable of running up to 48 parallel experiments (2mag) at a liquid handling station (Tecan). Comparison of high and low feed rate for MSM and 2xYT medium Through the connection to an additional liquid handling station (Hamilton) together with the Cedex HT Bio Analyzer (Roche), fast and accurate monitoring of critical parameters such as optical density (OD600) and glucose concentration is ensured. •Online measurements of pH and DOT •Atline OD600 determination (every 1-2 hours) •Offline: glucose, acetate, NH3, Mg2+, PO4-, Fe2+, pDNA