An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

Abstract

BackgroundHigh-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest.ResultsTo increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale.ConclusionsThe optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation.

Detection of growth rate‐dependent product formation in miniaturized parallel fed‐batch cultivations

“…For future studies, automation can be further advanced using on-line biomass sensing or automated cell separation, as they are used by others [28,29]. The μ-dependent protein secretion could be performed in pH-controlled minibioreactor systems, which could improve the predictive power of the obtained results [30].…”

Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

“…The higher transformation efficiency makes MB001 an ideal strain for metabolic engineering because the initial chromosomal integration of a plasmid is often the critical step. Finally, the significantly increased eYFP productivity is an interesting feature and could be relevant for all applications requiring high expression of proteins, such as protein production or the tuning of natural or synthetic metabolic pathways required for small-molecule production (42,43).…”

Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of d-xylose containing substrates

“…Medium preparation and inoculation were performed manually under a sterile bench. For the estimation of substrate-specific biomass yields (Y X/S ) the corresponding backscatter (BS) measurements were recalculated to cell dry weight (CDW) data using the calibration model CDW [g l −1 ] = 0.048 × BS g l −1 − 0.78 g l −1 (Rohe et al, 2012).…”