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.
Highlights
Determination of quantitative process parameters for optimizing production strains and cultivation conditions are key elements during bioprocess development for almost every new microbial product
The JuBOS is embedded in a laminar downflow housing, in which the air flow can be set to 0.45 m.s-1 (“operating mode”) or to 0.2 m.s-1 (“night mode”)
The operational laminar flow conditions of this clean bench can be negatively affected by the installed hardware of the cultivation platform
Summary
Determination of quantitative process parameters for optimizing production strains and cultivation conditions are key elements during bioprocess development for almost every new microbial product. The challenge is that the number of possible parameter combinations exponentially grows with every additional parameter. On the one hand there is a strong demand for significantly increased cultivation throughput in early process development. On the other hand process monitoring and process control should be as high as possible to allow substantial and reliable parameter optimization to ensure scalability of results. Achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. A high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest
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