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Abstract Background High-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. Results To 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. Conclusions The 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.

The metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae are described. The analysis has shown that metabolites at key branch points of metabolic pathways are affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates are specifically affected by time.

The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl‐β‐ d‐1‐thiogalactopyranoside concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. Based on a comprehensive data set of about 150 bench‐scale bioreactor cultivations in fed‐batch mode and choosing the cutinase yield as objective, it was shown that relative secretion performance for bioprocesses remains very similar, irrespective of the applied SP enabling Sec‐mediated cutinase secretion. However, to achieve the maximal absolute cutinase yield, careful adjustment of bioprocess conditions was found to be necessary. A model‐based, two‐step multiple regression approach resembled the collected data in a comprehensive way. The corresponding results suggest that the choice of the heterologous Sec SP and its interaction with the adjusted exponential feeding profile is highly relevant to maximize absolute cutinase yield in this study. For example, the impact of Sec SP is high at low growth rates and low at high growth rates. However, promising Sec SPs could be inferred from less complex batch cultivations. The extensive data were also evaluated in terms of cutinase productivity, highlighting the well‐known trade‐off between yield and productivity in bioprocess development in detail. Conclusively, only the right combination of target protein, Sec SP, and bioprocess conditions is the key to success. The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl‐β‐ d‐1‐thiogalactopyranoside (IPTG) concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. [ABSTRACT FROM AUTHOR]

Recently, a comprehensive screening workflow for the filamentous bacterium Streptomyces lividans, a highly performant source for pharmaceutically active agents was introduced. This framework used parallelized cultivation in microtiter plates to efficiently accelerate early upstream process development. Focusing on growth performance, cultivation was successfully scaled‐up to 1 L stirred tank reactors. However, metabolic adaptation was observed on the transcriptomic level as among others, several genes incorporated in light response were upregulated during bioreactor cultivation. Despite it was assumed that this was attributed to the fact that reactor cultivations were performed in glass vessels exposed to daylight and artificial room light, this setup did not allow distinguishing exclusively between light and other effects. Upon that, the present study directly investigates the influence of light by defined illumination of microtiter plate cultures. Almost identical growth performance was observed for cultures grown in the dark or with illumination. Transcriptomics revealed the upregulation of seven genes of which 6 have previously been described to be relevant for carotenoid synthesis and its regulation. These pigments are effective quenchers of reactive oxygen species. The seventh transcript coded for a photo‐lyase incorporated in UV‐damage repair of DNA further confirming induced light response. However, this was fully compensated by metabolic adaptation on the transcriptomic level and overall process performance was maintained. Consequently, environmental conditions need extremely careful control and evaluation during in‐depth omics analysis of bioprocesses. Otherwise metabolic adaptation induced by such issues can easily be misinterpreted, especially during studies addressing cultivation system comparisons. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:287–292, 2018 [ABSTRACT FROM AUTHOR]

Modern bioprocess development includes extensive screening tasks, but many tools tackling the resulting combinatorial explosion do not provide production scale relevant conditions. Thus, novel technologies are needed for scalable data acquisition via incorporation of miniaturization, automation and digitalization. The Microbial Bioprocess Lab-a Helmholtz Innovation Lab strives at designing such disruptive technologies and to catalyze their translation from science into industrial application. [ABSTRACT FROM AUTHOR]

ABSTRACT Performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. Especially, Corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. This study focuses on the identification of metabolic key mechanisms for this robustness to get a deeper insight and provide future targets for process orientated strain development. A 1,5-diaminopentane producing C. glutamicum strain was cultivated in a two compartment scale-down device to create short-term environmental changes simulating industrial scale cultivation conditions. Using multi omics based methods, it is shown, that central metabolism is flexibly rearranged under short-term oxygen depletion and carbon source excess to overcome shortage in NAD+ recycling. In order to balance the redox state, key enzymes for the non-oxygen dependent fermentative NAD+ regeneration were significantly up-regulated while parts of non-essential pathways were down-regulated. The transfer of the cells back into the well aerated zones with low substrate concentration triggers an additional upregulation of genes for the re-assimilation of previously formed side products, showing L-lactate forming and utilizing reactions being active at the same time. Especially L-lactate as reversible and flexible external buffer for carbon and redox equivalents puts C. glutamicum in a robust position to deal with inhomogeneity in large scale processes. Biotechnol. Bioeng. 2017;114: 560-575. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Microalgae offer great potential for the industrial production of numerous compounds, but most of the currently available processes fail on economic aspects. Due to the lack of appropriate microcultivation systems, especially screening and early stage laboratory process characterization limit throughput in process development. Consequently, a demand for high throughput photobioreactors has recently been identified upon which some prototype systems emerged. However, compared to microbial microbioreactors, the systems so far introduced suffer from at least one of several drawbacks, that is, inhomogeneous conditions, poor mixing or excessive evaporation. In this context, a microtiter plate based micro-photobioreactor was developed enabling 48-fold parallelized cultivation. Strict control of the process conditions enabled a high comparability between the distinct wells of one plate (±5% fluctuation in biomass formation). The small scale, resulting in a beneficial surface to volume ratio, as well as the fast mixing due to rigorous orbital shaking, ensured an excellent light supply of the cultures. Moreover, non-invasive online biomass quantification was implemented via a scattered light analyzer that is capable of biomass measurements during continuous illumination of the cultures. The system was shown to be especially qualified for parallelized laboratory screening applications like for instance media optimization. Easy automation via integration into a liquid handling platform is given by design. Thereby, the presented micro-photobioreactor system significantly contributes to improving the time efficiency during the development of phototrophic bioprocesses. Biotechnol. Bioeng. 2017;114: 122-131. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]