Improved microscale cultivation of Pichia pastoris for clonal screening

Abstract

BackgroundExpanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts. They combine high cultivation throughput with reproducibility and scalability, and represent promising tools for bioprocess development.ResultsParallelized microplate cultivation of the eukaryotic protein production host Pichia pastoris was applied effectively to support miniaturized phenotyping of clonal libraries in batch as well as fed-batch mode. By tailoring a chemically defined growth medium, we show that growth conditions are scalable from microliter to 0.8 L lab-scale bioreactor batch cultivation with different carbon sources. Thus, the set-up allows for a rapid physiological comparison and preselection of promising clones based on online data and simple offline analytics. This is exemplified by screening a clonal library of P. pastoris constitutively expressing AppA phytase from Escherichia coli. The protocol was further modified to establish carbon-limited conditions by employing enzymatic substrate-release to achieve screening conditions relevant for later protein production processes in fed-batch mode.ConclusionThe comparison of clonal rankings under batch and fed-batch-like conditions emphasizes the necessity to perform screenings under process-relevant conditions. Increased biomass and product concentrations achieved after fed-batch microscale cultivation facilitates the selection of top producers. By reducing the demand to conduct laborious and cost-intensive lab-scale bioreactor cultivations during process development, this study will contribute to an accelerated development of protein production processes.Electronic supplementary materialThe online version of this article (10.1186/s40694-018-0053-6) contains supplementary material, which is available to authorized users.

Extension of the yeast metabolic model to include iron metabolism and its use to estimate global levels of iron‐recruiting enzyme abundance from cofactor requirements

“…Genome‐scale models of the metabolic network are of central importance since they allow us to achieve a better understanding of how the fluxes can be rewired in response to an internal or external input, and thus have been frequently used in biotechnological applications as a prediction tool, since the aim is often to obtain a preferred flux distribution (Kerkhoven, Lahtvee, & Nielsen, ). Iron has an important role in a range of biotechnological applications from improving human nutrition (Puig, Andres‐Ccolas, Garcia‐Molina, & Penarrubia, ) to the production of recombinant proteins (Eck et al, ), and healthcare applications concerning iron storage abnormalities (Valerio, ). The incorporation of an extensive, well‐curated iron metabolism in metabolic networks may greatly enhance the opportunities metabolic models offer for applications in red, green, or white biotechnology.…”

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

“…The GFP fluorescent output of the JUB1 D BD -derived ATF/BS ( Figure 3 , green line) reached to the level of GFP was expressed from the GAPDH promoter ( Figure 3 , gray line) 12 h post-induction and was stable for at least 24 h. In contrast, the strong ANAC102-derived ATF/BS ( Figure 3 , black line) mediated a much higher expression output 12 h post-induction as compared with the GAPDH promoter. However, the reporter expression decreased dramatically and reached the level derived from the GAPDH promoter after 48 h. Growing P. pastoris to a high density is of great benefit when producing a heterologous protein ( Karbalaei et al, 2020 ); however, a high level of oxygen is required to achieve a high yield, which demands a culture vessel to deliver a sufficient amount of oxygen to the yeast ( Eck et al, 2018 ). Due to the inadequate essential nutrients (amino acids, carbohydrates, minerals, vitamins), hormones, growth factors, and change of the physicochemical environment (pH, osmotic pressure), as well as limited oxygen availability ( Krause et al, 2016 ), the reporter expression derived from plant-derived ATFs could be negatively affected in our study.…”

A closer look at Aspergillus: online monitoring via scattered light enables reproducible phenotyping

“…After 37 h, the Aspergillus cell structures reach the measurement threshold and an increase in backscatter can be detected throughout the cultivation process. However, compared to BioLector cultivations with other microorganisms [20, 25, 26], a highly variable and nonstable backscatter increase can be observed. Furthermore, large standard deviations of the scattered light signal indicate poor reproducibility of the growth and the optical measurement itself (Additional file 1).…”