Improved microscale cultivation of Pichia pastoris for clonal screening

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.

[1]  Andreas Schmid,et al.  Carbon metabolism limits recombinant protein production in Pichia pastoris , 2011, Biotechnology and bioengineering.

[2]  G. Letchworth,et al.  High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. , 2004, BioTechniques.

[3]  M. Wubbolts,et al.  Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. , 2004, FEMS yeast research.

[4]  R. Bill,et al.  Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime , 2009, Microbial cell factories.

[5]  Francisco Valero,et al.  Comprehensive clone screening and evaluation of fed-batch strategies in a microbioreactor and lab scale stirred tank bioreactor system: application on Pichia pastoris producing Rhizopus oryzae lipase , 2014, Microbial Cell Factories.

[6]  Xiangyang Wang,et al.  Defining Process Design Space for Biotech Products: Case Study of Pichia pastoris Fermentation , 2008, Biotechnology progress.

[7]  B. Byrne,et al.  Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures. , 2008, Microbial cell factories.

[8]  D. Hanahan,et al.  Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Büchs,et al.  Fed-batch operation in special microtiter plates: a new method for screening under production conditions , 2014, Journal of Industrial Microbiology & Biotechnology.

[10]  Ashraf Amanullah,et al.  Twenty‐four‐well plate miniature bioreactor high‐throughput system: Assessment for microbial cultivations , 2007, Biotechnology and bioengineering.

[11]  Carl-Fredrik Mandenius,et al.  Bioprocess optimization using design‐of‐experiments methodology , 2008, Biotechnology progress.

[12]  L. Harvey,et al.  Heterologous protein production using the Pichia pastoris expression system , 2005, Yeast.

[13]  Francisco Valero,et al.  Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. , 2007, Journal of biotechnology.

[14]  William C. Raschke,et al.  Recent Advances in the Expression of Foreign Genes in Pichia pastoris , 1993, Bio/Technology.

[15]  Frank Kensy,et al.  Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates , 2009, Microbial cell factories.

[16]  Alexandra B. Graf,et al.  Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level , 2015, BMC Genomics.

[17]  J Büchs,et al.  Fed‐batch mode in shake flasks by slow‐release technique , 2006, Biotechnology and bioengineering.

[18]  M. Inan,et al.  Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase , 2006, Biotechnology and bioengineering.

[19]  Peter Neubauer,et al.  The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli , 2016, Microbial Cell Factories.

[20]  P. Neubauer,et al.  Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations , 2014, Bioprocess and Biosystems Engineering.

[21]  Francisco Valero,et al.  Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter , 2013 .

[22]  D. Fotiadis,et al.  Cultivation strategies to enhance productivity of Pichia pastoris: A review. , 2015, Biotechnology advances.

[23]  Michael Sauer,et al.  Stress in recombinant protein producing yeasts. , 2004, Journal of biotechnology.

[24]  Wolfgang Wiechert,et al.  Bioprocess automation on a Mini Pilot Plant enables fast quantitative microbial phenotyping , 2015, Microbial Cell Factories.

[25]  M. Alting-Mees,et al.  Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. , 2003, Protein expression and purification.

[26]  Ulrich Schwaneberg,et al.  To get what we aim for – progress in diversity generation methods , 2013, The FEBS journal.

[27]  C. Brooks,et al.  Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris , 2013, Protein science : a publication of the Protein Society.

[28]  J Büchs,et al.  Introduction to advantages and problems of shaken cultures. , 2001, Biochemical engineering journal.

[29]  X. Lei,et al.  Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris , 2003, Biotechnology Letters.

[30]  A. Ferreira,et al.  Analysis of culture media screening data by projection to latent pathways: The case of Pichia pastoris X-33. , 2016, Journal of biotechnology.

[31]  G. Rao,et al.  A study of oxygen transfer in shake flasks using a non‐invasive oxygen sensor , 2003, Biotechnology and bioengineering.

[32]  G. Mathiesen,et al.  Expression of endoglucanases in Pichia pastoris under control of the GAP promoter , 2014, Microbial Cell Factories.

[33]  R. Fischer,et al.  Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations. , 2001, Biotechnology and bioengineering.

[34]  Brigitte Gasser,et al.  Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris , 2006, Microbial cell factories.

[35]  A. Pessoa,et al.  Applications of recombinant Pichia pastoris in the healthcare industry , 2013, Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology].

[36]  Robert Huber,et al.  Microbial Cell Factories Robo-lector – a Novel Platform for Automated High-throughput Cultivations in Microtiter Plates with High Information Content , 2022 .

[37]  Michael Sauer,et al.  The effect of temperature on the proteome of recombinant Pichia pastoris. , 2009, Journal of proteome research.

[38]  Wolfgang Wiechert,et al.  Framework for Kriging‐based iterative experimental analysis and design: Optimization of secretory protein production in Corynebacterium glutamicum , 2016 .

[39]  Frank Kensy,et al.  High-throughput screening of Hansenula polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale. , 2010, FEMS yeast research.

[40]  Zisheng Zhang,et al.  Medium Optimization for the Production of Phytase by Recombinant Pichia pastoris Grown on Glycerol , 2011 .

[41]  Mudassar Ahmad,et al.  Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production , 2014, Applied Microbiology and Biotechnology.

[42]  P. Bobrowicz,et al.  High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies , 2010, Journal of Industrial Microbiology & Biotechnology.

[43]  S. Roth,et al.  Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors , 2015, Journal of biological engineering.

[44]  Guadalupe de la Rosa,et al.  Kinetic and Thermodynamic Modeling of Cd+2 and Ni+2 Biosorption by Raw Chicken Feathers , 2011 .

[45]  Dirk Weuster-Botz,et al.  Milliliter-scale stirred tank reactors for the cultivation of microorganisms. , 2010, Advances in applied microbiology.

[46]  Karen M Polizzi,et al.  Can too many copies spoil the broth? , 2013, Microbial Cell Factories.

[47]  Y. Zhuang,et al.  Efficient generation of multi‐copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris , 2009, Journal of applied microbiology.

[48]  A. Neal,et al.  Mechanisms of Fatty Acid Toxicity for Yeast , 1965, Journal of bacteriology.

[49]  D. Haltrich,et al.  Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris , 2011, Applied Microbiology and Biotechnology.

[50]  D. Weuster‐Botz,et al.  Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors. , 2014, Biotechnology journal.

[51]  J. Short,et al.  Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement , 2004, Applied and Environmental Microbiology.

[52]  G J Lye,et al.  Scale‐up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched kLa , 2008, Biotechnology and bioengineering.

[53]  David Pollard,et al.  A review of advanced small‐scale parallel bioreactor technology for accelerated process development: Current state and future need , 2011, Biotechnology progress.

[54]  Matthias G Steiger,et al.  In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response , 2013, Biotechnology journal.

[55]  V. Sahai,et al.  Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production. , 2008, Bioresource technology.

[56]  Diethard Mattanovich,et al.  Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. , 2008, Biotechnology and bioengineering.

[57]  P. Czermak,et al.  Expression of enzymes for the usage in food and feed industry with Pichia pastoris. , 2015, Journal of biotechnology.

[59]  Jayanta Sinha,et al.  Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau. , 2005, Biotechnology and bioengineering.

[60]  Brigitte Gasser,et al.  Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion , 2014, Microbial Cell Factories.

[61]  Jochen Büchs,et al.  Microscale and miniscale fermentation and screening. , 2015, Current opinion in biotechnology.

[62]  M. Romanos,et al.  High-Level Expression of Tetanus Toxin Fragment C in Pichia Pastoris Strains Containing Multiple Tandem Integrations of the Gene , 1991, Bio/Technology.

[63]  J. Cregg,et al.  Posttransformational vector amplification in the yeast Pichia pastoris. , 2008, FEMS yeast research.

[64]  Francisco Valero,et al.  Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review , 2006, Microbial cell factories.

[65]  Nicolas Szita,et al.  Oxygen Transfer Characteristics of Miniaturized Bioreactor Systems , 2013, Biotechnology and bioengineering.

[66]  Sven-Olof Enfors,et al.  Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes , 2005, Bioprocess and biosystems engineering.

[67]  Marco Oldiges,et al.  An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform , 2012, Microbial Cell Factories.

[68]  J. Kalinowski,et al.  Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization , 2016, Microbial Cell Factories.