Transcription profiling by array of Pichia pastoria strains with different gene dosage of Rhizopus oryzae lipase (Rol) as a model protein
ABSTRACT: Pichia pastoris is a widely used yeast platform for heterologous protein production. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden. In order to find the bottlenecks of this strategy, here we present a transcriptome profile comparison of a series of Pichia pastoris strains carrying a different gene dosage of the Rhizopus oryzae lipase (Rol) as a model protein. Cells were grown on a mixture of glycerol:methanol in chemostat cultures. 2 color experiment in reference design.
Project description:Comparison of transcription profile of Pichia pastoris cells grown on Glucose medium with Pichia pastoris cells grown on Methanol/Glycerol medium, the fermentations were done in a chemostat. 2 color experiment in reference design. Pichia pastoris reference mix [mixed pool of Pichia pastoris cells sampled from various conditions including cells grown on glycerine, glucose and methanol, on full andminimal medium, in stationary and exponential growth phase, and in different stress states]
Project description:Pichia pastoris was grown in carbon limited chemostat cultures on mineral medium with glucose as carbon source. 3 different osmolarities were selected (140, 850, 1400mOsmol kg-1). Samples were taken at steady-state, thus the effect of osmolarity in adapted cells was monitored.
Project description:Changes in the proteome of Pichia pastoris cultures either grown on glucose or methanol were analysed using a 2D-chromatography based TMT labeling LC-ESI-MS workflow. Three biological replicates, with two technical replicates each, had been analyzed leading to six replicate data sets of both growth conditions. The data is supported by peroxisomal protein data.
Project description:We have identified a methanol- and biotin-starvation-inducible zinc finger protein named ROP [repressor of phosphoenolpyruvate carboxykinase (PEPCK)] in the methylotrophic yeast Pichia pastoris. When P.pastoris strain GS115 (wild-type, WT) is cultured in biotin-deficient, glucose ammonium (Bio-) medium, growth is suppressed due to the inhibition of anaplerotic synthesis of oxaloacetate, catalysed by the biotin-dependent enzyme pyruvate carboxylase (PC). Deletion of ROP results in a strain (∆ROP) that can grow under biotin-deficient conditions due to derepression of a biotin- and PC-independent pathway of anaplerotic synthesis of oxaloacetate. Northern analysis as well as microarray expression profiling of RNA isolated from WT and ∆ROP strains cultured in Bio(-) medium indicate that expression of the phosphoenolpyruvate carboxykinase gene (PEPCK) is induced in ∆ROP during biotin- or PC-deficiency even under glucose-abundant conditions. There is an excellent correlation between PEPCK expression and growth of ∆ROP in Bio(-) medium, suggesting that ROP-mediated regulation of PEPCK may have a crucial role in the biotin- and PC-independent growth of the ∆ROP strain. To our knowledge, ROP is the first example, of a zinc finger transcription factor involved in the catabolite repression of PEPCK in yeast cells cultured under biotin- or PC-deficient and glucose-abundant conditions. Agilent one-color experiment,Organism: Pichia pastoris ,Custom Pichia pastoris Gene Expression 8x15k array designed by Genotypic Technology Pvt. Ltd. (Agilent -AMADID: 25088 ) , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an ingrained aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. In order to evaluate the prevalence and underlying causes of clonal variation, nine strains were selected, each transformed with a single copy of the human serum albumin (HSA) gene. All strains were subjected to a wide-ranging evaluation to understand the implications of this phenomenon.