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.
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 (Komagataella phaffi) transcription factor Mxr1p is known to regulate multiple metabolic pathways by binding to Mxr1p response elements of target genes. Mxr1p possesses a transactivation domain within the N-terminal 400 amino acids (Mxr1N400) which is functional during methanol metabolism. (Parua et al; 2012) Studies from our lab have shown that delta_mxr1 complemented with Mxr1N400 fails to reverse the growth defect of delta_mxr1 cultured in a medium containing methanol suggesting that region beyond N-terminal 400 amino acids has an essential function. We employed DNA microarray to identify genes whose expression is regulated by Mxr1N400 and full length Mxr1 during methanol metabolism. Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction. Pabitra K. Parua, Paul M. Ryan and Elton T. Young. Molecular microbiology, 2012
Project description:Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris.
Project description:Over the past three decades, due to the universal application of Pichia yeast in the fermentation industry as well as the establishment of Pichia pastoris fermentation process for more than 30 years, the technology of the whole process has become very mature and has now reached a stagnated period of growth. However, studies and research conducted on the genomics of the classic fermentation process and the uncovering of the biological phenomena in the fermentation process from the point of view of high-throughput gene or protein is still in its early stages, and there is still insufficient data within this field. First, in collaboration with Agilent Company, we designed and prepared an expression microarray that could be used for the detection of Pichia pastoris transcriptomics. The transcriptomic changes in the five key technology steps (time points), during the fermentation process of Pichia pastoris would then be detected with the aid of an expression microarray. The five key steps of technology described above formed two important biological processes, namely, the limiting carbon source replacement and secondly, the fermentative production of exogenous proteins. The biological phenomena involved in these two processes were displayed and analyzed at the transcriptional level. In addition to this, with regard to the most important function in the fermentation process of Pichia pastoris, oxid-reduction, the metabolic drift process was analyzed and the important genes that might dominate the changes in the metabolic flux were discovered creatively by using the function tree method in this paper. This study was undertaken from the point of view of the transcriptome and the biological phenomena in the fermemntation process of Pichia pastoris. Both of which, were thoroughly explained during this study. The hope is for many more researchers to optimize the strain fermentation process, to produce proteins at the genetic level, as well as providing and obtaining new perspectives and detailed scientific data for the continued development within this field.
Project description:Transcriptional profiling of Pichia pastoris cultivated at different specific growth rates in carbon and energy-limited aerobic chemostats and retentostats
Project description:Over the past three decades, due to the universal application of Pichia yeast in the fermentation industry as well as the establishment of Pichia pastoris fermentation process for more than 30 years, the technology of the whole process has become very mature and has now reached a stagnated period of growth. However, studies and research conducted on the genomics of the classic fermentation process and the uncovering of the biological phenomena in the fermentation process from the point of view of high-throughput gene or protein is still in its early stages, and there is still insufficient data within this field. First, in collaboration with Agilent Company, we designed and prepared an expression microarray that could be used for the detection of Pichia pastoris transcriptomics. The transcriptomic changes in the five key technology steps (time points), during the fermentation process of Pichia pastoris would then be detected with the aid of an expression microarray. The five key steps of technology described above formed two important biological processes, namely, the limiting carbon source replacement and secondly, the fermentative production of exogenous proteins. The biological phenomena involved in these two processes were displayed and analyzed at the transcriptional level. In addition to this, with regard to the most important function in the fermentation process of Pichia pastoris, oxid-reduction, the metabolic drift process was analyzed and the important genes that might dominate the changes in the metabolic flux were discovered creatively by using the function tree method in this paper. This study was undertaken from the point of view of the transcriptome and the biological phenomena in the fermemntation process of Pichia pastoris. Both of which, were thoroughly explained during this study. The hope is for many more researchers to optimize the strain fermentation process, to produce proteins at the genetic level, as well as providing and obtaining new perspectives and detailed scientific data for the continued development within this field. The transcriptomic changes in the five key technology steps (time points), during the fermentation process of Pichia pastoris would then be detected with the aid of an expression microarray.
Project description:Pichia pastoris is widely used as a host for recombinant protein production. More than 500 proteins have been expressed in the organism at a variety of cultivation scales, from small shake flasks to large bioreactors. Large-scale fermentation strategies typically employ chemically-defined growth media because of its greater batch-to-batch consistency and in many cases, lower cost compared to complex media. For biopharmaceuticals, defined growth media may also simplify downstream purification and regulatory documentation. Standard formulations of defined media for Pichia are minimal ones that lack the metabolic intermediates provided by complex components such as peptone and yeast extract. As a result, growth rates and per-cell productivities are significantly lower than in complex media. We have designed an improved defined media for Pichia pastoris, rich defined medium (RDM), by systematically evaluating nutrients of increasing complexity and identifying those that are most critical for growth. We have also demonstrated the use of RDM for expression of three heterologous proteins, at titers comparable to or higher than in standard complex medium. Rich defined medium has the potential to improve productivity of Pichia pastoris fermentations and accelerate process development for new molecules.
Project description:D-lactic acid is a three-carbon organic acid with a chiral structure and can improve the thermostability of polylactic acid. Microorganisms such as the methylotrophic yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of D-lactic acid, have been engineered to produce it in high titers. However, tolerance to D-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to D-lactic acid and leads to increased D-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high D-lactic acid concentrations. Furthermore, integrating a D-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that can produce D-lactic acid at a titer of 5.12 0.35 g/L in 48 hours , a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to D-lactic acid including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of D-lactic acid by manipulating yeast flocculation.
Project description:The regulation of gene expression in Pichia pastoris has so far involved study of transcription factors. However, there are factors other than transcription factors affecting mRNA synthesis such as coactivators, enhancers, and chromatin modifiers. This study is a first ever report on epigenetic regulation in Pichia pastoris where, a histone acetyltransferase is reported to regulate transcription of genes involved in utilization of alternate carbon sources. We employed RNA Seq to identify new targets of Gcn5 in methanol medium.