Abstract
BackgroundThe methylotrophic yeast Pichia pastoris is widely used as a bioengineering platform for producing industrial and biopharmaceutical proteins, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis and peroxisome biogenesis. With the development of DNA microarray and mRNA sequence technology, the P. pastoris transcriptome has become a research hotspot due to its powerful capability to identify the transcript structures and gain insights into the transcriptional regulation model of cells under protein production conditions. The study of the P. pastoris transcriptome helps to annotate the P. pastoris transcript structures and provide useful information for further improvement of the production of recombinant proteins.ResultsWe used a massively parallel mRNA sequencing platform (RNA-Seq), based on next-generation sequencing technology, to map and quantify the dynamic transcriptome of P. pastoris at the genome scale under growth conditions with glycerol and methanol as substrates. The results describe the transcription landscape at the whole-genome level and provide annotated transcript structures, including untranslated regions (UTRs), alternative splicing (AS) events, novel transcripts, new exons, alternative upstream initiation codons (uATGs), and upstream open reading frames (uORFs). Internal ribosome entry sites (IRESes) were first identified within the UTRs of genes from P. pastoris, encoding kinases and the proteins involved in the control of growth. We also provide a transcriptional regulation model for P. pastoris grown on different carbon sources.ConclusionsWe suggest that the IRES-dependent translation initiation mechanism also exists in P. pastoris. Retained introns (RIs) are determined as the main AS event and are produced predominantly by an intron definition (ID) mechanism. Our results describe the metabolic characteristics of P. pastoris with heterologous protein production under methanol induction and provide rich information for further in-depth studies of P. pastoris protein expression and secretion mechanisms.
Highlights
The methylotrophic yeast Pichia pastoris is widely used as a bioengineering platform for producing industrial and biopharmaceutical proteins, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis and peroxisome biogenesis
Summary of RNA-Seq data To provide an analysis of the P. pastoris transcriptome at single base-pair resolution, cDNA libraries were constructed from poly (A)-enriched mRNA of P. pastoris chemostat cultures and analyzed using high-throughput Illumina sequencing
RNA samples were prepared from the chemostat cultures of P. pastoris GS115 transformed with different expression vectors bearing a lipase gene from Rhizomucor miehei (RML), including a secretory expression plasmid, a surface display expression plasmid, and a plasmid without the R. miehei lipase (RML) gene (Additional file 1: Figure S1)
Summary
The methylotrophic yeast Pichia pastoris is widely used as a bioengineering platform for producing industrial and biopharmaceutical proteins, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis and peroxisome biogenesis. With the development of DNA microarray and mRNA sequence technology, the P. pastoris transcriptome has become a research hotspot due to its powerful capability to identify the transcript structures and gain insights into the transcriptional regulation model of cells under protein production conditions. The methylotrophic yeast P. pastoris is widely used as a heterologous expression platform for the industrial production of a series of valuable proteins due to its excellent characteristics, such as highly inducible gene expression, highdensity cell growth, and high secretory capability [1]. The first DNA microarray for P. pastoris was produced using commercial sequence data (Integrated Genomics, Chicago, IL, USA), containing partial P. pastoris genes, and examined the unfolded protein response during protein production [6]. Transcriptomics, proteomics, and metabolic flux analysis data for P. pastoris will benefit from this now-public sequence information
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