Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing

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.

Characterization of Natural Antisense Transcript, Sclerotia Development and Secondary Metabolism by Strand-Specific RNA Sequencing of Aspergillus flavus

“…4A ), including retained introns (RIs), skipped exons (SEs), alternative 5′-splice sites (A5SSs) and alternative 3′-splice sites (A3SSs). About 12.78% of the A. flavus multi-exon genes produced AS isoforms, similar to A. oryzae RIB40 (11.10%) [43] and many more than Pichia pastoris (4.78%) [68]. This is in agreement with the conclusion that the frequency of AS events is proportional to the ratio of multi-exon genes in a genome (76.98% for A. oryzae and 11.91% for P. pastoris ) [69].…”

High-density linkage map construction and QTL analysis for earliness-related traits in Gossypium hirsutum L

“…The quality of our genetic map was evaluated by heat map analysis, which confirmed the accuracy of the 26 linkage groups because the recombination frequency among adjacent markers was relatively low (Additional file 13). In addition, linkage information for the 139 SSR loci was validated by a comparison with genetic maps reported by Liang et al [14] and Blenda et al [48]; in particular, the distribution of loci in their maps confirmed the common-marker-based assignment of the 26 linkage groups in our study (Additional file 14). Most of the marker collinearity between the genetic map and the genome sequence was clear, thus illustrating that the markers were accurately located on the present genetic map and had sufficient coverage on the 26 chromosomes (Fig.…”

“…Yu et al reported a genetic map containing 2,072 loci with an average marker interval of 1.63 cM [13]. Liang et al reported an updated map consisting of 3,414 loci with an average marker interval of 1.08 cM [14]. Shi et al constructed a genetic map with 2,292 loci and 2.23 cM between adjacent markers [15].…”

GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris

“…TSS sites are ranked based on expression in the SDEG condition [55]. GC-ARS and AT-ARS sites are ranked by the strength of the best match to the G/C- and the A/T-rich motif respectively.…”