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Open Access Highly Accessed Research article

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

Shuli Liang1, Bin Wang1, Li Pan1, Yanrui Ye1, Minghui He3, Shuangyan Han1, Suiping Zheng1, Xiaoning Wang12* and Ying Lin1*

Author affiliations

1 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong 510006, China

2 School of life Science, General Hospital of PLA, Beijing 100853, China

3 Beijing Genomics Institute at Shenzhen, Shenzhen 518000, China

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Citation and License

BMC Genomics 2012, 13:738  doi:10.1186/1471-2164-13-738

Published: 31 December 2012

Abstract

Background

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 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.

Results

We 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.

Conclusions

We 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.

Keywords:
RNA-Seq; Transcriptome; Pichia pastoris; Methanol induction; Internal ribosome entry site (IRES); Translation initiation mechanism