School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong, China.
BMC Genomics. 2012 Dec 31;13:738. doi: 10.1186/1471-2164-13-738.
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.
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.
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.
甲醇营养型酵母毕赤酵母被广泛用作生产工业和生物制药蛋白、研究蛋白表达和分泌机制以及分析代谢物合成和过氧化物酶体生物发生的生物工程平台。随着 DNA 微阵列和 mRNA 序列技术的发展，毕赤酵母转录组由于其强大的能力，即鉴定转录结构并深入了解蛋白生产条件下细胞的转录调控模型，成为了一个研究热点。研究毕赤酵母转录组有助于注释毕赤酵母转录结构，并为进一步提高重组蛋白的生产提供有用信息。
我们使用大规模平行 mRNA 测序平台（RNA-Seq），基于下一代测序技术，在以甘油和甲醇为底物的生长条件下，以基因组规模对毕赤酵母的动态转录组进行了图谱绘制和定量。结果描述了全基因组水平的转录景观，并提供了注释的转录结构，包括非翻译区（UTRs）、选择性剪接（AS）事件、新转录本、新外显子、选择性上游起始密码子（uATGs）和上游开放阅读框（uORFs）。在毕赤酵母基因的 UTR 内首次鉴定到内部核糖体进入位点（IRESes），这些基因编码激酶和参与生长控制的蛋白。我们还为毕赤酵母在不同碳源上生长提供了一个转录调控模型。
我们认为 IRES 依赖的翻译起始机制也存在于毕赤酵母中。保留内含子（RIs）被确定为主要的 AS 事件，主要由内含子定义（ID）机制产生。我们的结果描述了甲醇诱导下毕赤酵母异源蛋白生产的代谢特征，并为进一步深入研究毕赤酵母蛋白表达和分泌机制提供了丰富的信息。
