Background Pichia pastoris is becoming a promising chassis cell for metabolic engineering and synthetic biology after its whole genome and transcriptome sequenced. However, the current systems for multigene co-expression in P. pastoris are not efficient. The internal ribosome entry site (IRES) has an ability to recruit the ribosome to initiate protein synthesis by cap-independent translation manner. This study seeks to screen IRES sequences that are functional in P. pastoris, which will allow P. pastoris to express multiple proteins in a single mRNA and increase its efficacy as a platform for metabolic engineering and synthetic biology. Results In order to efficiently screen the IRES sequences, we first set out to create a screening system using LacZ gene. Due to the cryptic transcription of the LacZ gene, we established the α-complementation system of β-galactosidase in P. pastoris with the optimum length of the α-complementing peptide at ~ 92 amino acids. The optimal α-complementing peptide was then used as the second reporter to screen IRESes in the engineered GS115 expressing the corresponding ω-peptide. A total of 34 reported IRESes were screened. After ruling out all false positive or negative IRESes, only seven IRESes were functional in P. pastoris, which were from TEV, PVY, RhPV, TRV, KSHV, crTMV viruses and the 5'-UTR of the YAP1 gene of S. cerevisiae. Conclusions We showed here that α-complementation also works in P. pastoris and it can be used in a variety of in vivo studies. The functional IRESes screened in this study can be used to introduce multiple genes into P. pastoris via a prokaryotic-like polycistronic manner, which provided new efficient tools for metabolic engineering and synthetic biology researches in P. pastoris.

中文翻译：

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使用毕赤酵母中β-半乳糖苷酶的α-互补系统筛选功能性IRES。

背景毕赤酵母在其全基因组和转录组测序后，正在成为代谢工程和合成生物学的有前途的底盘细胞。然而，目前在巴斯德毕赤酵母中进行多基因共表达的系统效率不高。内部核糖体进入位点 (IRES) 具有募集核糖体以通过不依赖帽的翻译方式启动蛋白质合成的能力。本研究旨在筛选在毕赤酵母中具有功能的 IRES 序列，这将使毕赤酵母能够在单个 mRNA 中表达多种蛋白质，并提高其作为代谢工程和合成生物学平台的功效。结果为了有效筛选IRES序列，我们首先着手创建使用LacZ基因的筛选系统。由于 LacZ 基因的隐秘转录，我们在巴斯德毕赤酵母中建立了β-半乳糖苷酶的α-互补系统，α-互补肽的最佳长度约为92个氨基酸。然后将最佳的 α-互补肽用作第二个报告基因，以筛选表达相应 ω-肽的工程化 GS115 中的 IRESes。共筛选了 34 个报告的 IRESes。排除所有假阳性或假阴性IRES后，只有7个IRES在巴斯德毕赤酵母中起作用，分别来自TEV、PVY、RhPV、TRV、KSHV、crTMV病毒和酿酒酵母YAP1基因的5'-UTR。结论 我们在此表明​​，α-补体也适用于巴斯德毕赤酵母，它可用于各种体内研究。本研究中筛选的功能性 IRES 可用于通过类原核多顺反子方式将多个基因引入巴斯德毕赤酵母中，