Screening for functional IRESes using α-complementation system of β-galactosidase in Pichia pastoris

Abstract

AbstractBackgroundPichia pastorisis 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 inP. pastorisare 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 inP. pastoris, which will allowP. pastoristo express multiple proteins in a single mRNA and increase its efficacy as a platform for metabolic engineering and synthetic biology.ResultsIn order to efficiently screen the IRES sequences, we first set out to create a screening system usingLacZgene. Due to the cryptic transcription of theLacZgene, we established the α-complementation system of β-galactosidase inP. pastoriswith 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 inP. pastoris, which were from TEV, PVY, RhPV, TRV, KSHV, crTMV viruses and the 5′-UTR of theYAP1gene ofS. cerevisiae.ConclusionsWe showed here that α-complementation also works inP. pastorisand 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 intoP. pastorisvia a prokaryotic-like polycistronic manner, which provided new efficient tools for metabolic engineering and synthetic biology researches inP. pastoris.