Screening for functional IRESes using α-complementation system of β-galactosidase in 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 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 , which will allow to express multiple proteins in a single mRNA and increase its efficacy as a platform for metabolic engineering and synthetic biology. In order to efficiently screen the IRES sequences, we first set out to create a screening system using gene. Due to the cryptic transcription of the gene, we established the α-complementation system of β-galactosidase in 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 , which were from TEV, PVY, RhPV, TRV, KSHV, crTMV viruses and the 5'-UTR of the gene of . We showed here that α-complementation also works in 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 via a prokaryotic-like polycistronic manner, which provided new efficient tools for metabolic engineering and synthetic biology researches in .