Abstract
BackgroundPichia 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.ResultsIn 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.ConclusionsWe 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.
Highlights
Pichia pastoris is becoming a promising chassis cell for metabolic engineering and synthetic biology after its whole genome and transcriptome sequenced
The microorganism expressing β-galactosidase forms blue colonies on the solid medium bearing 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-gal), which makes β-galactosidase a convenient and effective reporter in microbial research [26]. Based on this property of β-galactosidase, we planned to use β-galactosidase as a reporter in bicistronic system to indicate the function of internal ribosome entry sites (IRESes) in P. pastoris
If the reporter gene in reporter constructs can be transcribed by cryptic promoter in plasmid backbone or reporter gene itself, it will reduce the reliability of the experimental results especially in screening for functional IRESes
Summary
Pichia pastoris is becoming a promising chassis cell for metabolic engineering and synthetic biology after its whole genome and transcriptome sequenced. 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. The. In metabolic engineering and synthetic biology, the production of synthetic molecules often involves integration of multiple genes or even entire metabolic pathways into the host. In the production of riboflavin, a total of six key genes in the riboflavin biosynthesis pathway were integrated into P. pastoris genome, which effectively increased the yield of riboflavin [9]. How to better solve co-expression of multiple genes is important for P. pastoris to become a highly efficient platform for the application of metabolic engineering and synthetic biology
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