Engineering The UPR Pathway To Improve Heterologous Expression In Yeast

Many natural proteins were developed into drugs and produced for direct analysis, requiring improved hosts to achieve high-level heterologous proteins production. The unfolded protein response (UPR) is an evolutionarily conserved mechanism (especially the yeast and filamentous fungi) by which all eukaryotic cells adapt to the accumulation of unfolded proteins in endoplasmic reticulum (ER). Hac1p, the transcription factor can activate the UPR signal transduction, which regulates genes related the protein folding, degradation and etc.To investigate the effects of engineering UPR pathway to heterologous expression, hen egg white lysozyme (HEL) cDNA cloned from tissue of mature hen oviduct fused toα-signal peptide was inserted into pYES2 and expressed as a modle protein. The shuttle plasmids copy number was assessed using a novel method based on the E.coli transformation and plate count technique, meanwhile, the plasmids stability was estimated. Results proposed one-step cultivation strategy was a feasible alternative.HAC1icDNA was gained by RT-PCR and cloned into different plasmids or substituted the HAC1locus on genome. The results indicated the effect of UPR pathway activation on heterologous expression was dose-dependent, and yeast strain W303 harboring HAC1icDNA on a multi-copy plasmid YEplac181 achieved the highest yields, 2.7 mg HEL/L.A novel high-throughput screening (HTS) system to obtain heterologous over-expression in Saccharomyces cerevisiae strains was developed. The protocol designed here was based on bio-macromolecular physical interaction between CBD or leucine zipper displayed on the surface of hosts and the cellulose substrate. Using the screening system, directed evolution of Hac1p was carried out in Saccharomyces cerevisiae, and an improved mutant (Hac1pm1, Arg48Gly, Ser111Gly, 3.9 mg HEL/L yields) were selected. Results proposed that through selective pressure, this assay may afford a more effective screening system compared with previous selection system. Moreover, it could be employed in general biochemical analysis without utilization of flow cytometry or well plate reader.At last, through UPR pathway analysis, we found that the reduction-oxidation (Redox) balance disruption of strains by UPR activation was the main reason for growth deficit. UPRE (UPR element) mediating the signal augmentation of UPR pathway was performed to improve the production. Truncated form, MAE1s under the control of the promoter of KAR2, harboring UPRE sequence was amplified into the ura3 locus, and 1.4 fold yield was gained compared with the control (W303 +Hac1pm1) when UPR pathway was activated by Hac1pm1. Further, GPD2 was knocked out to balance the Redox and achieved more large heterologous expression.Engineering UPR pathway is expected to improve the heterologous expression in other microorganisms (P.pastoris, K.lactis, filamentous fungi, etc.).