Yeast Synthetic Minimal Biosensors for Evaluating Protein Production.

Kai Peng,Heinrich Kroukamp,Ian T Paulsen,Isak S Pretorius

doi:10.1021/acssynbio.0c00633

Abstract

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer, the induction of the UPR has a significant impact on the recombinant protein production in eukaryotic cell factories, including the industrial workhorseSaccharomyces cerevisiae. Being able to accurately detect and measure this ER stress response in single cells, enables the rapid optimization of protein production conditions and high-throughput strain selection strategies. Current methodologies to monitor the UPR in S. cerevisiae are often temporally and spatially removed from the cultivation stage or lack updated systematic evaluation. To this end, we constructed and systematically evaluated a series of high-throughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The native promoters of DER1 and ERO1 were identified to have suitable UPR biosensor properties and served as an expression level guide for orthogonal sensor benchmarking. Our best synthetic minimal sensor is only 98 bp in length, has minimal homology to other native yeast sequences and displayed superior sensor characteristics. The synthetic minimal UPR sensor was able to accurately distinguish between cells expressing different heterologous proteins and between the different secretion levels of the same protein. This work demonstrated the potential of synthetic UPR biosensors as high-throughput tools to predict the protein production capacity of strains, interrogate protein properties hampering their secretion, and guide rational engineering strategies for optimal heterologous protein production.

Highlights

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation
Disruption in the ER membrane morphology or lipid composition has been shown to trigger the UPR.[3−5] The UPR relieves the ER stress and restores the ER homeostasis by modulating the transcription of UPRresponsive genes (UPR genes)[6] involved in processes to modulate the protein translocation rates in-and-out of the ER, fine-tune the protein chaperone level, expand the volume of the ER, and enhance the ER associated degradation (ERAD) of terminally misfolded proteins.[6−10] The importance of this ER quality control mechanism is highlighted in mammalian cells by the presence of multiple UPR pathways, namely, the PERK, ATF6, and IRE1 pathways, allowing for more precise control of metabolism, translation, and apoptosis to resolve the ER stress.[2,11]
The mature HAC1 mRNA produced via this alternative splicing can be translated into Hac1p, which subsequently gets imported into the nucleus, where it binds to the upstream activating sequence, called the UPR element (UPRE) to regulate the expression of UPR genes.[13,19−21]

Summary

Introduction

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. Current methodologies to monitor the UPR in S. cerevisiae are often temporally and spatially removed from the cultivation stage or lack updated systematic evaluation To this end, we constructed and systematically evaluated a series of highthroughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to maintain the endoplasmic reticulum (ER) folding homeostasis and acts as a protective buffer against excessive ER stress.[1,2] When the protein folding workload in the ER exceeds the capacity of its folding machinery, the accumulation of misfolded proteins typically results in the UPR activation. Sensors exploited the Hac1p-dependent UPRE of the UPR genes, with constructs consisting of one or multiple copies of the 22 bp redundant UPRE consensus sequence 1 (rUPRE1,19 later refined to the CAGCGTG core sequence20), placed upstream of a CYC1 core promoter, and used enzymatic reporters like βgalactosidas,[13,19,26] which are mostly superseded by fluorescent proteins.[8,27,28] In addition to the UPR transcription mechanism, several UPR sensor designs tapped into other UPR related non-transcriptional cellular processes as an indirect reflection of the UPR, like using redox-sensitive GFP to detect the redox change in the ER lumen,[27] using fluorescent tagged ER lumen chaperone to monitor its mobility caused by UPR activation,[29] and making an UPR-specific alternative splicing reporter by substituting GFP for the first exon of the HAC1 gene, which only produces GFP when the alternative splicing occurs.[30]

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Conclusion