The in vivo detection and measurement of the unfolded protein response in recombinant cellulase producing Saccharomyces cerevisiae strains

Abstract

The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and has been engineered for consolidated bioprocessing (CBP) of lignocellulosic biomass through heterologous cellulase expression. However, S. cerevisiae produces low titers of cellulases and one suspected reason for this is that heterologous proteins induce the unfolded protein response (UPR). Current methods of measuring the UPR are RNA based and can be inconsistent and cumbersome. We developed vector‐based biosensors that will detect and quantify UPR activation. The vector consisted of either the Trichoderma reesei xylanase 2 or codon optimized green fluorescent protein (eGFP) reporter genes under the control of the S. cerevisiae PHAC1 or PKAR2 promoters. The eGFP reporter under control of PKAR2 was identified as the preferred combination due to its superior dynamic range and its greater sensitivity when measuring UPR induction in cellulase producing strains. To our knowledge, we show for the first time that significant UPR activation differences could consistently be observed for different cellulase candidate genes unlike previous RNA‐based tests, which were unable to detect these differences. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them favorable for use in CBP yeasts.

Yeast synthetic minimal biosensors for evaluating protein production

“…We first investigated if and which native UPR promoters could be effective synthetic parts in building UPR sensors, which also helps to establish a more holistic view of the expression characteristics of the native UPR genes in baker’s yeast. Except for some commonly used UPR genes like KAR2 , ERO1 and HAC1 (Cedras 2020, Pincus 2014), the transcriptional activation profiles of many native UPR promoters have not been documented. To this end we selected and characterised the expression profiles of 9 putative promoters of known UPR genes in S. cerevisiae (Travers 2000), covering different functional categories of the secretory pathway: DER1 for misfolded protein degradation, ERO1 , EUG1 , KAR2 and PDI1 for polypeptide folding, PMT1 for protein modification, SEC12 and SEC62 for protein trafficking, plus HAC1 the UPR effector.…”

“…The previous UPR transcription-based sensors have relied upon either the 22bp redundant UPRE1 (Mori 1992) plus native non-UPR promoter like CYC1 ; or upon limited choices of native UPR promoters like that of HAC1 , KAR2 (Cedras 2020) and ERO1 (Patil 2004, Pincus 2014), that could be subjected to additional non-UPR specific regulation. While in the last decade substantial advances have been made in both UPRE (Fordyce et al 2012) and synthetic minimal promoter in S. cerevisiae (Redden et al 2015).…”

Surface tethered xylosidase activity improved xylan conversion in engineered strains of Saccharomyces cerevisiae

“…After the amplification procedures, the PCR products were separated on 1% agarose gels. Putative transformants were also spot plated onto 0.1% AZCL‐xylan (Megazyme, Bray, Ireland) SC ‐URA plates to test for xylanase activity, along with the background strains as negative controls and Y294( XYN2 ) as a positive control 31,32 …”

“…Putative transformants were also spot plated onto 0.1% AZCL-xylan (Megazyme, Bray, Ireland) SC -URA plates to test for xylanase activity, along with the background strains as negative controls and Y294(XYN2) as a positive control. 31,32 Enzymatic assays YP media (10 g/L yeast extract and 20 g/L peptone) was prepared and aliquoted into volumetric flasks at a volume of 10 mL and autoclaved. Media was supplemented either with sterile glucose (20 g/L -YPD) or with glucose (10 g/L) and xylose (10 g/L -YPDX).…”

Editorial for the special issue on “Metabolic engineering”

“…The system developed by Cedras et al. [8] benefits the selection of candidate cellulases with reduced induction of the unfolded protein response. The third‐generation biorefinery, based on microalgae, is the focus of three reviews in this special issue.…”