Overexpression of native Saccharomyces cerevisiae ER-to-Golgi SNARE genes increased heterologous cellulase secretion

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs) are essential components of the yeast protein-trafficking machinery and are required at the majority of membrane fusion events in the cell, where they facilitate SNARE-mediated fusion between the protein transport vesicles, the various membrane-enclosed organelles and, ultimately, the plasma membrane. We have demonstrated an increase in secretory titers for the Talaromyces emersonii Cel7A (Te-Cel7A, a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (Sf-Cel3A, a β-glucosidase) expressed in Saccharomyces cerevisiae through single and co-overexpression of some of the endoplasmic reticulum (ER)-to-Golgi SNAREs (BOS1, BET1, SEC22 and SED5). Overexpression of SED5 yielded the biggest improvements for both of the cellulolytic reporter proteins tested, with maximum increases in extracellular enzyme activity of 22 % for the Sf-Cel3A and 68 % for the Te-Cel7A. Co-overexpression of the ER-to-Golgi SNAREs yielded proportionately smaller increases for the Te-Cel7A (46 %), with the Sf-Cel3A yielding no improvement. Co-overexpression of the most promising exocytic SNARE components identified in literature for secretory enhancement of the cellulolytic proteins tested (SSO1 for Sf-Cel3A and SNC1 for Te-Cel7A) with the most effective ER-to-Golgi SNARE components identified in this study (SED5 for both Sf-Cel3A and Te-Cel7A) yielded variable results, with Sf-Cel3A improved by 131 % and Te-Cel7A yielding no improvement. Improvements were largely independent of gene dosage as all strains only integrated single additional SNARE gene copies, with episomal variance between the most improved strains shown to be insignificant. This study has added further credence to the notion that SNARE proteins fulfil an essential role within a larger cascade of secretory machinery components that could contribute significantly to future improvements to S. cerevisiae as protein production host.

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

“…The S. cerevisiae yENO1 reference strain was used in this study as it was a strain in which the UPR was uninduced and was thus used to compare UPR induction levels of recombinant cellulase secreting S. cerevisiae Y294 strains. The S. cerevisiae Y294 CBH and β‐glucosidase (BGL) producing strains used here were previously described .…”

“…The yeast Saccharomyces cerevisiae has many desirable traits for CBP such as high ethanol productivity, high ethanol yield, and comparatively high resistance to inhibitors found in the lignocellulosic hydrolysate . S. cerevisiae is also an extensively studied host for heterologous protein production and has thus been the subject of much research to engineer and enhance the expression of necessary cellulases to accomplish the ultimate goal of being able to break down lignocellulose for second‐generation production of bioethanol . Despite significant success achieved in cellulase expression in yeast, the secretion titers of cellobiohydrolases (CBHs) have been relatively low, and one of the reasons for this phenomenon is the induction of the unfolded protein response (UPR) .…”

Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol

“…In addition to the CBHs, we compared the amounts of BGL and EGL secreted into the culture supernatant by the signal peptide MFα, the native signal sequence, and TFPs for SfBGL1 21 and TrEGL2 27, 28 . For EGL activity analysis, the secretion of EGL by the TFP system was analysed by incubating the cell-free culture supernatants of S. cerevisiae harbouring YGaTFPn-EGL with carboxymethylcellulose (CMC) and determining the amount of the reducing sugars formed.…”

“…In contrast, the free cellulase secretion strategy offers a method to avoid an argument about the physical restrictions associated with cell-surface display. Cellulolytic yeasts were constructed by the secretion of individual cellulases into the medium 19–21 ; however, complete conversion of the insoluble cellulosic substrate by recombinant yeasts has not yet been achieved 15 . The key element to determining the performance of this system is the amount of enzymes secreted, because enzymes might be diffused away in a reactor 15 .…”

Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid

“…For example, the S. fibuligera BGL1 gene, encoding β-glucosidase 1, has been heterologously expressed in S. cerevisiae to construct cellobiose-growing and fermenting strains [22–24]. Several studies aimed at overexpressing S. fibuligera β-glucosidase in heterologous hosts have been also conducted to provide the recombinant enzyme as supplement in cellulase mixtures to enhance the saccharification of cellulose [25, 26]. Although the production of lignocellulosic ethanol is expected to increase rapidly as advanced biofuel in the renewable fuel industry, starch is still the most commonly used feedstock for the production of conventional biofuel (http://www.afdc.energy.gov/laws/RFS).…”