Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiaeidentifies mutants with improved heterologous exocellulase activity and host secretion

Abstract

BackgroundAs a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability.ResultsSaccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae.ConclusionsSystematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives

“…Additionally, the application of extra EGs to complement the debilitated activity of glycosylated CBHs should be avoided to limit external enzyme supplementation and develop cost-effective biorefineries [98]. Another strategy includes the heterologous expression of CBHs in glycosylation-deficient strains, as was reported for a Saccharomyces cerevisiae mutant, in which the knockout of glycosylation-related genes resulted in significantly higher activity of heterologously expressed P. chrysosporium exocellulase [99]. In contrast, the inhibition of O-glycosylation in T. reesei [100] and mutation of O-glycosylation in A. nidulans [101] impaired native glycoprotein secretion, indicating that the exact correlation between glycosylation and protein secretion should be investigated before applying glycosylation-deficient strains in heterologous protein expression.…”

Rational design and evolutional fine tuning of Saccharomyces cerevisiae for biomass breakdown

“…Increased N-glycosylation of recombinant cellulases reduces their activity and might also reduce their secretion by yeast. Knockout of the inherent glycosylation-related genes mnn10 and pmt5 increases the extracellular activity of Phanerochaete crysosporium excocellulase PCX up to 6.0-fold and 4.3-fold, respectively [29]. Golgi-to-endosome transport gene vps21 also improves the PCX activity up to 6.5-fold.…”

“…Golgi-to-endosome transport gene vps21 also improves the PCX activity up to 6.5-fold. On the other hand, the double knockout of mnn10 and vps21 slows yeast cell growth [29]. Whereas correctly modified proteins are trafficked to the plasma membrane for secretion, misfolded proteins are either translocated through the ER back to the cytosol and degraded by ERassociated protein degradation machinery or transported to vacuoles and degraded by proteolytic enzymes.…”

Filamentous fungi-like secretory pathway strayed in a yeast system: peculiarities of Yarrowia lipolytica secretory pathway underlying its extraordinary performance

“…Individual traffic steps and specificity of vesicular fusion at the destination membrane at the traffic cross-roads are subjected to stringent control by numerous intracellular membrane proteins (Idiris et al 2010a). Nevertheless, several studies conducted on Schizosaccharomyces pombe (Idiris et al 2010b), S. cerevisiae (Kitagawa et al 2011; Wang et al 2013), Hansenula polymorpha (Agaphonov et al 2005), or Pichia pastoris ( Komagataella spp) (Marsalek et al 2017) demonstrate significant contribution of mis-sorting events to the overall secretory efficiency of the yeast cell, which could be overcome to some extent by specific genetic manipulations presented in those works. In reference to the scope of this review, occurrence of mis-sorting events has not been studied in Y. lipolytica , so far.…”