Metabolic engineering strategies for improvement of ethanol production in cellulolytic Saccharomyces cerevisiae

Abstract

As a traditional ethanol-producing microorganism, Saccharomyces cerevisiae is an ideal host for consolidated bioprocessing. However, expression of heterologous cellulase increases the metabolic burden in yeast, which results in low cellulase activity and poor cellulose degradation efficiency. In this study, cellulase-expressing yeast strains that could efficiently degrade different cellulosic substrates were created by optimizing cellulase ratios through a POT1-mediated δ-integration strategy. Metabolic engineering strategies, including optimization of codon usage, promoter and signal peptide, were also included in this system. We also confirmed that heterologous cellulase expression in cellulosic yeast induced the unfolded protein response. To enhance protein folding capacity, the endoplasmic reticulum chaperone protein BiP and the disulfide isomerase Pdi1p were overexpressed, and the Golgi membrane protein Ca2+/Mn2+ ATPase Pmr1p was disrupted to decrease the glycosylation of cellulase. The resultant strain, SK18-3, could produce 5.4 g L-1 ethanol with carboxymethyl-cellulose. Strain SK12-50 achieved 4.7 g L-1 ethanol production with phosphoric acid swollen cellulose hydrolysis. When Avicel was used as the substrate, 3.8 g L-1 ethanol (75% of the theoretical maximum yield) was produced in SK13-34. This work will significantly increase our knowledge of how to engineer optimal yeast strains for biofuel production from cellulosic biomass.

Metabolic engineering strategies for consolidated production of lactic acid from lignocellulosic biomass

“…Moreover, the choice of enzyme candidates for RCSs is often limited to those who are efficiently secreted by the microbial host . So far, the number of strategies to solve or reduce issues in protein secretion is relatively little . In most cases, they consist of engineering the signal peptide of cellulases by replacing it with host‐specific signal peptides .…”

“…The minimal requirement for efficient depolymerization of cellulosic substrate is a system consisting of three enzyme activities (i.e., an exoglucanase, an endoglucanase, and a β‐glucosidase) and, additionally, a scaffoldin for cellulosome‐inspired complexes . However, expression of heterologous cellulases is often toxic because of saturation of protein secretion pathways in the host . These issues have severely hampered advances of RCSs.…”

Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

“…As a result, S. cerevisiae strains capable of simultaneous saccharification and fermentation (SSF) on a range of simple cellulosic substrates are available [ 8 – 10 ], but the ratios of the enzymes required for optimal hydrolysis changes depending on the available substrate and mostly remains undefined [ 11 ]. Therefore, many efforts have now turned to the optimisation of enzyme ratios by harnessing different promoters [ 12 ], combining specific ratios of yeast strains displaying different enzymes [ 13 ] and manipulating gene copy numbers one by one [ 14 ].…”

Rapid Optimisation of Cellulolytic Enzymes Ratios in Saccharomyces Cerevisiae using in Vitro SCRaMbLE

“…Therefore, many efforts have now turned to the optimisation of enzyme ratios by harnessing different promoters [12], combining specific ratios of yeast strains displaying different enzymes [13] and manipulating gene copy numbers one by one [14].…”