Engineering Saccharomyces cerevisiae for application in integrated bioprocessing biorefineries.

After decades of research and development, no organism - natural or engineered - has been described that can produce commodity products through direct microbial conversion to meet industry demands in terms of rates and yields. Variation in lignocellulosic biomass (LCB) feedstocks, the lack of a widely applicable pretreatment method, and the limited economic value of energy products further complicates second-generation biofuel production. Nevertheless, the emergence of advanced genomic editing tools and a more comprehensive understanding of yeast metabolic systems offer promising avenues for the creation of yeast strains tailored to LCB biorefineries.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks.

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses.

Increasing extracellular cellulase activity of the recombinant Saccharomyces cerevisiae by engineering cell wall-related proteins for improved consolidated processing of carbon neutral lignocellulosic biomass.

Sustainable bioproduction usingcarbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinantyeast of Saccharomyces cerevisiaeis a promising strategy forlignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels.

Improvement of cell-tethered cellulase activity in recombinant strains of Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) remains an attractive option for the production of commodity products from pretreated lignocellulose if a process-suitable organism can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second-generation (2G) bioethanol. A promising strategy for heterologous cellulase production in yeast entails displaying enzymes on the cell surface by means of glycosylphosphatidylinositol (GPI) anchors.

Fluorescence-Based Biosensors for the Detection of the Unfolded Protein Response.

The unfolded protein response (UPR) is a highly conserved protein quality control mechanism of eukaryotic cells. Aberrations in this response have been linked to several human diseases, including retinitis pigmentosa and several cancers, and have been shown to have a drastic impact on recombinant protein yields in fungal, insect, and mammalian cell lines. Here, we describe the use of in vivo biosensors to measure and characterize this dynamic cellular response, specifically for detecting the UPR induced by protein overproduction stress in the model cell factory Saccharomyces cerevisiae.

Heterologous production of cellulose- and starch-degrading hydrolases to expand Saccharomyces cerevisiae substrate utilization: Lessons learnt.

Selected strains of Saccharomyces cerevisiae are used for commercial bioethanol production from cellulose and starch, but the high cost of exogenous enzymes for substrate hydrolysis remains a challenge. This can be addressed through consolidated bioprocessing (CBP) where S. cerevisiae strains are engineered to express recombinant glycoside hydrolases during fermentation.

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited.

Omics analysis reveals mechanism underlying metabolic oscillation during continuous very-high-gravity ethanol fermentation by Saccharomyces cerevisiae.

During continuous very-high-gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae, the process exhibits sustained oscillation in residual glucose, ethanol, and biomass, raising a question: how do yeast cells respond to this phenomenon? In this study, the oscillatory behavior of yeast cells was characterized through transcriptome and metabolome analysis for one complete oscillatory period. By analyzing the accumulation of 26 intracellular metabolites and the expression of 90 genes related to central carbon metabolism and stress response, we confirmed that the process oscillation was attributed to intracellular metabolic oscillation with phase difference, and the expression of HXK1, HXT1,2,4, and PFK1 was significantly different from other genes in the Embden-Meyerhof-Parnas pathway, indicating that glucose transport and phosphorylation could be key nodes for regulating the intracellular metabolism under oscillatory conditions. Moreover, the expression of stress response genes was triggered and affected predominately by ethanol inhibition in yeast cells.

Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains.

The yeast cell surface provides space to display functional proteins. Heterologous proteins can be covalently anchored to the yeast cell wall by fusing them with the anchoring domain of glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs). In the yeast cell-surface display system, the anchorage position of the target protein in the cell wall is an important factor that maximizes the capabilities of engineered yeast cells because the yeast cell wall consists of a 100- to 200-nm-thick microfibrillar array of glucan chains.

Improved cellulase expression in diploid yeast strains enhanced consolidated bioprocessing of pretreated corn residues.

In an effort to find a suitable genetic background for efficient cellulolytic secretion, genetically diverse strains were transformed to produce core fungal cellulases namely, β-glucosidase (BGLI), endoglucanase (EGII) and cellobiohydrolase (CBHI) in various combinations and expression configurations. The secreted enzyme activity levels, gene copy number, substrate specificities, as well as hydrolysis and fermentation yields of the transformants were analysed. The effectiveness of the partially cellulolytic yeast transformants to convert two different pre-treated corn residues, namely corn cob and corn husk was then explored.

Improved cellulase production in recombinant Saccharomyces cerevisiae by disrupting the cell wall protein-encoding gene CWP2.

Budding yeast Saccharomyces cerevisiae has been widely used for heterologous protein production. However, low protein production titer and secretion levels continue to challenge its practical applications. The yeast cell wall plays important roles in yeast cell growth and environmental responses.

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

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).

Overexpression of endogenous stress-tolerance related genes in Saccharomyces cerevisiae improved strain robustness and production of heterologous cellobiohydrolase.

To enable Saccharomyces cerevisiae to produce renewable fuels from lignocellulose in a consolidated bioprocess, a heterologous cellulase system must be engineered into this yeast. In addition, inherently low secretion titers and sensitivity to adverse environmental conditions must be overcome. Here, two native S.

Identification of superior cellulase secretion phenotypes in haploids derived from natural Saccharomyces cerevisiae isolates.

The yeast Saccharomyces cerevisiae is considered an important host for consolidated bioprocessing and the production of high titres of recombinant cellulases is required for efficient hydrolysis of lignocellulosic substrates to fermentable sugars. Since recombinant protein secretion profiles vary highly among different strain backgrounds, careful selection of robust strains with optimal secretion profiles is of crucial importance. Here, we construct and screen sets of haploid derivatives, derived from natural strain isolates YI13, FINI and YI59, for improved general cellulase secretion.

Effect of N-linked glycosylation on the activity and stability of a β-glucosidase from Putranjiva roxburghii.

In this study, the effect of N-glycosylation on the conformational and functional stability of Putranjiva roxburghii family 1 β-glucosidase (PRGH1) enzyme was investigated. The deglycosylation of PRGH1 was carried out by using PNGase F enzyme and confirmed by SDS-PAGE and carbohydrate estimation. Comparative analysis with respect to enzyme activity, stability and aggregation behaviour was carried out for the glycosylated and deglycosylated PRGH1.

Strain Breeding Enhanced Heterologous Cellobiohydrolase Secretion by Saccharomyces cerevisiae in a Protein Specific Manner.

The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels.

Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae.

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface.

Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used.

Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production.

Cellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic feasibility is the recalcitrance of lignocellulose requiring large amount of enzyme to break. Several engineered yeast strains have been developed with cellulolytic activities to reduce the need for enzyme addition, but exhibiting limited effect.

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes.

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

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.

Combined cell-surface display- and secretion-based strategies for production of cellulosic ethanol with Saccharomyces cerevisiae.

Background: Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promising strategy for production of bioethanol from lignocellulose. The production of cellulase is usually pursued by one of the two strategies: displaying enzyme on the cell surface or secreting enzyme into the medium. However, to our knowledge, the combination of the two strategies in a yeast strain has not been employed.

Progress and challenges in the engineering of non-cellulolytic microorganisms for consolidated bioprocessing.

Lignocellulosic biomass is an abundant, renewable feedstock for the production of fuels and chemicals, if an efficient and affordable conversion technology can be established to overcome its recalcitrance. Consolidated bioprocessing (CBP) featuring enzyme production, substrate hydrolysis and fermentation in a single step is a biologically mediated conversion approach with outstanding potential if a fit-for-purpose microorganism(s) can be developed. Progress in developing CBP-enabling microorganisms is ongoing by engineering (i) naturally cellulolytic microorganisms for improved product-related properties or (ii) non-cellulolytic organisms exhibiting high product yields to heterologously produce different combinations of cellulase enzymes.

Over-expression of native Saccharomyces cerevisiae exocytic SNARE genes increased heterologous cellulase secretion.

SNAREs (soluble NSF [N-ethylmaleimide-sensitive factor] attachment receptor proteins) are required at the majority of fusion events during intracellular membrane transport and play crucial roles in facilitating protein trafficking between the various membrane-enclosed organelles and the plasma membrane. We demonstrate increases in the secretion of the Talaromyces emersonii Cel7A (a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (a β-glucosidase), through the separate and simultaneous over-expression of different components of the exocytic SNARE complex in Saccharomyces cerevisiae. Over-expression of SNC1 yielded the biggest improvement in Te-Cel7A secretion (71 %), whilst SSO1 over-expression lead to the highest increases in Sf-Cel3A secretion (43.