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. Here, we discuss recent advances toward developing yeast strains that could convert different LCB fractions into a series of economically viable commodity products in a biorefinery.

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. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Supplementation of recombinant cellulases with LPMOs and CDHs improves consolidated bioprocessing of cellulose.

The increased demand for energy has sparked a global search for renewable energy sources that could partly replace fossil fuel resources and help mitigate climate change. Cellulosic biomass is an ideal feedstock for renewable bioethanol production, but the process is not currently economically feasible due to the high cost of pretreatment and enzyme cocktails to release fermentable sugars. Lytic polysaccharide monooxygenases (LPMOs) and cellobiose dehydrogenases (CDHs) are auxiliary enzymes that can enhance cellulose hydrolysis. In this study, four LPMO and two CDH genes were subcloned and expressed in the Saccharomyces cerevisiae Y294 laboratory strain. SDS-PAGE analysis confirmed the extracellular production of the LPMOs and CDHs in the laboratory S. cerevisiae Y294 strain. A rudimentary cellulase cocktail (cellobiohydrolase 1 and 2, endoglucanase and ß-glucosidase) was expressed in the commercial CelluX™ 4 strain and extracellular production of the individual cellulases was confirmed by SDS-PAGE analysis. In vitro cooperation of the CDHs and LPMOs with the rudimentary cellulases produced by strain CelluX™ 4[F4-1] was demonstrated on Whatman filter paper. The significant levels of soluble sugars released from this crystalline cellulose substrate indicated that these auxiliary enzymes could be important components of the CBP yeast cellulolytic system.

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.For more efficient CBP, MIG1spsc01isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption ofCWP2, YGP1, andUTH1,which are functionally related toMIG1spsc01, also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption ofYGP1and overexpression ofSED5, which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass.

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. While strains producing a core set of cell-adhered cellulases that enabled crystalline cellulose hydrolysis have been created, secreted levels of enzyme were insufficient for complete cellulose hydrolysis. In fact, all reported recombinant yeast CBP candidates must overcome the drawback of generally low secretion titers. Rational strain engineering can be applied to enhance the secretion phenotype. This study aimed to improve the amount of cell-adhered cellulase activities of recombinant S. cerevisiae strains expressing a core set of four cellulases, through overexpression of genes that were previously shown to enhance cellulase secretion. Results showed significant increases in cellulolytic activity for all cell-adhered cellulase enzyme types. Cell-adhered cellobiohydrolase activity was improved by up to 101%, ß-glucosidase activity by up to 99%, and endoglucanase activity by up to 231%. Improved hydrolysis of crystalline cellulose of up to 186% and improved ethanol yields from this substrate of 40-50% in different strain backgrounds were also observed. In addition, improvement in resistance to fermentation stressors was noted in some strains. These strains represent a step towards more efficient organisms for use in 2G biofuel production. KEY POINTS: • Cell-surface-adhered cellulase activity was improved in strains engineered for CBP. • Levels of improvement of activity were strain and enzyme dependent. • Crystalline cellulose conversion to ethanol could be improved up to 50%.

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. Looking back at numerous strategies undertaken over the past four decades to improve recombinant protein production in S. cerevisiae, it is evident that various steps in the protein production "pipeline" can be manipulated depending on the protein of interest and its anticipated application. In this review, we briefly introduce some of the strategies and highlight lessons learned with regards to improved transcription, translation, post-translational modification and protein secretion of heterologous hydrolases. We examine how host strain selection and modification, as well as enzyme compatibility, are crucial determinants for overall success. Finally, we discuss how lessons from heterologous hydrolase expression can inform modern synthetic biology and genome editing tools to provide process-ready yeast strains in future. However, it is clear that the successful expression of any particular enzyme is still unpredictable and requires a trial-and-error approach.

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. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus ß-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

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. This progress not only contributes to the understanding of mechanisms underlying the process oscillation observed for continuous VHG ethanol fermentation, but also provides insights for understanding unsteady state that might develop in other continuous fermentation processes operated under VHG conditions to increase product titers for robust production.

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). 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.

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. Nevertheless, the effects of yeast cell wall proteins on heterologous protein production and secretion remain unclear. CWP2 encodes a mannoprotein that is the major component of the yeast cell wall. So far, studies on its function have been very limited. Here we show that CWP2 disruption improved extracellular cellobiohydrolase activity by 85.9%. A calcofluor white hypersensitivity assay revealed increased sensitivity of the mutant compared to the parental strain, indicating impaired cell wall integrity. However, no changes were observed in normal cell growth or growth stressed by tunicamycin and dithiothreitol, suggesting that the unfolded protein response pathway was not affected by the gene disruption. Comparative transcriptome analysis revealed changes in multiple genes involved in cell wall structure, biosynthesis, and cell wall integrity induced by CWP2 disruption, suggesting a pivotal role of Cwp2p in yeast cell wall organization. Notably, CWP2 disruption also led to elevated transcription of a large number of genes involved in ribosome biogenesis, which indicated that CWP2 is not only in yeast cell wall biosynthesis, but also in protein translation. This work reveals novel insights into the functions of CWP2 and also presents a new strategy to increase heterologous protein production in yeast strains by manipulating cell wall-related proteins.

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. However, knowledge is limited regarding the anchorage position of GPI-attached proteins in the yeast cell wall. Here, we report a comparative study on the effect of GPI-anchoring domain-heterologous protein fusions on yeast cell wall localization. GPI-anchoring domains derived from well-characterized GPI-CWPs, namely Sed1p and Sag1p, were used for the cell-surface display of heterologous proteins in the yeast Saccharomyces cerevisiae. Immunoelectron-microscopic analysis of enhanced green fluorescent protein (eGFP)-displaying cells revealed that the anchorage position of the GPI-attached protein in the cell wall could be controlled by changing the fused anchoring domain. eGFP fused with the Sed1-anchoring domain predominantly localized to the external surface of the cell wall, whereas the anchorage position of eGFP fused with the Sag1-anchoring domain was mainly inside the cell wall. We also demonstrate the application of the anchorage position control technique to improve the cellulolytic ability of cellulase-displaying yeast. The ethanol titer during the simultaneous saccharification and fermentation of hydrothermally-processed rice straw was improved by 30% after repositioning the exo- and endo-cellulases using Sed1- and Sag1-anchor domains. This novel anchorage position control strategy will enable the efficient utilization of the cell wall space in various fields of yeast cell-surface display technology.

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, ß-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. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants produced up to 1.34 fold higher glucose concentrations (g/L) than a control composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted ratio of 1:15 (cellulase activity unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments and yielded 4.05 g/L ethanol in fermentations. We demonstrate that the choice of optimal genetic background and cellulase activity secretion ratio can improve cellulosic ethanol production by consolidated bioprocessing yeast strains.

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. cerevisiae genes related to yeast stress tolerance, YHB1 and SET5, were overexpressed under transcriptional control of the constitutive PGK1 promoter and their effects on heterologous secretion of Talaromyces emersonii cel7A cellobiohydrolase was investigated. Transformants showed increased secreted enzyme activity that ranged from 22% to 55% higher compared to the parental strains and this did not lead to deleterious growth effects. The recombinant strains overexpressing either YHB1 or SET5 also demonstrated multi-tolerant characteristics desirable in bioethanol production, i.e. improved tolerance to osmotic and heat stress. Quantitative reverse transcriptase PCR analysis in these strains showed decreased transcription of secretion pathway genes. However, decreased unfolded protein response was also observed, suggesting novel mechanisms for enhancing enzyme production through stress modulation. Overexpression of YHB1 in an unrelated diploid strain also enhanced stress tolerance and improved ethanol productivity in medium containing acetic acid. To our knowledge, this is the first demonstration that improved heterologous secretion and environmental stress tolerance could be engineered into yeast simultaneously.

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. This report details a novel approach that combines secretion profiles of strains and phenotypic responses to stresses known to influence the secretion pathway for the development of a phenotypic screen to isolate strains with improved secretory capacities. A clear distinction was observed between the YI13 haploid derivatives and industrial and laboratory counterparts, Ethanol Red and S288c, respectively. By using sub-lethal concentrations of the secretion stressor tunicamycin and cell wall stressor Congo Red, YI13 haploid derivative strains demonstrated tolerance profiles related to their heterologous secretion profiles. Our results demonstrated that a new screening technique combined with a targeted mating approach could produce a pool of novel strains capable of high cellulase secretion.

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

In this study, the effect of N-glycosylation on the conformational and functional stability of Putranjiva roxburghii family 1 ß-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. The deglycosylation of PRGH1 affected enzyme activity to a certain extent only where Km was not affected but a slight reduction in Vmax for various substrates was observed. Circular dichroism, fluorescence studies and differential scanning calorimetry (DSC) analysis demonstrated the possible effect of glycosylation on local and/or global conformational dynamics of protein and its effect on the thermostability of PRGH1. DSC results showed deglycosylated form had lower Tm as compared to the glycosylated form of PRGH1. The PRGH1 was found to be more sensitive to proteolysis after deglycosylation suggesting that the glycosylated PRGH1 was quite compact and rigid. Mutagenesis studies revealed that out of seven potential N-linked glycosylation sites, only three were glycosylated. The results demonstrated that N-linked glycosylation played an important role in conformational stability of PRGH1; however, it did not affect the enzyme function drastically.

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. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast.

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. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201-1207. © 2017 Wiley Periodicals, Inc.

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. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (ß-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

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. Here, we report the successful engineering of a cellulose-adherent Saccharomyces cerevisiae displaying four different synergistic cellulases on the cell surface. The cellulase-displaying yeast strain exhibited clear cell-to-cellulose adhesion and a "tearing" cellulose degradation pattern; the adhesion ability correlated with enhanced surface area and roughness of the target cellulose fibers, resulting in higher hydrolysis efficiency. The engineered yeast directly produced ethanol from rice straw despite a more than 40% decrease in the required enzyme dosage for high-density fermentation. Thus, improved cell-to-cellulose interactions provided a novel strategy for increasing cellulose hydrolysis, suggesting a mechanism for promoting the feasibility of cellulosic biofuel production.