Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol

By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.Alternatives to fossil fuels for transportation are under extensive investigation due to the increasing concerns about energy security, sustainability, and global climate change (22, 24, 35). Lignocellulosic biofuels, such as bioethanol, have been widely regarded as a promising and the only foreseeable alternative to petroleum products currently used in transportation (11, 35, 39, 41). The central technological impediment to more widespread utilization of lignocellulose is the absence of low-cost technology to break down its major component, cellulose (19, 41). Cellulose (a linear homopolymer of glucose linked by β-1,4-glycosidic bonds) is insoluble, forms a distinct crystalline structure, and is protected by a complex plant cell wall structural matrix (10, 32). As a result, a separate processing step is required to produce large amounts of cellulases for the hydrolysis of cellulose into fermentable glucose, which makes cellulosic ethanol too expensive to compete with gasoline. Therefore, consolidated bioprocessing (CBP), which combines enzyme production, cellulose hydrolysis, and fermentation in a single step, has been proposed to significantly lower the cost of cellulosic ethanol production (23, 24). However, the great potential of CBP cannot be realized using microorganisms available today.One engineering strategy to construct CBP-enabling microbes is to endow ethanologenic microorganisms, such as Saccharomyces cerevisiae, with the ability to utilize cellulose by heterologously expressing a functional cellulase system. Nature has provided two ways of designing such systems: (i) noncomplexed cellulase systems, in which free enzymes are secreted and act discretely, and (ii) complexed cellulase systems, namely, cellulosomes, in which many enzymes are held together by a noncatalytic scaffoldin protein through high-affinity interactions between its cohesins and enzyme-borne dockerins (24). By mimicking the noncomplexed cellulase system, several groups successfully constructed cellulolytic S. cerevisiae strains that directly ferment amorphous cellulose to ethanol, although the titer and yield were relatively low (12, 16, 17). Compared to the noncomplexed cellulase systems, the cellulosome exhibits much greater degradative potential as a result of its highly ordered structural organization that enables enzyme proximity synergy and enzyme-substrate-microbe complex synergy (2, 13, 14, 21). Therefore, the second strategy could provide a “quantum leap” in development of biomass-to-biofuel technology (3).Recent studies revealed the modular nature of cellulosome assembly; by simply appending a dockerin domain, up to three enzymes (either cellulosomal or noncellulosomal) with different origins could be incorporated into a chimeric miniscaffoldin consisting of divergent cohesin domains to form a minicellulosome in vitro. The chimeric miniscaffoldin was in the form of either purified (7, 15, 26) or yeast surface-displayed protein (34). In both cases, the resulting recombinant minicellulosomes showed enhanced hydrolysis activity with cellulose. These results indicate that the high-affinity cohesin-dockerin interactions are sufficient to dictate assembly of a functional cellulosome. Therefore, in theory, the same results could also be achieved in vivo by coexpressing the cellulosomal components in a recombinant host. To date, in vivo production of recombinant cellulosomes has been limited to unifunctional complexes containing only one type of cellulolytic enzyme (1, 8, 27). Since complete enzymatic hydrolysis of cellulose requires synergistic action of three types of cellulases, endoglucanases (EGs) (EC 3.2.1.4), exoglucanases (including cellodextrinases [EC 3.2.1.74] and cellobiohydrolases [CBHs] [EC 3.2.1.91]), and β-glucosidases (BGLs) (EC 3.2.1.21) (24), none of the engineered microorganisms were shown to utilize cellulose directly.In this study, we report the first successful assembly of trifunctional minicellulosomes in S. cerevisiae. The resulting recombinant strain was able to simultaneously hydrolyze and ferment amorphous cellulose to ethanol, demonstrating the feasibility of constructing cellulolytic and fermentative yeasts by displaying recombinant minicellulosomes on the cell surface. We chose the cell surface display format over secretory proteins to potentially incorporate the cellulose-enzyme-microbe complex synergy unique to native cellulolytic microorganisms (21). Coupled with flow cytometry, yeast surface display provides a more convenient engineering platform, avoiding labor-intensive protein purification steps. Such a cell-bound format is also amenable to analysis of enzyme activity with insoluble substrates (31). Therefore, the system described here could be a useful tool for studying and engineering recombinant cellulosomes for various industrial and biotechnological applications.     

Cell Wall Assembly in Saccharomyces cerevisiae

An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.     

Engineering of Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae for Improved Cyclodextrin Production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

Production of Ethanol from Starch by Respiration-Deficient Recombinant Saccharomyces cerevisiae

A 100%-respiration-deficient nuclear petite amylolytic Saccharomyces cerevisiae NPB-G strain was generated, and its employment for direct fermentation of starch into ethanol was investigated. In a comparison of ethanol fermentation performances with the parental respiration-sufficient WTPB-G strain, the NPB-G strain showed an increase of ca. 48% in both ethanol yield and ethanol productivity.     

Ethanol Production by Saccharomyces cerevisiae Immobilized in Hollow-Fiber Membrane Bioreactors

Saccharomyces cerevisiae ATCC 4126 was grown within the macroporous matrix of asymmetric-walled polysulfone hollow-fiber membranes and on the exterior surfaces of isotropic-walled polypropylene hollow-fiber membranes. Nutrients were supplied and products were removed by single-pass perfusion of the fiber lumens. Growth of yeast cells within the macrovoids of the asymmetric-walled membranes attained densities of greater than 10¹⁰ cells per ml and in some regions accounted for nearly 100% of the available macrovoid volume, forming a tissue-like mass. A radial distribution of cell packing existed across the fiber wall, indicating an inadequate glucose supply to cells located beyond 100 μm from the lumen surface. By comparison, yeast cell growth on the exterior surfaces of the isotropic-walled membranes resulted in an average density of 3.5 × 10⁹ viable cells per ml. Ethanol production by reactors containing isotropic polypropylene fibers reached a maximum value of 26 g/liter-h based on the total reactor volume. Reactor performance depended on the fiber packing density and on the glucose medium flow rate and was limited by low nutrient and product transport rates. The inhibition of ethanol production and the reduction in fermentation efficiency arose primarily from the accumulation of CO₂ gas within the sealed reactor shell space.     

Genetic Engineering of Inhibitor-Tolerant Saccharomyces cerevisiae for Improved Xylose Utilization in Ethanol Production

For economical lignocellulose-to-ethanol production, a desirable biocatalyst should tolerate inhibitors derived from preteatment of lignocellulose and be able to utilize heterogeneous biomass sugars of hexoses and pentoses. Previously, we developed an inhibitor-tolerant Saccharomyces cerevisiae strain NRRL Y-50049 that is able to in situ detoxify common aldehyde inhibitors such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF). In this study, we genetically engineered Y-50049 to enable and enhance its xylose utilization capability. A codon-optimized xylose isomerase gene for yeast (YXI) was synthesized and introduced into a defined chromosomal locus of Y-50049. Two newly identified xylose transport related genes XUT4 and XUT6, and previously reported xylulokinase gene (XKS1), and xylitol dehydrogenase gene (XYL2) from Scheffersomyces stipitis were also engineered into the yeast resulting in strain NRRL Y-50463. The engineered strain was able to grow on xylose as sole carbon source and a minimum ethanol production of 38.6?g?l^?1 was obtained in an anaerobic fermentation on mixed sugars of glucose and xylose in the presence of furfural and HMF.     

Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface

Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.     

Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting Saccharomyces cerevisiae Improves Ethanol Production

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol⁻¹, mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.     

Assembly of F0 in Saccharomyces cerevisiae

Respiratory deficient mutants of Saccharomyces cerevisiae have been instrumental in identifying an increasing number of nuclear gene products that promote pre- and post-translational steps of the pathway responsible for biogenesis of the mitochondrial ATP synthase. In this article we have attempted to marshal current information about the functions of such accessory factors and the roles they play in expression and assembly of the mitochondrially encoded subunits of the ATP synthase. We also discuss evidence that the ATP synthase may be built up from three separate modules corresponding to the F1 ATPase, the stator and F0.     

Expression of a Fungal Hydrophobin in the Saccharomyces cerevisiae Cell Wall: Effect on Cell Surface Properties and Immobilization

The aim of this work was to modify the cell surface properties of Saccharomyces cerevisiae by expression of the HFBI hydrophobin of the filamentous fungus Trichoderma reesei on the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C_12-18EO₅.     

利用米曲霉和酿酒酵母高效转化大豆糖浆为酒精

目的：提高大豆糖浆转化为酒精的生产强度和转化率。方法：利用米曲霉L-09和酿酒酵母Z-06作为混合菌株,采用同步糖化与发酵的工艺,对补料方式和发酵条件进行优化。结果：筛选出一株α-半乳糖苷酶酶活（12.8IU/mL）较高的米曲霉与酿酒酵母混合发酵,糖浆最佳发酵条件为：初始发酵浓度50%,摇瓶发酵,流加补料。结论：经30℃、60h发酵后,发酵醪酒精浓度达到15.2%（V/V）,转化率为理论值的96.3%。     

Regulation of Sugar and Ethanol Metabolism in Saccharomyces cerevisiae

Abstract

This review briefly surveys the literature on the nature, regulation, genetics, and molecular biology of the major energy-yielding pathways in yeasts, with emphasis on Sacchuromyces cerevisiae. While sugar metabolism has received the lion's share of attention from workers in this field because of its bearing on the production of ethanol and other metabolites, more attention is now being paid to ethanol metabolism and the regulation of aerobic metabolism by fermentable and non-fermentable substrates. The utility of yeast as a highly manipulable organism and the discovery that yeast metabolic pathways are subject to the same types of control as those of higher cells open up many opportunities in such diverse areas as molecular evolution and cancer research.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60-3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8-13% vs. 33-47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Ethanol tolerance in free and sol-gel immobilised Saccharomyces cerevisiae

The tolerance of sol-gel immobilised and free Saccharomyces cerevisiae to ethanol was studied. The effects of ethanol preincubation time showed that the specific death velocity decreased from 2×10⁵ c.f.u. min^–1 for free cells to 2×10⁴ c.f.u. min^–1 for immobilised cells thus indicating that immobilised yeast was far less sensitive to the ethanol damage. The specific glucose consumption of immobilised and free cells on a per cell basis was 3×10^–12 g cell^–1 h^–1 and 9×10^–12 g cell^–1 h^–1, respectively.     

Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae

In this study, we expressed two cellulase encoding genes, an endoglucanase of Trichoderma reesei (EGI) and the beta-glucosidase of Saccharomycopsis fibuligera (BGL1), in combination in Saccharomyces cerevisiae. The resulting strain was able to grow on phosphoric acid swollen cellulose (PASC) through simultaneous production of sufficient extracellular endoglucanase and beta-glucosidase activity. Anaerobic growth (0.03h(-1)) up to 0.27gl(-1) DCW was observed on medium containing 10gl(-1) PASC as sole carbohydrate source with concomitant ethanol production of up to 1.0gl(-1). We have thus demonstrated the construction of a yeast strain capable of growth on and one-step conversion of amorphous cellulose to ethanol, representing significant progress towards realization of one-step processing of cellulosic biomass in a consolidated bioprocessing configuration. To our knowledge, this is the first report of a recombinant strain of S. cerevisiae growing on pure cellulose.     

Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae

The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.     

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.