Synergies in coupled hydrolysis and fermentation of cellulose using a Trichoderma reesei enzyme preparation and a recombinant Saccharomyces cerevisiae strain.

We describe a procedure by which filter paper is digested with a cellulolytic enzyme preparation, obtained from Trichoderma reesei cultivated under solid state fermentation conditions and then fermented by a recombinant Saccharomyces cerevisiae strain. The yeast strain produces a ß-glucosidase encoded by the BGL1 gene from Saccharomycopsis fibuligera that quantitatively and qualitatively complements the limitations that the Trichoderma enzyme complex shows for this particular activity. The supplemental ß-glucosidase activity fuels the progression of cellulose hydrolysis and fermentation by decreasing the inhibitory effects caused by the accumulation of cellobiose and glucose. Fermentation of filter paper by this procedure yields ethanol concentrations above 70 g/L.

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 ß-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. Co-overexpression of the ER-to-Golgi SNAREs yielded proportionately smaller increases for the Te-Cel7A (46 %), with the Sf-Cel3A yielding no improvement. Co-overexpression of the most promising exocytic SNARE components identified in literature for secretory enhancement of the cellulolytic proteins tested (SSO1 for Sf-Cel3A and SNC1 for Te-Cel7A) with the most effective ER-to-Golgi SNARE components identified in this study (SED5 for both Sf-Cel3A and Te-Cel7A) yielded variable results, with Sf-Cel3A improved by 131 % and Te-Cel7A yielding no improvement. Improvements were largely independent of gene dosage as all strains only integrated single additional SNARE gene copies, with episomal variance between the most improved strains shown to be insignificant. This study has added further credence to the notion that SNARE proteins fulfil an essential role within a larger cascade of secretory machinery components that could contribute significantly to future improvements to S. cerevisiae as protein production host.

Characterization and identification of three novel aldo-keto reductases from Lodderomyces elongisporus for reducing ethyl 4-chloroacetoacetate.

Lodderomyces elongisporus LH703 isolated from soil samples contained three novel aldo-keto reductases (AKRs) (LEAKR 48, LEAKR 49, and LEAKR 50). The three enzymes were cloned, expressed, and purified to homogeneity for characterization. These three AKRs shared <40% amino acid identity with each other. LEAKR 50 was identified as a member of AKR3 family, whereas the other two LEAKRs were identified as members of two novel AKR families, respectively. All the three AKRs required nicotinamide adenine dinucleotide phosphate as a cofactor. However, they showed diverse characteristics, including optimum catalyzing conditions, resistance to adverse reaction conditions, and substrate specificity. LEAKR 50 was estimated to be a promising biocatalyst that could reduce ethyl 4-chloroacetoacetate with high enantiomeric excess (98% e. e.) and high activity residue under adverse conditions.

Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11.

The MIG1 gene of Saccharomycopsis fibuligera A11 was cloned from its genomic DNA using the degenerated primers and inverse PCR. The MIG1 gene (1152bp, accession number: HM450676) encoded a 384-amino acid protein very similar to Mig1s from other fungi. Besides their highly conserved zinc fingers, the Mig1 proteins displayed short conserved motifs of possible significance in glucose repression. The MIG1 gene in S. fibuligera A11 was disrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF (Open Reading Frame) of the MIG1 gene. The disruptant A11-c obtained could grow in the media containing hygromycin and 2-deoxy-d-glucose, respectively. α-Amylase, glucoamylse, acid protease and ß-glucosidase production by the disruptant and expression of their genes in the disruptant were greatly enhanced. This confirms that Mig1, the transcriptional repressor, indeed regulates expression of the genes and production of the extracellular enzymes in S. fibuligera A11. At the same time, it was found that cell budding was enhanced and mycelial formation was reduced in the disruptant.

Coexpression of α-l-arabinofuranosidase and ß-glucosidase in Saccharomyces cerevisiae.

Monoterpenes are important aroma compounds in grape varieties such as Muscat, Gewürztraminer and Riesling, and are present as either odourless, glycosidically bound complexes or free aromatic monoterpenes. Commercial enzymes can be used to release the monoterpenes, but they commonly consist of crude extracts that often have unwanted and unpredictable side-effects on wine aroma. This project aims to address these problems by the expression and secretion of the Aspergillus awamoriα-l-arabinofuranosidase in combination with either the ß-glucosidases from Saccharomycopsis fibuligera or from Aspergillus kawachii in the industrial yeast Saccharomyces cerevisiae VIN13. The concentration of five monoterpenes was monitored throughout alcoholic fermentation of Gewürztraminer grapes. The recombinant yeast strains that caused an early boost in the geraniol concentration led to a reduction in the final geraniol levels due to the downregulation of the sterol biosynthetic pathway. Monoterpene concentrations were also analysed 9 and 38 days after racking and the performance of the VB2 and VAB2 recombinant strains was similar, and in many cases, better than that of a commercial enzyme used in the same experiment. The results were backed by sensorial analysis, with the panel preferring the aroma of the wines produced by the VAB2 strain.

Structural and functional analysis of hybrid enzymes generated by domain shuffling between Saccharomyces cerevisiae (var. diastaticus) Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 ß-glucosidase.

Saccharomyces cerevisiae Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 ß-glucosidase, two relevant enzymes from a biotechnological point of view, are proteins with multidomain structure. Starting with homology-based structural models of Sta1 and Bgl1, we have constructed a series of hybrid enzymes by interchanging domains of the two proteins. The first purpose of these constructs was to check available hypotheses about the uncertain biological functions of two domains: the serine/threonine-rich domain (STRD) of Sta1 and a ß-sandwich domain present in Bgl1 that we have designated fibronectin-like domain (FLD). While, according to the initial hypothesis, proteins carrying the FLD tend to adhere to the cell wall, our results argued against the idea of an involvement of the STRD in protein secretion that stemmed from the presence of similar domains in different proteins secreted by yeast. The second objective of this work was to increase the enzymatic repertoire by generating enzymes with new structural and functional properties.

Arabinoxylo- and Arabino-Oligosaccharides-Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast Saccharomycopsis fibuligera.

Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45°C and pH 7.0 in sodium phosphate buffer and at 50°C and pH 6.0 in sodium acetate buffer, respectively. These exo-acting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)-and α-(1,3)-L-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)-and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

Molecular characterization of the Saccharomycopsis fibuligera ATF genes, encoding alcohol acetyltransferase for volatile acetate ester formation.

Aroma ester components produced by fermenting yeast cells via alcohol acetyltransferase (AATase)-catalyzed intracellular reactions are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. Acetate esters are reportedly produced at relatively high concentrations by non-Saccharomyces species. Here, we identified 12 ATF orthologues (SfATFs) encoding putative AATases, in the diploid genome of Saccharomycopsis fibuligera KJJ81, an isolate from wheat-based Nuruk in Korea. The identified SfATF proteins (SfAtfp) display low sequence identities with S. cerevisiae Atf1p (between 13.3 and 27.0%). All SfAtfp identified, except SfAtf(A)4p and SfAtf(B)4p, contained the activation domain (HXXXD) conserved in other Atf proteins. Culture supernatant analysis using headspace gas chromatography mass spectrometry confirmed that the recombinant S. cerevisiae strains expressing SfAtf(A)2p, SfAtf(B)2p, and SfAtf(B)6p produced high levels of isoamyl and phenethyl acetates. The volatile aroma profiles generated by the SfAtf proteins were distinctive from that of S. cerevisiae Atf1p, implying difference in the substrate preference. Cellular localization analysis using GFP fusion revealed the localization of SfAtf proteins proximal to the lipid particles, consistent with the presence of amphipathic helices at their N- and C-termini. This is the first report that systematically characterizes the S. fibuligera ATF genes encoding functional AATases responsible for acetate ester formation using higher alcohols as substrate, demonstrating their biotechnological potential for volatile ester production.

Surface display of acid protease on the cells of Yarrowia lipolytica for milk clotting.

The acid protease structural gene was amplified from the genomic DNA of Saccharomycopsis fibuligera A11. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid protease activity. The cells displaying the acid protease can be used to effectively clot skimmed milk. The highest clotting milk activity (1,142.9 U/ml) was observed under the conditions of pH 3.0, 40 degrees C, 20 mM of CaCl(2), and 10% skimmed milk powder. We found that the acid protease displayed on the cells of Y. lipolytica which has generally regarded as safe status could be easily isolated and concentrated compared to the free acid protease. Therefore, the displayed acid protease may have many potential applications in food and cheese industries. This is the first report that the yeast cells displaying the acid protease were used to clot milk.

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor.

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.

Glycerol accumulation in the dimorphic yeast Saccharomycopsis fibuligera: cloning of two glycerol 3-phosphate dehydrogenase genes, one of which is markedly induced by osmotic stress.

Glycerol plays an important role in the osmoadaptation responses of Saccharomyces cerevisiae. However, there is no detailed investigation about the role of glycerol in the osmoadaptation responses of Saccharomycopsis fibuligera. Here we show that both intra- and extracellular glycerol concentrations in Sm. fibuligera cells responded very quickly when they were subjected to osmotic stress. We then cloned two isogenes encoding putative NAD(+)-dependent glycerol 3-phosphate dehydrogenase (GPD) from Sm. fibuligera PD70 by degenerate PCR and subsequent chromosome walking methods. Those two genes, designated SfGPD1 and SfGPD2, respectively, exhibited 86.6% pairwise identity in their encoding regions, while there was no obvious homology in their non-coding regions. Either SfGPD1 or SfGPD2 could complement the salt tolerance characteristics of the gpd1gpd2 double mutant strain of S. cerevisiae, further demonstrating that both of those genes are functional homologues of S. cerevisiae GPD1 and GPD2. Northern blot analysis revealed that SfGPD1 was induced markedly by osmotic stress, while SfGPD2 was not. In consistency with the observation that there was no obvious glycerol content change when the cells were transferred to anoxic conditions, neither SfGPD1 nor SfGPD2 was induced when the cells were transferred to anoxic conditions, thus suggesting a functional splitting of glycerol 3-phosphate dehydrogenase between S. cerevisiae and Sm. fibuligera.

Utilization of acorn fringe for ellagic acid production by Aspergillus oryzae and Endomyces fibuliger.

Conversion of acorn fringe extract into ellagic acid production by Aspergillus oryzae and Endomyces fibuliger were investigated. The results showed that ellagic acid production was maximized when co-fermentation of the two fungi was performed at 30 degrees C and pH 5.0 with 5.7 g/l of initial substrate concentration, which were close to the optimal values for both fungi to yield an appropriate consortium of hydrolytic enzymes. Meanwhile, it was found that the co-fermentation could compensate the deficiencies in the level of polyphenol oxidase activity from pure A. oryzae and the levels of ellagitannin acyl hydrolase and beta-glucosidase activities from pure E. fibuliger, resulting in. 0.91 g/l of biomass concentration containing 1.84 g/l of ellagic acid. The research not only demonstrates that the co-fermentation is an effective approach to utilize forest byproduct for ellagic acid production, but also provides more evidences for understanding evolution of ellagic acid production with enzymes actions, which is important for process control of ellagic acid production in industrial application.

Starch degradation by glucoamylase Glm from Saccharomycopsis fibuligera IFO 0111 in the presence and absence of a commercial pullulanase.

This study describes the course of enzymatic hydrolysis of the native corn starches Maritena 100 and Maritena 300. Hydrolyses were carried out with glucoamylase Glm produced by Saccharomycopsis fibuligera IFO 0111, which degrades also native starch, with the purpose to substitute a two-step hydrolysis (amylase followed by glucoamylase) by a one-step process (glucoamylase only). Hydrolysis generally became more effective by adding the pullulanase Promozyme D, which cleaves alpha-1,6-glycosidic bonds more effectively than glucoamylase Glm does. The time course (kinetics) of hydrolysis was followed by determination of the glucose concentration and calculation of dextrose equivalents.

Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain.

Most glucoamylases (alpha-1,4-D-glucan glucohydrolase, EC 3.2.1.3) have structures consisting of both a catalytic and a starch binding domain. The structure of a glucoamylase from Saccharomycopsis fibuligera HUT 7212 (Glu), determined a few years ago, consists of a single catalytic domain. The structure of this enzyme with the resolution extended to 1.1 A and that of the enzyme-acarbose complex at 1.6 A resolution are presented here. The structure at atomic resolution, besides its high accuracy, shows clearly the influence of cryo-cooling, which is manifested in shrinkage of the molecule and lowering the volume of the unit cell. In the structure of the complex, two acarbose molecules are bound, one at the active site and the second at a site remote from the active site, curved around Tyr464 which resembles the inhibitor molecule in the 'sugar tongs' surface binding site in the structure of barley alpha-amylase isozyme 1 complexed with a thiomalto-oligosaccharide. Based on the close similarity in sequence of glucoamylase Glu, which does not degrade raw starch, to that of glucoamylase (Glm) from S. fibuligera IFO 0111, a raw starch-degrading enzyme, it is reasonable to expect the presence of the remote starch binding site at structurally equivalent positions in both enzymes. We propose the role of this site is to fix the enzyme onto the surface of a starch granule while the active site degrades the polysaccharide. This hypothesis is verified here by the preparation of mutants of glucoamylases Glu and Glm.

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression.

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.

Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11.

Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.

An increase in the transglycosylation activity of Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.

The 84th tryptophan residue in Saccharomycopsis alpha-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.

Subcellular distribution of enzymes in the yeast saccharomycopsis lipolytica, grown on n-hexadecane, with special reference to the omega-hydroxylase.

The subcellular localization of the omega-hydroxylase of Saccharomycopsis lipolytica was assessed by the analytical fractionation technique, originally described by de Duve C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F., and hitherto little, if at all, applied to yeasts. Protoplasts were separated in six fractions by differential centrifugation. Some of these fractions were further fractionated by density gradient centrifugation. The distribution of omega-hydroxylase and 15 other constituents chosen as possible markers of its subcellular entities. (1) Mitochondria were characterized by particulate malate dehydrogenase, particulate Antimycin A-insensitive NADH-cytochrome c reductase, oligomycin-sensitive and K+-stimulated ATPase pH 9. (2) Most if not all of the catalase and urate oxidase is peroxisomal. (3) Free ribosomes account for most RNA. (4) Nucleoside diphosphatase is for the first time reported in a yeast and appears to belong to an homogeneous population of small membranes. (5) The soluble compartment contains magnesium pyrophosphatase, alkaline, 5'-nucleotidase and part of the NADH-cytochrome c reductase. Latent arylesterase and ATPase pH 7 have an unspecific distribution. Alkaline phosphodiesterase I has not been detected.

Construction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains.

Beta-glucosidase genes of fungal origins were isolated and heterologously expressed in Saccharomyces cerevisiae to enable growth on the disaccharide, cellobiose. To promote secretion of the beta-glucosidases, the genes were fused to the secretion signal of the Trichoderma reesei xyn2 gene and constitutively expressed from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The two most promising enzymes, BGL1 from Saccharomycopsis fibuligera and BglA from Aspergillus kawachii, were anchored to the yeast cell surface by fusing the mature proteins to the alpha-agglutinin (AGalpha1) or cell wall protein 2 (Cwp2) peptides. The maximum specific growth rates (mu(max)) of the recombinant S. cerevisiae strains were determined in batch cultivation. S. cerevisiae secreting the recombinant S. fibuligera BGL1 enzyme sustained growth aerobically and anaerobically, in minimal medium containing 5g L(-1) cellobiose at 0.23 h(-1) (compared to 0.29 h(-1) on glucose) and 0.18 h(-1) (compared to 0.25 h(-1) on glucose), respectively. Substrate consumption and product formation were determined to evaluate product yields in glucose and cellobiose.

[Expression of B-glucosidase gene of Sacchromycopsis fibuligera in Pichia pastoris].

A beta-Glucosidase gene (BGL 1) was amplified with PCR from the total DNA of Sacchromycopsis fibuligera, and was linked with pGEM-T vector. After cut down by restriction enzyme from pGEM-T vector, BGL 1 was inserted into the expression vector pPIC9K of Pichia pastoris in reading frame with alpha-factor secreting signal peptide sequence to construct the recombinant plasmid pSHL9K. The recombinant plasmid pSHL9K was transformed into Pichia pastoris GS115 with electroporation. The recombinant Pichia pastoris strains which could efficiently secret recombinant beta-Glucosidase were selected. The optimum temperature of the recombinant beta-Glucosidase was 50degreesC, and the optimum pH was 5.4. The activity of beta-Glucosidase could reach to 47U/mL in the culture medium.