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Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy- l-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway . Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% ( w/ v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae. [ABSTRACT FROM AUTHOR]

Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate-degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

The main sterol of the human cell membrane is cholesterol, whereas in yeast it is ergosterol. In this study, we constructed a cholesterol-producing yeast strain by disrupting the genes related to ergosterol synthesis and inserting the genes related to cholesterol synthesis. The total sterols of the mutant yeast were extracted and the sterol composition was analyzed by GC-MS. We confirmed that cholesterol was produced instead of ergosterol in yeast and subsequently examined the activity of the yeast G-protein-coupled receptor (GPCR) Ste2p. Ste2p signaling was assessed in wild type (WT) with ergosterol and the cholesterol-producing yeast instead of ergosterol to determine whether sterol composition affects the activity of the yeast GPCR. Our results demonstrated that Ste2p could transduce a signal even in the cholesterol-rich membrane, but the maximum signal intensity was weaker than that transduced in the ergosterol-rich original (WT) membrane. This result indicates that sterol composition affects the activity of yeast GPCRs, and thus, this provides new insight into GPCR-mediated transduction using yeast for future fundamental and applied studies on GPCRs from yeast to other organisms. [ABSTRACT FROM AUTHOR]

Pyrosequencing system utilizing luciferase is one of the next-generation DNA sequencing systems. However, there is a crucial problem with the current pyrosequencing system: luciferase cannot discriminate between ATP and dATP completely, and dATPαS must be used as the dATP analogue. dATPαS is expensive and has low activity for the enzyme. If luciferase can clearly recognize the difference between ATP and dATP, dATP could be used instead of the expensive dATPαS in the pyrosequencing system. We attempted to prepare a novel luciferase with improved specific activity and dATP discrimination with the molecular display method. First, we selected two amino acid residues, Ser440 and Ser456, as target residues for mutation from the whole sequence of Photinus pyralis luciferase; we comprehensively mutated these two amino acids. A mutant luciferase library was constructed using yeast cell surface engineering. Through three step-wide screenings with individual conditions, we easily and speedily isolated three candidate mutants from 1,152 candidates and analyzed the properties of these mutants. Consequently, we succeeded in obtaining interesting mutant luciferases with improved specific activity and dATP discrimination more conveniently than with other methods. [ABSTRACT FROM AUTHOR]

By cell surface display of ModE protein that is a transcriptional regulator of operons involved in the molybdenum metabolism in Escherichia coli, we have constructed a molybdate-binding yeast (Nishitani et al., Appl Microbiol Biotechnol 86:641-648, ). In this study, the binding specificity of the molybdate-binding domain of the ModE protein displayed on yeast cell surface was improved by substituting the amino acids involved in oxyanion binding with other amino acids. Although the displayed S126T, R128E, and T163S mutant proteins adsorbed neither molybdate nor tungstate, the displayed ModE mutant protein (T163Y) abolished only molybdate adsorption, exhibiting the specific adsorption of tungstate. The specificity of the displayed ModE mutant protein (T163Y) for tungstate was increased by approximately 9.31-fold compared to the displayed wild-type ModE protein at pH 5.4. Therefore, the strategy of protein design and its cell surface display is effective for the molecular breeding of bioadsorbents with metal-specific adsorption ability based on a single species of microorganism without isolation from nature. [ABSTRACT FROM AUTHOR]

A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase. [ABSTRACT FROM AUTHOR]

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved. [ABSTRACT FROM AUTHOR]

Carnosine (β-alanyl- l-histidine) is one of the bioactive dipeptides and has antioxidant, antiglycation, and cytoplasmic buffering properties. In this study, to synthesize carnosine from nonprotected amino acids as substrates, we cloned the carnosinase (CN1) gene and constructed a whole-cell biocatalyst displaying CN1 on the yeast cell surface with α-agglutinin as the anchor protein. The display of CN1 was confirmed by immunofluorescent labeling, and CN1-displaying yeast cells showed hydrolytic activity for carnosine. When carnosine was synthesized by the reverse reaction of CN1, organic solvents were added to the reaction mixture to reduce the water content. The CN1-displaying yeast cells were lyophilized and examined for organic solvent tolerance. Results showed that the CN1-displaying yeast cells retained their original hydrolytic activity in hydrophobic organic solvents. In the hydrophobic organic solvents and hydrophobic ionic liquids, the CN1-displaying yeast cells catalyzed carnosine synthesis, and carnosine was synthesized from nonprotected amino acids in only one step. The results of this research suggest that the whole-cell biocatalyst displaying CN1 on the yeast cell surface can be used to synthesize carnosine with ease and convenience. [ABSTRACT FROM AUTHOR]

In modern industrial society, molybdenum is one of the important metals for development of the industry of rare metals. It is important to recycle the rare metals from wastes because they are technically and economically difficult to be dug and be purified, and they exist in only a few regions in the world. In this study, ModE protein derived from Escherichia coli, which is a molybdate-dependent transcriptional regulator with the ability to bind molybdate as a form of soluble molybdenum, was displayed on the yeast cell surface by α-agglutinin-based cell surface display system for the adsorption and recovery of molybdate. Displayed ModE, confirmed by immunofluorescence labeling, caught molybdate more preferably at pH 3.0 than at basic pH. Yeast cells displaying C-terminal domain of ModE, which lacks N-terminal DNA binding domain, more effectively adsorbed molybdate than those displaying full-length ModE, suggesting that the deletion of the domain unrelated to metal binding enhanced the binding ability. Our results indicated that the adsorption system on cell surface of yeast cells displaying ModE is effective not only for adsorption of molybdate as a rare metal bioadsorbent but also for the easy recovery of molybdate located on the cell surface. [ABSTRACT FROM AUTHOR]

Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and β-1, 4 glucanase activities. In our previous study, we generated P. fukuinensis chitosanase-displaying yeast cells using a yeast cell surface-displaying system. Chitosanase-displaying yeast can be utilized as a chitosanase cluster without many time-consuming purification steps. In this study, using the system, we have investigated whether Glu302, which is supposed as a putative proton acceptor, is an essential amino acid residue for exhibiting chitosanase activity and analyzed the contribution of mutual interaction between Glu302 and Asn312 to the activity. A mutant library in which Glu302 and Asn312 were comprehensively substituted by the other amino acid residues was constructed on the yeast cell surface. From the results of chitosanase and β-1, 4 glucanase activity assays, we demonstrated that Glu302 was a proton acceptor for chitosanase activity, and Asn312 also participated in the hydrolysis of chitosan and cellulose. [ABSTRACT FROM AUTHOR]

The whole-cell biocatalyst displaying Candida antarctica lipase B (CALB) on the yeast cell surface with α-agglutinin as the anchor protein was easy to handle and possessed high stability. The lyophilized CALB-displaying yeasts showed their original hydrolytic activity and were applied to an ester synthesis using ethanol and l-lactic acid as substrates. In water-saturated heptane, CALB-displaying yeasts catalyzed ethyl lactate synthesis. The synthesis efficiency increased depending on temperature and reached approximately 74% at 50°C. The amount of l-ethyl lactate increased gradually. l-Ethyl lactate synthesis stopped at 200 h and restarted after adding of l-lactic acid at 253 h. It indicated that CALB-displaying yeasts retained their synthetic activity under such reaction conditions. In addition, CALB-displaying yeasts were able to recognize l-lactic acid and d-lactic acid as substrates. l-Ethyl lactate was prepared from l-lactic acid and d-ethyl lactate was prepared from d-lactic acid using the same CALB-displaying whole-cell biocatalyst. These findings suggest that CALB-displaying yeasts can supply the enantiomeric lactic esters for preparation of useful and improved biopolymers of lactic acid. [ABSTRACT FROM AUTHOR]

Vector engineering and gene disruption in host cells were attempted for the enhancement of α-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2- d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology. [ABSTRACT FROM AUTHOR]

We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First, we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced by GM yeast were targeted to non-GM yeast ( Saccharomyces cerevisiae BY4741 ΔCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible. [ABSTRACT FROM AUTHOR]

The cell wall of Saccharomyces cerevisiae plays an essential role in the biophysical characteristics of the cell surface. The modification of the cell wall property is an important factor for cellular adaptation to a stressful environment. In this study, we randomly modified the cell wall by displaying combinatorial random peptides on the yeast cell surface, and by screening, we successfully obtained a novel peptide, Scr35, that endowed yeasts with acid tolerance. The yeast, surface-modified by Scr35, was able to grow well under acidic condition and low glucose condition and showed high glucose uptake activity. However, the growth of the modified yeast became inferior as extracellular pH became higher. This inferiority was rescued by decreasing glucose concentration in a medium. Our results suggest that the optimum pH of a medium becomes low when the newly created Scr35 affects glucose uptake activity through cell-surface modification. Therefore, such artificial modification of the cell surface has a great potential as a useful tool for breeding acid-tolerant yeasts for industrial applications of S. cerevisiae as a biocatalyst. [ABSTRACT FROM AUTHOR]

For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of β-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin. Three β-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these β-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit β-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying β-glucosidase. [ABSTRACT FROM AUTHOR]

To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry. [ABSTRACT FROM AUTHOR]

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae mutant strain, KK-211, isolated from serial culture in medium containing isooctane showed an extremely higher tolerance to the hydrophobic organic-solvents, which are toxic to yeast cells compared to the wild-type parent strain, DY-1. To detect genes that are related to this tolerance, a DNA microarray analysis was performed using mRNAs isolated from strains DY-1 and KK-211. Fourteen genes were identified as being related to the tolerance. The expression of 12 genes including ICT1, YNL190W, and PRY3, was induced while the expression of two genes including PHO84 was repressed in strain KK-211. Two genes, ICT1 and YNL190W showed the same profile in the DNA microarray analysis and a differential display-polymerase chain reaction analysis. But, there is no detectable difference in the expression profile of KK-211 cells cultured with or without isooctane. The results suggest that change in expression levels of multiple genes that confer the modification function of the cell surface, not by a single gene, might be required for yeast cell tolerance to organic solvents. [ABSTRACT FROM AUTHOR]

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)–protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein–protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP–protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP–protein A and EYFP–Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein–protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei. [ABSTRACT FROM AUTHOR]

To increase the level of adsorption of heavy metal ions in surface-engineered yeasts, a yeast metallothionein (YMT) was tandemly fused and displayed by means of an α-agglutinin-based display system. The display of the YMT and its tandem repeats was examined by immunofluorescent labeling. The adsorption and recovery of Cd2+ on the cell surface was increasingly enhanced with increasing number of tandem repeats. All Cd2+-binding sites in the YMT tandem repeats were suggested to be completely occupied. To investigate the relationship between cell-surface adsorption and protection against heavy metal ion toxicity, the tolerance of these surface-engineered yeasts to Cd2+ was examined by growing in Cd2+-containing liquid medium. The rate of growth was found to be dependent on the number of displayed tandem repeats of YMT. These results suggest that the characteristics of surface-engineered yeasts as a bioadsorbent were dependent on the ability of the displayed proteins to bind metal ions, and the adsorption of heavy metal ions on the cell surface plays a major role in the ability of the cells to resist the toxic effects of metal ions. [ABSTRACT FROM AUTHOR]