Coproduction of acetic acid and electricity by application of microbial fuel cell technology to vinegar fermentation.

The coproduction of a useful material and electricity via a novel application of microbial fuel cell (MFC) technology to oxidative fermentation was investigated. We focused on vinegar production, i.e.

Sugar consumption and ethanol fermentation by transporter-overexpressed xylose-metabolizing Saccharomyces cerevisiae harboring a xyloseisomerase pathway.

Four kinds of transporters, HXT1 and HXY7 from Saccharomyces cerevisiae, and GXF1 and GXS1 from Candida intermedia, were overexpressed in xylose-metabolizing S. cerevisiae harboring a xyloseisomerase-based pathway. Overexpression of transporter enhanced sugar consumption and ethanol production, and GXF1 was efficient for ethanol fermentation from both glucose and xylose.

Construction of a xylose-metabolizing yeast by genome integration of xylose isomerase gene and investigation of the effect of xylitol on fermentation.

A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.

Evaluation of the biodegradability of polyurethane and its derivatives by using lipase-displaying arming yeast.

Candida antarctica lipase B (CALB) has been used to polymerize and degrade polyesters. We developed a convenient method for investigating the biodegradability of plastics that involves the use of CALB-displaying "arming yeast." Polyurethane containing dulcitol units was prepared and used as the model material.

Ester synthesis reaction with CALB displaying yeast whole cell biocatalyst: effect of organic solvent and initial water content.

The effect of the reaction conditions on the ester synthesis reaction with CALB displaying yeast whole cells was determined. Utilization of hydrophobic organic solvent improved the efficiency of the ester synthesis reaction. Also the initial water content was important for the expression of the ester synthesis activity of CALB displaying yeast whole cells.

Development of an enzyme activity screening system for beta-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting.

Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying beta-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls.

Evaluation of cell surface-displayed protein stability against simulated gastric fluid.

A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase.

A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast.

To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay.

Improvement of a Candida antarctica lipase B-displaying yeast whole-cell biocatalyst and its application to the polyester synthesis reaction.

A Candida antarctica lipase B (CALB)-displaying yeast whole-cell biocatalyst was constructed with the integration of the CALB cell-surface display expression cassette in the yeast genome and cell fusion by mating. Lipase hydrolytic activity of the yeast whole-cell biocatalyst subsequently increased, in both a- and alpha-type yeast cells, with the number of copies of the CALB cell-surface display expression cassette introduced, and reached 43.6 and 32.

Development of yeast cells displaying Candida antarctica lipase B and their application to ester synthesis reaction.

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface.

Preparation of a whole-cell biocatalyst of mutated Candida antarctica lipase B (mCALB) by a yeast molecular display system and its practical properties.

To prepare a whole-cell biocatalyst of a stable lipase at a low price, mutated Candida antarctica lipase B (mCALB) constructed on the basis of the primary sequences of CALBs from C. antarctica CBS 6678 strain and from C. antarctica LF 058 strain was displayed on a yeast cell surface by alpha-agglutinin as the anchor protein for easy handling and stability of the enzyme.

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica.

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A.

Development of novel yeast cell surface display system for homo-oligomeric protein by coexpression of native and anchored subunits.

Streptavidin derived from Streptomyces avidinii was displayed on the cell surface of the yeast Saccharomyces cerevisiae by cell-surface engineering using two types of plasmid for the expression of a native subunit and an anchored subunit fused with the C-terminus of 318 amino acids of Flo1p containing a glycosylphosphatidylinositol anchor attachment signal. The displayed streptavidin had the binding ability for biotinylated compounds. This was confirmed by fluorescence microscopy after the adsorption of yeast cells displaying streptavidin and biotinylated fluorescein isothiocyanate.

Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system.

A Pichia pastoris cell-surface display system was constructed using a Flo1p anchor system, which was developed in Saccharomyces cerevisiae. The lipase from Rhizopus oryzae with a pro sequence (ProROL) was used as the model protein and was genetically fused to the anchor consisting of amino acids 1-1099 of Flo1p (FS anchor). The resulting fusion protein FSProROL was expressed under the control of the alcohol oxidase 1 promoter (pAOX1).

Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion.

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica.