Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

Bile salt biotransformations by human intestinal bacteria.

In recent years, growing attention has been devoted to the conversion of biomass into fuel ethanol, considered the cleanest liquid fuel alternative to fossil fuels. Significant advances have been made towards the technology of ethanol fermentation. This review provides practical examples and gives a broad overview of the current status of ethanol fermentation including biomass resources, microorganisms, and technology. Also, the promising prospects of ethanol fermentation are especially introduced. The prospects included are fermentation technology converting xylose to ethanol, cellulase enzyme utilized in the hydrolysis of lignocellulosic materials, immobilization of the microorganism in large systems, simultaneous saccharification and fermentation, and sugar conversion into ethanol.

Ethanol fermentation from biomass resources: current state and prospects.

Xylanases are hydrolytic enzymes which randomly cleave the β 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented.

Xylanases, xylanase families and extremophilic xylanases

Despite an increased knowledge of microbial xylanolytic systems in the past few years, further studies are required to achieve a complete understanding of the mechanism of xylan degradation by microorganisms and their enzymes. The enzyme system used by microbes for the metabolism of xylan is the most important tool for investigating the use of the second most abundant polysaccharide (xylan) in nature. Recent studies on microbial xylanolytic systems have generally focussed on induction of enzyme production under different conditions, purification, characterization, molecular cloning and expression, and use of enzyme predominantly for pulp bleaching. Rationale approaches to achieve these goals require a detailed knowledge of the regulatory mechanism governing enzyme production. This review will focus on complex xylan structure and the microbial enzyme complex involved in its complete breakdown, studies on xylanase regulation and production and their potential industrial applications, with special reference to biobleaching.

Microbial xylanases and their industrial applications: a review.

Yeast Saccharomyces cerevisiae has a rigid cell wall outside its cell membrane (21). The cell wall is the largest organelle of the yeast cell and protects the cell from mechanical injury, hypotonic lysis, and chemical substances that could damage the cell. The cell wall is composed of glucan, mannoproteins, and a small amount of chitin. Mannoproteins are proteins with a large amount of N- and/or O-linked mannoses and compose the outermost layer of the yeast cell wall (42). Mannoproteins in the yeast cell wall are grouped into two classes (9). The first class is proteins that can be extracted with sodium dodecyl sulfate (SDS). These proteins are considered to be noncovalently entrapped or associated in the cell wall. The other class is cell wall proteins that cannot be extracted with SDS (3, 36). These proteins are considered to be covalently bound to cell wall glucan and can be solubilized by incubation with β-1,3-glucanase (36). Among these proteins, α-agglutinin and Aga1p are involved in sexual agglutination (14) and Flo1p is related to flocculation (40), but there are many other proteins whose functions are not clear. These proteins are considered as structural cell wall proteins and some of them (Cwp1p, Cwp2p, and Tip1p) have been previously identified from a glucanase extract of SDS-treated cell walls (36). These proteins are rich in serine and threonine residues and contain signals for the addition of a glycosylphosphatidylinositol (GPI) anchor. This anchor is transferred to cell wall proteins in the endoplasmic reticulum, and GPI-anchored proteins are transported through the Golgi apparatus to the plasma membrane. Although some GPI-anchored proteins remain to bind to the plasma membrane (19), cell wall proteins are transferred to β-1,6-glucan that is bound to β-1,3-glucan by an unknown mechanism (7).

In previous studies, we have isolated cell wall proteins solubilized from SDS-treated cell walls with Rarobacter faecitabidus protease I (RPI) and characterized them as Cwp1p (31) and Tir1p (8). Cwp1p was coincidentally identified from a laminarinase extract of cell walls (36). Cwp1p is a putative GPI-anchored protein. The C-terminal hydrophobic sequence of Cwp1p is needed for attaching the molecule to cell walls because a mutant Cwp1p deficient in this sequence was secreted into the culture medium (31). Tir1p is a cell wall protein specifically expressed in cells cultured anaerobically (8) and also has a GPI anchor signal. Since these proteins are solubilized from cell walls with β-1,3-glucanase, these proteins were considered to be bound to cell wall glucan. RPI is a yeast-lytic protease that specifically recognizes mannose chains of mannoproteins and cleaves their peptide bonds (29, 30). Therefore, solubilization of cell wall proteins by digestion with RPI is very useful since extracted proteins show less heterogeneity in size than do cell wall proteins prepared by glucanase digestion (8, 31).

Here we report the isolation of another cell wall protein by using RPI. This protein was prepared from aerobic culture and was identified as Sed1p by amino acid sequence analysis. SED1 was highly expressed in the stationary phase, and its disruptant was more sensitive to Zymolyase than the wild-type cells in the stationary phase. We believe that Sed1p is required for stress resistance in stationary-phase cells.

Sed1p Is a Major Cell Wall Protein of Saccharomyces cerevisiae in the Stationary Phase and Is Involved in Lytic Enzyme Resistance

Crystal Structure of Aminopeptidase N (Proteobacteria Alanyl Aminopeptidase) from Escherichia coli and Conformational Change of Methionine 260 Involved in Substrate Recognition

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2002.03244.x

Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae

Tolerance Mechanism of the Ethanol-Tolerant Mutant of Sake Yeast

Five extracellular endo-xylanases were recognized in the culture broth of shochu koji mold (Aspergillus kawachii, IFO 4308), and three major xylanases (XylA, XylB, and XylC) were purified and characterized. The molecular masses of XylA, XylB, and XylC were 35,000, 26,000, and 29,000, and isoelectric points were pH 6.7, 4.4, and 3.5, respectively. Amino acid compositions and other properties were studied and these three xylanases were found to be greatly different in their properties. These three xylanases, XylA, XylB, and XylC, were stable between pH 3-10, 3-10, and 1-9 and the optimum pHs were 5.5, 4.5, and 2.0, respectively. Consequently, these xylanases were acid stable xylanases, especially XylC was an acidophilic xylanase (acid xylanase). These xylanases produced various xylooligosaccharides including xylose from xylan and the main product was xylobiose in all xylanases. The production of acid xylanase (XylC) was enhanced with a low initial pH of the medium.

Kiyoshi Ito

Papers

Sed1p Is a Major Cell Wall Protein of Saccharomyces cerevisiae in the Stationary Phase and Is Involved in Lytic Enzyme Resistance

Crystal Structure of Aminopeptidase N (Proteobacteria Alanyl Aminopeptidase) from Escherichia coli and Conformational Change of Methionine 260 Involved in Substrate Recognition

Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae

Tolerance Mechanism of the Ethanol-Tolerant Mutant of Sake Yeast

Purification and Properties of Acid Stable Xylanases from Aspergillus kawachii