Fumiyoshi Okazaki

A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology

The biorefinery manufacturing process for producing chemicals and liquid fuels from biomass is a promising approach for securing energy and resources. To establish cost-effective fermentation of lignocellulosic biomass, the consolidation of sacccharification and fermentation processes is a desirable strategy, but requires the development of microorganisms capable of cellulose/hemicellulose hydrolysis and target chemical production. Such an endeavor requires a large number of prerequisites to be realized, including engineering microbial strains with high cellulolytic activity, high product yield, productivities, and titers, ability to use many carbon sources, and resistance to toxic compounds released during the pretreatment of lignocellulosic biomass. Researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product yields to become cellulolytic. This article reviews recent advances in the development of microorganisms for the production of renewable chemicals and advanced biofuels, as well as ethanol, from lignocellulosic materials through consolidated bioprocessing.

Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

A beta-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the beta-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed beta-1,3-xylan but not other polysaccharides such as beta-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or beta-1,4-mannan. TxyA was capable of binding specifically to beta-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the K(d) and the maximum amount of protein bound to beta-1,3-xylan were 4.2 microM and 18.2 micromol/g of beta-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of beta-1,3-xylan.

Enzymatic glutathione production using metabolically engineered Saccharomyces cerevisiae as a whole-cell biocatalyst

We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.

From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae.

BackgroundMannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source.ResultsParental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate.ConclusionsWe successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

The first crystal structure of a family 31 carbohydrate-binding module with affinity to β-1,3-xylan

Here, we present the crystal structure of the family 31 carbohydrate‐binding module (CBM) of β‐1,3‐xylanase from Alcaligenes sp. strain XY‐234 (AlcCBM31) determined at a resolution of 1.25 Å. The AlcCBM31 shows affinity with only β‐1,3‐xylan. The AlcCBM31 molecule makes a β‐sandwich structure composed of eight β‐strands with a typical immunoglobulin fold and contains two intra‐molecular disulfide bonds. The folding topology of AlcCBM31 differs from that of the large majority of other CBMs, in which eight β‐strands comprise a β‐sandwich structure with a typical jelly‐roll fold. AlcCBM31 shows structural similarity with CBM structures of family 34 and family 9, which also adopt structures based on immunoglobulin folds.

Complex carriers of affibody-displaying bio-nanocapsules and composition-varied liposomes for HER2-expressing breast cancer cell-specific protein delivery.

A bio-nanocapsule (BNC), a hollow particle composed of hepatitis B virus (HBV) surface antigen (HBsAg), and liposome (LP) conjugation method (BNC/LP) has been recently developed by . The BNC/LP complex carrier could successfully deliver fluorescence-labeled beads (100 nm) into liver cells. In this study, we report the promising delivery of proteins incorporated in the complex carriers, which were prepared by the BNC/LP conjugation method with specificity-altered BNC and composition-varied LPs. The specificity-altered BNC, ZHER2-BNC was developed by replacing the hepatocyte recognition site of BNC with ZHER2 binding to HER2 receptor specifically. Using green fluorescent protein (GFP; 27 kDa) and cellular cytotoxic protein (exotoxin A; 66 kDa) for the delivery, we herein present the impact of different charges attributed to the composition of the LP on specific cell targeting and cellular uptake of the complex carriers. In addition, we demonstrate that the mixture prepared by mixing LPs with helper lipid possessing endosomal escaping ability boosts the functional expression of the cellular cytotoxic exotoxin A activity specifically. Finally, we further show the blending ratio of the LP mixture and ZHER2-BNC is a critical factor in determining the highly-efficient expression of the cytotoxic activity of exotoxin A.

Effective saccharification of kraft pulp by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae

Kraft pulp is a promising feedstock for bioproduction. The efficiency of kraft pulp saccharification was improved by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae. Application of the cellulase cocktail was demonstrated by simultaneous saccharification and fermentation, using kraft pulp and non-cellulolytic yeast. Such application would make possible to do an efficient production of other chemicals from kraft pulp.

Thermal stability and starch degradation profile of α-amylase from Streptomyces avermitilis.

Amylases from Streptomyces are useful in the production of maltooligosaccharides, but they have weak thermal stability at temperatures higher than 40 °C. In this study, α-amylase (SAV5981 gene of Streptomyces avermitilis) was expressed from Streptomyces lividans 1326 and purified by ammonium sulfate fractionation followed by anionic chromatography (Q-HP sepharose). The properties of the purified SAV5981 amylase were determined by the starch-iodine method. The effect of metal ions on amylase activity was investigated. The optimal temperature shifted from 25 to 50 °C with the addition of the Ca2+ ion. The thermal stability of SAV5981 was also dramatically enhanced by the addition of 10 mM CaCl2. Improvement of the thermal stability of SAV5981 was examined by CD spectra in the presence and the absence of the Ca2+ ion. Thin-layer chromatography (TLC) analysis and HPLC analysis of starch degradation revealed that SAV5981 mainly produced maltose and maltotriose, not glucose. The maltoorigosaccharide-producing amylase examined in this study has the potential in the industrial application of oligosaccharide production.

Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, β-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.

Expression, crystallization and preliminary X-ray diffraction studies of thermostable β-1,3-xylanase from Thermotoga neapolitana strain DSM 4359

Crystals of β-1,3-xylanase (1,3-β-D-xylan xylanohydrolase; EC 3.2.1.32) from Thermotoga neapolitana strain DSM 4359 with maximum dimensions of 0.2×0.1×0.02 mm were grown using the sitting-drop vapour-diffusion method at 293 K over 24 h. The crystals diffracted to a resolution of 1.82 Å, allowing structure determination. The crystals belonged to space group P2(1), with unit-cell parameters a=39.061, b=75.828, c=52.140 Å; each asymmetric unit cell contained a single molecule.