Direct Ethanol Production from Ionic Liquid-Pretreated Lignocellulosic Biomass by Cellulase-Displaying Yeasts.

Among the many types of lignocellulosic biomass pretreatment methods, the use of ionic liquids (ILs) is regarded as one of the most promising strategies. In this study, the effects of four kinds of ILs for pretreatment of lignocellulosic biomass such as bagasse, eucalyptus, and cedar were evaluated. In direct ethanol fermentation from biomass incorporated with ILs by cellulase-displaying yeast, 1-butyl-3-methylimidazolium acetate ([Bmim][OAc]) was the most effective IL. The ethanol production and yield from [Bmim][OAc]-pretreated bagasse reached 0.81 g/L and 73.4% of the theoretical yield after fermentation for 96 h. The results prove the initial concept, in which the direct fermentation from lignocellulosic biomass effectively promoted by the pretreatment with IL.

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

BACKGROUND: Mannans 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 ß-mannanase and ß-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. RESULTS: Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered ß-mannanase and ß-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-ß-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-ß-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. CONCLUSIONS: We successfully displayed ß-mannanase and ß-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying ß-mannanase and ß-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering ß-mannanase and ß-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.

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.

Expression of cold-adapted ß-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

Cold-adapted ß-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes.

L-lactic acid production from starch by simultaneous saccharification and fermentation in a genetically engineered Aspergillus oryzae pure culture.

Lactic acid is a commodity chemical that can be produced biologically. Lactic acid-producing Aspergillus oryzae strains were constructed by genetic engineering. The A. oryzae LDH strain with the bovine L-lactate dehydrogenase gene produced 38 g/L of lactate from 100g/L of glucose. Disruption of the wild-type lactate dehydrogenase gene in A. oryzae LDH improved lactate production. The resulting strain A. oryzae LDHΔ871 produced 49 g/L of lactate from 100g/L of glucose. Because A. oryzae strains innately secrete amylases, A. oryzae LDHΔ871 produced approximately 30 g/L of lactate from various starches, dextrin, or maltose (all at 100 g/L). To our knowledge, this is the first report describing the simultaneous saccharification and fermentation of lactate from starch using a pure culture of transgenic A. oryzae. Our results indicate that A. oryzae could be a promising host for the bioproduction of useful compounds such as lactic acid.

Aspergillus oryzae-based cell factory for direct kojic acid production from cellulose.

BACKGROUND: Kojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications. Thus, an efficient and cost-effective kojic acid production process would be valuable. However, little is known about the complete set of genes for kojic acid production. Currently, kojic acid is produced from glucose. The efficient production of kojic acid using cellulose as an inexpensive substrate would help establish cost-effective kojic acid production. RESULTS: A kojic acid transcription factor gene over-expressing the A. oryzae strain was constructed. Three genes related to kojic acid production in this strain were transcribed in higher amounts than those found in the wild-type strain. This strain produced 26.4 g/L kojic acid from 80 g/L glucose. Furthermore, this strain was transformed with plasmid harboring 3 cellulase genes. The resultant A. oryzae strain successfully produced 0.18 g/L of kojic acid in 6 days of fermentation from the phosphoric acid swollen cellulose. CONCLUSIONS: Kojic acid was produced directly from cellulose material using genetically engineered A. oryzae. Because A. oryzae has efficient protein secretion ability and secondary metabolite productivity, an A. oryzae-based cell factory could be a platform for the production of various kinds of bio-based chemicals.