Category Archives: Biotechnology

Background:
The trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in dried distillers grains with solubles (DDGS), a co-product of fuel ethanol fermentation, when grain containing DON is used to produce fuel ethanol. Even low levels of DON ([less than or equal to] 5ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON) and reduce its toxicity in plants and animals.
Results:
Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (Saccharomyces cerevisiae) during a series of small-scale barley (Hordeum vulgare) ethanol fermentations. DON was concentrated 1.6 to 8.2 times in DDGS compared to the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON when Virginia winter barley cultivars Eve, Thoroughbred, and Price and experimental line VA06H-25 were used. Barley mashes from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, while FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red (R), which also consumed galactose when present in the mash.
Conclusions:
This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.Source:
http://www.biotechnologyforbiofuels.com/rss/

Background:
Biochemical conversion of lignocellulose hydrolysates remains challenging largely because most microbial processes have significantly reduced efficiency in the presence of both hexoses and pentoses. Thus, identification of microorganisms capable of efficient and simultaneous utilization of both glucose and xylose is pivotal.
Results:
In this work, we showed that the oleaginous yeast Trichosporon cutaneum AS 2.571 assimilated glucose and xylose simultaneously, and accumulated intracellular lipid up to 59 wt% with a lipid coefficient up to 0.17 g/g sugar, upon cultivation on a 2:1 glucose/xylose mixture in a 3-liter stirred tank bioreactor. In addition, no classical diauxic growth behavior was observed as microbial cell mass was increasing during the whole culture process without any lag periods. During shake flask cultures with different initial glucose/xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. Simultaneous utilization of glucose and xylose was also observed during corn stover hydrolysate fermentation with lipid content and coefficient of 39.2% and 0.15 g/g sugar, respectively. Lipid produced herein had fatty acid compositional profile similar to those of conventional vegetable oil, indicating that it could be explored as raw material for biodiesel production.
Conclusion:
Efficient lipid production with simultaneous consumption of glucose and xylose was achieved in this study. It provides an exciting opportunity to transform lignocellulosic materials into biofuel molecules and should also provoke further study to elucidate this unique sugar assimilation mechanism.Source:
http://www.biotechnologyforbiofuels.com/rss/

Background:
Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tract of ruminants that can actively digest cellulosic materials and its cellulases are of great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus.
Results:
We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches on N. patriciarum to accelerate gene identification, enzyme classification, and application in rice straw degradation. By complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative glycosyl hydrolase (GH) contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: beta-glucosidase, endo-1,4-beta-xylanase, xylanase B and Cel48A exo-glucanase. From the sequences of assembled contigs we cloned 19 putative cellulase genes, including GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks.
Conclusions:
These GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One beta-glucosidases (W5-16143) and one exo-cellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.Keywordsanaerobic fungi, biomass, rice straw, sugarcane, napiergrass, GH, next-generation sequencingSource:
http://www.biotechnologyforbiofuels.com/rss/

Background:
To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never-been-cultivated microorganisms.
Results:
From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologues. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in E. coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-alpha-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-beta-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-beta-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids.
Conclusions:
Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

Background:
Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials.
Results:
The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by two times by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by three times. In aerobic cultivations in shake flasks in mineral medium the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They were increased to 4.12 and 2.4 mg per g glucose in YPD complex medium under aerobic conditions, respectively.
Conclusions:
Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production.Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial improvement in isobutanol production for the reference strain, but not for the ILV2 ILV3 ILV5 overexpression strain. This result implies that other constraints besides the enzyme activities for the supply of 2-ketoisovalerate may become bottlenecks for isobutanol production after ILV2 ILV3 and ILV5 have been overexpressed, and it most probably includes the valine inhibition to Ilv2.

Background:
As the supply of starch grain and sugar cane, currently the main feedstocks for bioethanol production, become limited, lignocelluloses will be sought as alternative materials for bioethanol production. Production of cellulosic ethanol is still cost-inefficient due to the low final ethanol concentration and the addition of nutrients. Here, simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic residues from commercial furfural production (furfural residue, FR) and corn kernels were carried out to compare different nutritional media. The final ethanol concentration, the yield, the amount of live yeast cells and yeast cell death ratio were investigated to evaluate the effectiveness of integrating cellulosic and starch ethanol.
Results:
Both the ethanol yield and amount of live yeast cells increased with increasing corn kernels concentration, while the yeast cell death ratio decreased in the SSCF of FR and corn kernels. An ethanol concentration of 73.1 g/L at 120 h, which corresponded to a 101.1% ethanol yield based on FR cellulose and corn starch, was obtained in the SSCF of 7.5% FR and 14.5% corn kernels with mineral salt medium. SSCF could simultaneously convert cellulose into ethanol from both corn kernels and FR, and the SSCF ethanol yield was similar between the organic and mineral salt media.
Conclusions:
Starch ethanol promotes cellulosic ethanol by providing important nutrients for fermentative organisms whereby cellulosic ethanol promotes starch ethanol by providing cellulosic enzymes that convert the cellulosic polysaccharides in starch materials into additional ethanol. It is feasible to produce ethanol in SSCF of FR and corn kernels with mineral salt medium. It would be cost-efficient to produce ethanol in SSCF of high water insoluble solid (WIS) of lignocellulosic materials and corn kernels. Compared with prehydrolysis and fed-batch strategy using lignocellulosic materials, addition of starch hydrolysates to cellulosic ethanol production is a more suitable method to improve the final ethanol concentration.