Phenotypic selection of a wild Saccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEXTM pretreated corn stover

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Background:
Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover, degradation products resulting from biomass pretreatment impair fermentation of sugars, especially xylose, and can synergize with high temperature stress. One approach to resolve both concerns is to utilize a strain background with innate tolerance to both elevated temperatures and degradation products.
Results:
In this study, we screened a panel of 108 wild and domesticated Saccharomyces cerevisiae strains isolated from a wide range of environmental niches. One wild strain was selected based on its growth tolerance to simultaneous elevated temperature and AFEXTM (Ammonia Fiber Expansion) degradation products. After engineering the strain with two copies of the Scheffersomyces stipitis xylose reductase, xylitol dehydrogenase and xylulokinase genes, we compared the ability of this engineered strain to the benchmark 424A(LNH-ST) strain in ethanol production and xylose fermentation in standard lab medium and AFEX pretreated corn stover (ACS) hydrolysates, as well as in SSCF of ACS at different temperatures. In SSCF of 9% (w/w) glucan loading ACS at 35[degree sign]C, the engineered strain showed higher cell viabilities and produced a similar amount of ethanol (51.3 g/L) compared to the benchmark 424A(LNH-ST) strain.
Conclusion:
These results validate our approach in the selection of wild Saccharomyces cerevisiae strains with thermo-tolerance and degradation products tolerance properties for lignocellulosic biofuel production. The wild and domesticated yeast strains phenotyped in this work are publically available for others to use as genetic backgrounds for fermentation of their pretreated biomass at elevated temperatures.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/108

The building that breathes ,biotechnology researching center, Eng Mohammed Tarek, – Video

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The building that breathes ,biotechnology researching center, Eng Mohammed Tarek,
The project is The building that breathes,Eng Mohammed Tarek,cairo,egypt .It is Biotechnology Research Center. It is working on the discovery of the remedial...

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The building that breathes ,biotechnology researching center, Eng Mohammed Tarek, - Video

Selecting beta-glucosidases to support cellulases in cellulose saccharification

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Background:
Enzyme end-product inhibition is a major challenge in the hydrolysis of lignocellulose at a high dry matter consistency. beta-glucosidases (BGs) hydrolyze cellobiose into two molecules of glucose, thereby relieving the product inhibition of cellobiohydrolases (CBHs). However, BG inhibition by glucose will eventually lead to the accumulation of cellobiose and the inhibition of CBHs. Therefore, the kinetic properties of candidate BGs must meet the requirements determined by both the kinetic properties of CBHs and the set-up of the hydrolysis process.
Results:
The kinetics of cellobiose hydrolysis and glucose inhibition of thermostable BGs from Acremonium thermophilum (AtBG3) and Thermoascus aurantiacus (TaBG3) was studied and compared to Aspergillus sp. BG purified from Novozyme(R)188 (N188BG). The most efficient cellobiose hydrolysis was achieved with TaBG3, followed by AtBG3 and N188BG, whereas the enzyme most sensitive to glucose inhibition was AtBG3, followed by TaBG3 and N188BG. The use of higher temperatures had an advantage in both increasing the catalytic efficiency and relieving the product inhibition of the enzymes. Our data, together with data from a literature survey, revealed a trade-off between the strength of glucose inhibition and the affinity for cellobiose; therefore, glucose-tolerant BGs tend to have low specificity constants for cellobiose hydrolysis. However, although a high specificity constant is always an advantage, in separate hydrolysis and fermentation, the priority may be given to a higher tolerance to glucose inhibition.
Conclusions:
The specificity constant for cellobiose hydrolysis and the inhibition constant for glucose are the most important kinetic parameters in selecting BGs to support cellulases in cellulose hydrolysis.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/105

Integrated OMICS guided engineering of biofuel butanol-tolerance in photosynthetic Synechocystis sp. PCC 6803

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Background:
Photosynthetic cyanobacteria have been recently proposed as a 'microbial factory' to produce butanol due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources, respectively. However, to improve the productivity, one key issue needed to be addressed is the low tolerance of the photosynthetic hosts to butanol.
Results:
In this study, we first applied a quantitative transcriptomics approach with a next-generation RNA sequencing technology to identify gene targets relevant to butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803. The results showed that 278 genes were induced by the butanol exposure at all three sampling points through the growth time course. Genes encoding heat-shock proteins, oxidative stress related proteins, transporters and proteins involved in common stress responses, were induced by butanol exposure. We then applied GC-MS based metabolomics analysis to determine the metabolic changes associated with the butanol exposure. The results showed that 46 out of 73 chemically classified metabolites were differentially regulated by butanol treatment. Notably, 3-phosphoglycerate, glycine, serine and urea related to general stress responses were elevated in butanol-treated cells. To validate the potential targets, we constructed gene knockout mutants for three selected gene targets. The comparative phenotypic analysis confirmed that these genes were involved in the butanol tolerance.
Conclusion:
The integrated OMICS analysis provided a comprehensive view of the complicated molecular mechanisms employed by Synechocystis sp. PCC 6803 against butanol stress, and allowed identification of a series of potential gene candidates for tolerance engineering in cyanobacterium Synechocystis sp. PCC 6803.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/106

Improving the performance of enzymes in hydrolysis of high solids paper pulp derived from MSW

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Background:
The research aimed to improve the overall conversion efficiency of the CTec(R) family of enzymes by identifying factors that lead to inhibition and seeking methods to overcome these through process modification and manipulation. The starting material was pulp derived from municipal solid waste and processed in an industrial-scale washing plant.
Results:
Analysis of the pulp by acid hydrolysis showed a ratio of 55 : 12 : 6 : 24 : 3 of glucan : xylan : araban/galactan/mannan : lignin : ash. At high total solids content (>18.5% TS) single-stage enzyme hydrolysis gave a maximum glucan conversion of 68%. It was found that two-stage hydrolysis could give higher conversion if sugar inhibition was removed by an intermediate fermentation step between hydrolysis stages. This, however, was not as effective as direct removal of the sugar products, including xylose, by washing of the residual pulp at pH 5. This improved the water availability and allowed reactivation of the pulp-bound enzymes. Inhibition of enzyme activity could further be alleviated by replenishment of beta-glucosidase which was shown to be removed during the wash step.
Conclusions:
The two-stage hydrolysis process developed could give an overall glucan conversion of 88%, with an average glucose concentration close to 8% in 4 days, thus providing an ideal starting point for ethanol fermentation with a likely yield of 4 wt%. This is a significant improvement over a single-step process. This hydrolysis configuration also provides the potential to recover the sugars associated with residual solids which are diluted when washing hydrolysed pulp.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/107

Microevolution from shock to adaptation revealed strategies improving ethanol tolerance and production in Thermoanaerobacter

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IntroductionThe molecular links between shock-response and adaptation remain poorly understood, particularly for extremophiles. This has hindered rational engineering of solvent tolerance and correlated traits (e.g., productivity) in extremophiles. To untangle such molecular links, here we established a model that tracked the microevolution from shock to adaptation in thermophilic bacteria.MethodTemporal dynamics of genomes and transcriptomes was tracked for Thermoanaerobacter sp. X514 which under increasing exogenous ethanol evolved from ethanol-sensitive wild-type (Strain X) to tolerance of 2%- (XI) and eventually 6%-ethanol (XII). Based on the reconstructed transcriptional network underlying stress tolerance, genetic engineering was employed to improve ethanol tolerance and production in Thermoanaerobacter.
Results:
The spontaneous genome mutation rate (mug) of Thermoanaerobacter sp. X514, calculated at 0.045, suggested a higher mutation rate in thermophile than previously thought. Transcriptomic comparison revealed that shock-response and adaptation were distinct in nature, whereas the transcriptomes of XII resembled those of the extendedly shocked X. To respond to ethanol shock, X employed fructose-specific phosphotransferase system (PTS), Arginine Deiminase (ADI) pathway, alcohol dehydrogenase (Adh) and a distinct mechanism of V-type ATPase. As an adaptation to exogenous ethanol, XI mobilized resistance-nodulation-cell division (RND) efflux system and Adh, whereas XII, which produced higher ethanol than XI, employed ECF-type [coptic small letter shima]24, an alcohol catabolism operon and phase-specific heat-shock proteins (Hsps), modulated hexose/pentose-transport operon structure and reinforced membrane rigidity. Exploiting these findings, we further showed that ethanol productivity and tolerance can be improved simultaneously by overexpressing adh or [coptic small letter shima]24 in X.
Conclusion:
Our work revealed thermophilic-bacteria specific features of adaptive evolution and demonstrated a rational strategy to engineer co-evolving industrial traits. As improvements of shock-response, stress tolerance and productivity have been crucial aims in industrial applications employing thermophiles, our findings should be valuable not just to the production of ethanol but also to a wide variety of biofuels and biochemicals.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/103

iShares NASDAQ Biotechnology Index (IBB) Shares Sold by Neumann Capital Management LLC – The Cerbat Gem

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iShares NASDAQ Biotechnology Index (IBB) Shares Sold by Neumann Capital Management LLC
The Cerbat Gem
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iShares NASDAQ Biotechnology Index (IBB) Shares Sold by Neumann Capital Management LLC - The Cerbat Gem

Product inhibition of cellulases studied with 14C-labeled cellulose substrates

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Background:
As a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products.
Results:
The cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates.
Conclusions:
GH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of product inhibition.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/104