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Category Archives: Biotechnology

Arc the Lad OST Biotechnology Research Institute – Video

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Arc the Lad OST Biotechnology Research Institute
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Arc the Lad OST Biotechnology Research Institute - Video

Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment

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Background:
Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases.
Results:
In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of beta-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases.
Conclusion:
The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/115

RNA-Seq analysis and targeted mutagenesis for improved free fatty acid production in an engineered cyanobacterium

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Background:
High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs.
Results:
Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response.
Conclusions:
A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/113

High levels of variation in Salix lignocellulose genes revealed using poplar genomic resources

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Background:
Little is known about the levels of variation in lignin or other wood related genes in Salix, a genus that is being increasingly used for biomass and biofuel production. The lignin biosynthesis pathway is well characterized in a number of species, including the model tree Populus. We aimed to transfer the genomic resources already available in Populus to its sister genus Salix to assess levels of variation within genes involved in wood formation.
Results:
Amplification trials for 27 gene regions were undertaken in 40 Salix taxa. Twelve of these regions were sequenced. Alignment searches of the resulting sequences against reference databases, combined with phylogenetic analyses, showed the close similarity of these Salix sequences to Populus, confirming homology of the primer regions and indicating a high level of conservation within the wood formation genes. However, all sequences were found to vary considerably among Salix species, mainly as SNPs with a smaller number of insertions-deletions. Between 25 and 176 SNPs per kbp per gene region (in predicted exons) were discovered within Salix.
Conclusions:
The variation found is sizeable but not unexpected as it is based on interspecific and not intraspecific comparison; it is comparable to interspecific variation in Populus. The characterisation of genetic variation is a key process in pre-breeding and for the conservation and exploitation of genetic resources in Salix. This study characterises the variation in several lignocellulose gene markers for such purposes.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/114

The synergistic action of accessory enzymes enhances the hydrolytic potential of a ¿cellulase mixture¿ but is highly substrate specific

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Background:
Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still too high. One way to reduce the amount of protein/enzyme required is to formulate a more efficient enzyme cocktail by adding so-called accessory enzymes such as xylanase, lytic polysaccharide monooxygenase (AA9, formerly known as GH61), etc., to the cellulase mixture. Previous work has shown the strong synergism that can occur between cellulase and xylanase mixtures during the hydrolysis of steam pretreated corn stover, requiring lower protein loading to achieve effective hydrolysis. However, relatively high loadings of xylanases were required. When family 10 and 11 endo-xylanases and family 5 xyloglucanase were supplemented to a commercial cellulase mixture varying degrees of improved hydrolysis over a range of pretreated, lignocellulosic substrates were observed.
Results:
The potential synergistic interactions between cellulase monocomponents and hemicellulases from family 10 and 11 endo-xylanases (GH10 EX and GH11 EX) and family 5 xyloglucanase (GH5 XG), during hydrolysis of various steam pretreated lignocellulosic substrates, were assessed. It was apparent that the hydrolytic activity of cellulase monocomponents was enhanced by the addition of accessory enzymes although the “boosting” effect was highly substrate specific. The GH10 EX and GH5 XG both exhibited broad substrate specificity and showed strong synergistic interaction with the cellulases when added individually. The GH10 EX was more effective on steam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective on softwood substrates. The synergistic interaction between GH10 EX and GH5 XG when added together further enhanced the hydrolytic activity of the cellulase enzymes over a range of pretreated lignocellulosic substrates. GH10 EX addition could also stimulate further cellulose hydrolysis when added to the hydrolysis reactions when the rate of hydrolysis had levelled off.
Conclusions:
Endo-xylanases and xyloglucanases interacted synergistically with cellulases to improve the hydrolysis of a range of pretreated lignocellulosic substrates. However, the extent of improved hydrolysis was highly substrate dependent. It appears that those accessory enzymes, such as GH10 EX and GH5 XG, with broader substrate specificities promoted the greatest improvements in the hydrolytic performance of the cellulase mixture on all of the pretreated biomass substrates.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/112


  1. Biotechnology