Special issue: Application of biotechnology for biofuels: transforming biomass to biofuels

Mittal, Ashutosh; Decker, Stephen R.

doi:10.1007/s13205-013-0122-8

Cited by 12 publications

(10 citation statements)

References 17 publications

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“…Extensive efforts have been made during the last decades for the development and assembly of efficient cellulolytic enzymatic cocktails. However, cellulose degradation is still not efficient enough to be cost effective [3,16]. One of the key bottlenecks for achieving cost-effective degradation of plant cell wall biomass is the requirement for large amounts of cellulases (about 100–200 g of cellulase per gallon of cellulosic ethanol) [17].…”

Section: Introductionmentioning

confidence: 99%

Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

Yoav

Stern

Salama-Alber

et al. 2019

IJMS

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β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

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Section: Introductionmentioning

confidence: 99%

Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

Yoav

Stern

Salama-Alber

et al. 2019

IJMS

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“…Cellulosic ethanol, one of the suggested solutions of this global issue, meets the necessary requirements of being renewable and environmentally friendly [ 2 , 3 ]. Unlike the production process of the first-generation bioethanol alternative, which utilizes the edible parts of plants, the cellulosic ethanol alternative exploits the inedible polysaccharides of the plant, notably the cellulose, found in the cell walls of lignocellulosic biomasses [ 4 , 5 ]. Agriculture or industrial lignocellulosic wastes can be used as sources of biomass, although removal of plant residues from the field could also have negative effects on soil fertility and quality [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

How does cellulosome composition influence deconstruction of lignocellulosic substrates in Clostridium (Ruminiclostridium) thermocellum DSM 1313?

Yoav¹,

Barak²,

Shamshoum³

et al. 2017

Biotechnol Biofuels

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BackgroundBioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium (Ruminiclostridium) thermocellum, the cellulosome, was studied here. C. thermocellum is known to assemble cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated.ResultsGlucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different cellulosome samples. The most abundant catalytic subunits in all cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived cellulosome types.ConclusionThe results reported here highlight the finding that cellulosomes derived from cells grown on glucose and microcrystalline cellulose are more efficient in their action on cellulosic substrates than other cellulosome preparations. These results should be considered in the future development of C. thermocellum-based cellulolytic cocktails, designer cellulosomes, or engineering of improved strains for deconstruction of lignocellulosic biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0909-7) contains supplementary material, which is available to authorized users.

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“…Large modifications of tree form and architecture have been proposed as important steps in modifying trees for use in bioenergy plantations (Ragauskas et al 2006;Mittal and Decker 2013). Key goals include rapid growth rate, reduced stature, and increased allocation to stems in relation to other woody tissues.…”

Section: Introductionmentioning

confidence: 99%

Recombinant DNA modification of gibberellin metabolism alters growth rate and biomass allocation in Populus

Viswanath

et al. 2015

Tree Genetics & Genomes

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Overexpression of genes that modify gibberellin (GA) metabolism and signaling have been previously shown to produce trees with improved biomass production but highly disturbed development. To examine if more subtle types of genetic modification of GA could improve growth rate and modify tree architecture, we transformed a model poplar genotype (Populus tremula × P. alba) with eight genes, including two cisgenes (intact copies of native genes), four intragenes (modified copies of native genes), and two transgenes (from sexually incompatible species), and studied their effects under greenhouse and field conditions. In the greenhouse, four out of the eight tested genes produced a significant and often striking improvement of stem volume, and two constructs significantly modified the proportion of root or shoot biomass. Characterization of GA concentrations in the cisgenic population that had an additional copy of a poplar GA20-oxidase gene showed elevated concentrations of 13-hydroxylated GAs compared to wild-type poplars. In the field, we observed growth improvement for three of the six tested constructs, but it was significantly greater for only one of the constructs, a pRGL:GA20-oxidase intragene. The greenhouse and field responses were highly variable, possibly to due to cross-talk among the GA pathway and other stress response pathways, or due to interactions between the cisgenes and intragenes with highly similar endogenes. Our results indicate that extensive field trials, similar to those required for conventional breeding, will be critical to evaluating the value and pleiotropic effects of GA-modifying genes.

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Special issue: Application of biotechnology for biofuels: transforming biomass to biofuels

Cited by 12 publications

References 17 publications

Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

How does cellulosome composition influence deconstruction of lignocellulosic substrates in Clostridium (Ruminiclostridium) thermocellum DSM 1313?

Recombinant DNA modification of gibberellin metabolism alters growth rate and biomass allocation in Populus

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