synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates
BACKGROUND: Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaf...
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Veröffentlicht in: | Biotechnology for biofuels 2013-12, Vol.6 (1), p.182-182, Article 182 |
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description | BACKGROUND: Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. RESULTS: Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. CONCLUSIONS: The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning |
doi_str_mv | 10.1186/1754-6834-6-182 |
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The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. RESULTS: Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. CONCLUSIONS: The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.</description><identifier>ISSN: 1754-6834</identifier><identifier>EISSN: 1754-6834</identifier><identifier>DOI: 10.1186/1754-6834-6-182</identifier><identifier>PMID: 24341331</identifier><language>eng</language><publisher>England: Springer-Verlag</publisher><subject>amino acids ; Bacteria ; Bacteriology ; Biomass energy ; carbohydrate binding ; Carbohydrates ; cell walls ; Cellulase ; cellulases ; Cellulose ; Cellulose fibers ; cellulosome ; Design ; Enzymes ; ethanol production ; Health aspects ; Hydrolysis ; Pharmaceutical industry ; Physiological aspects ; polypeptides ; Scaffolding ; Science ; Synthetic biology ; Triticum aestivum ; wheat straw</subject><ispartof>Biotechnology for biofuels, 2013-12, Vol.6 (1), p.182-182, Article 182</ispartof><rights>COPYRIGHT 2013 BioMed Central Ltd.</rights><rights>2013 Vazana et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.</rights><rights>Copyright © 2013 Vazana et al.; licensee BioMed Central Ltd. 2013 Vazana et al.; licensee BioMed Central Ltd.</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-b676t-6129a9ba5657aa92b1baee372198aaa8b5bf52975c1dc48db0bb8f5dea743f3d3</citedby><cites>FETCH-LOGICAL-b676t-6129a9ba5657aa92b1baee372198aaa8b5bf52975c1dc48db0bb8f5dea743f3d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878649/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878649/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24341331$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vazana, Yael</creatorcontrib><creatorcontrib>Barak, Yoav</creatorcontrib><creatorcontrib>Unger, Tamar</creatorcontrib><creatorcontrib>Peleg, Yoav</creatorcontrib><creatorcontrib>Shamshoum, Melina</creatorcontrib><creatorcontrib>Ben-Yehezkel, Tuval</creatorcontrib><creatorcontrib>Mazor, Yair</creatorcontrib><creatorcontrib>Shapiro, Ehud</creatorcontrib><creatorcontrib>Lamed, Raphael</creatorcontrib><creatorcontrib>Bayer, Edward A</creatorcontrib><title>synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates</title><title>Biotechnology for biofuels</title><addtitle>Biotechnol Biofuels</addtitle><description>BACKGROUND: Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. RESULTS: Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. CONCLUSIONS: The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.</description><subject>amino acids</subject><subject>Bacteria</subject><subject>Bacteriology</subject><subject>Biomass energy</subject><subject>carbohydrate binding</subject><subject>Carbohydrates</subject><subject>cell walls</subject><subject>Cellulase</subject><subject>cellulases</subject><subject>Cellulose</subject><subject>Cellulose fibers</subject><subject>cellulosome</subject><subject>Design</subject><subject>Enzymes</subject><subject>ethanol production</subject><subject>Health aspects</subject><subject>Hydrolysis</subject><subject>Pharmaceutical industry</subject><subject>Physiological aspects</subject><subject>polypeptides</subject><subject>Scaffolding</subject><subject>Science</subject><subject>Synthetic biology</subject><subject>Triticum aestivum</subject><subject>wheat 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biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates</title><author>Vazana, Yael ; Barak, Yoav ; Unger, Tamar ; Peleg, Yoav ; Shamshoum, Melina ; Ben-Yehezkel, Tuval ; Mazor, Yair ; Shapiro, Ehud ; Lamed, Raphael ; Bayer, Edward A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-b676t-6129a9ba5657aa92b1baee372198aaa8b5bf52975c1dc48db0bb8f5dea743f3d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>amino acids</topic><topic>Bacteria</topic><topic>Bacteriology</topic><topic>Biomass energy</topic><topic>carbohydrate binding</topic><topic>Carbohydrates</topic><topic>cell walls</topic><topic>Cellulase</topic><topic>cellulases</topic><topic>Cellulose</topic><topic>Cellulose fibers</topic><topic>cellulosome</topic><topic>Design</topic><topic>Enzymes</topic><topic>ethanol production</topic><topic>Health aspects</topic><topic>Hydrolysis</topic><topic>Pharmaceutical industry</topic><topic>Physiological aspects</topic><topic>polypeptides</topic><topic>Scaffolding</topic><topic>Science</topic><topic>Synthetic biology</topic><topic>Triticum aestivum</topic><topic>wheat straw</topic><toplevel>online_resources</toplevel><creatorcontrib>Vazana, Yael</creatorcontrib><creatorcontrib>Barak, Yoav</creatorcontrib><creatorcontrib>Unger, Tamar</creatorcontrib><creatorcontrib>Peleg, Yoav</creatorcontrib><creatorcontrib>Shamshoum, Melina</creatorcontrib><creatorcontrib>Ben-Yehezkel, Tuval</creatorcontrib><creatorcontrib>Mazor, Yair</creatorcontrib><creatorcontrib>Shapiro, Ehud</creatorcontrib><creatorcontrib>Lamed, Raphael</creatorcontrib><creatorcontrib>Bayer, Edward A</creatorcontrib><collection>AGRIS</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central 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Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vazana, Yael</au><au>Barak, Yoav</au><au>Unger, Tamar</au><au>Peleg, Yoav</au><au>Shamshoum, Melina</au><au>Ben-Yehezkel, Tuval</au><au>Mazor, Yair</au><au>Shapiro, Ehud</au><au>Lamed, Raphael</au><au>Bayer, Edward A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates</atitle><jtitle>Biotechnology for biofuels</jtitle><addtitle>Biotechnol Biofuels</addtitle><date>2013-12-16</date><risdate>2013</risdate><volume>6</volume><issue>1</issue><spage>182</spage><epage>182</epage><pages>182-182</pages><artnum>182</artnum><issn>1754-6834</issn><eissn>1754-6834</eissn><abstract>BACKGROUND: Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. RESULTS: Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. CONCLUSIONS: The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.</abstract><cop>England</cop><pub>Springer-Verlag</pub><pmid>24341331</pmid><doi>10.1186/1754-6834-6-182</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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subjects | amino acids Bacteria Bacteriology Biomass energy carbohydrate binding Carbohydrates cell walls Cellulase cellulases Cellulose Cellulose fibers cellulosome Design Enzymes ethanol production Health aspects Hydrolysis Pharmaceutical industry Physiological aspects polypeptides Scaffolding Science Synthetic biology Triticum aestivum wheat straw |
title | synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates |
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