Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult...Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.展开更多
Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold.Through templating key protein domains(cohesin and dockerin...Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold.Through templating key protein domains(cohesin and dockerin),designer cellulosomes have been engineered from bacterial motifs to alter the activity,stability,and degradation efficiency of enzyme complexes.Recently a parts list for fungal cellulosomes from the anaerobic fungi(Neocallimastigomycota)was determined,which revealed sequence divergent fungal cohesin,dockerin,and scaffoldin domains that could be used to expand the available toolbox to synthesize designer cellulosomes.In this work,multi-domain carbohydrate active enzymes(CAZymes)from 3 cellulosome-producing fungi were analyzed to inform the design of chimeric proteins for synthetic cellulosomes inspired by anaerobic fungi.In particular,Piromyces finnis was used as a structural template for chimeric carbohydrate active enzymes.Recombinant enzymes with retained properties were engineered by combining thermophilic glycosyl hydrolase domains from Thermotoga maritima with dockerin domains from Piromyces finnis.By preserving the protein domain order from P.finnis,chimeric enzymes retained catalytic activity at temperatures over 80°C and were able to associate with cellulosomes purified from anaerobic fungi.Fungal cellulosomes harbor a wide diversity of glycoside hydrolases,each representing templates for the design of chimeric enzymes.By conserving dockerin domain position within the primary structure of each protein,the activity of both the catalytic domain and dockerin domain was retained in enzyme chimeras.Taken further,the domain positioning inferred from native fungal cellulosome proteins can be used to engineer multi-domain proteins with non-native favorable properties,such as thermostability.展开更多
文摘Production of economically viable bioethanol is potentially an environmentally and financially worthwhile endeavor.One major source for fermentable sugars is lignocellulose.However,lignocellulosic biomass is difficult to degrade,owing to its inherent structural recalcitrance.Cellulosomes are complexes of cellulases and associated polysaccharide-degrading enzymes bound to a protein scaffold that can efficiently degrade lignocellulose.Integration of the enzyme subunits into the complex depends on intermodular cohesin-dockerin interactions,which are robust but nonetheless non-covalent.The modular architecture of these complexes can be used to assemble artificial designer cellulosomes for advanced nanotechnological applications.Pretreatments that promote lignocellulose degradation involve high temperatures and acidic or alkaline conditions that could dismember designer cellulosomes,thus requiring separation of reaction steps,thereby increasing overall process cost.To overcome these challenges,we developed a means of covalently locking cohesin-dockerin interactions by integrating the chemistry of SpyCatcher-SpyTag approach to target and secure the interaction.The resultant cohesin-conjugated dockerin complex was resistant to high temperatures,SDS,and urea while high affinity and specificity of the interacting modular components were maintained.Using this approach,a covalently locked,bivalent designer cellulosome complex was produced and demonstrated to be enzymatically active on cellulosic substrates.The combination of affinity systems with SpyCatcher-SpyTag chemistry may prove of general use for improving other types of protein ligation systems and creating unconventional,biologically active,covalently locked,affinity-based molecular architectures.
基金The authors are grateful to funding sources from the Office of Science(BER),U.S.Department of Energy(DE-SC0010352)the National Science Foundation(MCB-1553721)+1 种基金the Institute for Collaborative Biotechnologies through grants W911NF-09-0001 and W911NF-19-D-0001 from the U.S.Army Research Officeand the Camille Dreyfus Teacher-Scholar Awards Program.All authors acknowledge support from the California NanoSystems Institute(CNSI)Challenge Grant Program,supported by the University of California,Santa Barbara and the University of California,Office of the President.SPG also acknowledges support from the National Science Foundation Graduate Research Fellowship Program under Grant DGE 1144085.We thank Prof.Robert Kelly for providing us with T.maritima DNA.The authors acknowledge the use of the Biological Nanostructures Laboratory within the California NanoSystems Institute,supported by the University of California,Santa Barbara and the University of California,Office of the President.
文摘Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold.Through templating key protein domains(cohesin and dockerin),designer cellulosomes have been engineered from bacterial motifs to alter the activity,stability,and degradation efficiency of enzyme complexes.Recently a parts list for fungal cellulosomes from the anaerobic fungi(Neocallimastigomycota)was determined,which revealed sequence divergent fungal cohesin,dockerin,and scaffoldin domains that could be used to expand the available toolbox to synthesize designer cellulosomes.In this work,multi-domain carbohydrate active enzymes(CAZymes)from 3 cellulosome-producing fungi were analyzed to inform the design of chimeric proteins for synthetic cellulosomes inspired by anaerobic fungi.In particular,Piromyces finnis was used as a structural template for chimeric carbohydrate active enzymes.Recombinant enzymes with retained properties were engineered by combining thermophilic glycosyl hydrolase domains from Thermotoga maritima with dockerin domains from Piromyces finnis.By preserving the protein domain order from P.finnis,chimeric enzymes retained catalytic activity at temperatures over 80°C and were able to associate with cellulosomes purified from anaerobic fungi.Fungal cellulosomes harbor a wide diversity of glycoside hydrolases,each representing templates for the design of chimeric enzymes.By conserving dockerin domain position within the primary structure of each protein,the activity of both the catalytic domain and dockerin domain was retained in enzyme chimeras.Taken further,the domain positioning inferred from native fungal cellulosome proteins can be used to engineer multi-domain proteins with non-native favorable properties,such as thermostability.
