Designing chimeric enzymes inspired by fungal cellulosomes

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...

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Main Authors: Sean P. Gilmore, Stephen P. Lillington, Charles H. Haitjema, Randall de Groot, Michelle A. O'Malley
Format: Article
Language:English
Published: KeAi Communications Co., Ltd. 2020-03-01
Series:Synthetic and Systems Biotechnology
Online Access:http://www.sciencedirect.com/science/article/pii/S2405805X2030003X
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spelling doaj-7a6faace2ac945d1a68098c2cb97b0bf2021-04-02T10:36:56ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2020-03-01512332Designing chimeric enzymes inspired by fungal cellulosomesSean P. Gilmore0Stephen P. Lillington1Charles H. Haitjema2Randall de Groot3Michelle A. O'Malley4Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA, 93106, United StatesDepartment of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA, 93106, United StatesDepartment of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA, 93106, United StatesDepartment of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA, 93106, United StatesCorresponding author. MC 5080, University of California, Santa Barbara, CA, 93106-5080, United States.; Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA, 93106, United StatesCellulosomes 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. Keywords: Cellulosome, Dockerin, Scaffoldin, Anaerobic fungi, Thermophile, Enzymehttp://www.sciencedirect.com/science/article/pii/S2405805X2030003X
collection DOAJ
language English
format Article
sources DOAJ
author Sean P. Gilmore
Stephen P. Lillington
Charles H. Haitjema
Randall de Groot
Michelle A. O'Malley
spellingShingle Sean P. Gilmore
Stephen P. Lillington
Charles H. Haitjema
Randall de Groot
Michelle A. O'Malley
Designing chimeric enzymes inspired by fungal cellulosomes
Synthetic and Systems Biotechnology
author_facet Sean P. Gilmore
Stephen P. Lillington
Charles H. Haitjema
Randall de Groot
Michelle A. O'Malley
author_sort Sean P. Gilmore
title Designing chimeric enzymes inspired by fungal cellulosomes
title_short Designing chimeric enzymes inspired by fungal cellulosomes
title_full Designing chimeric enzymes inspired by fungal cellulosomes
title_fullStr Designing chimeric enzymes inspired by fungal cellulosomes
title_full_unstemmed Designing chimeric enzymes inspired by fungal cellulosomes
title_sort designing chimeric enzymes inspired by fungal cellulosomes
publisher KeAi Communications Co., Ltd.
series Synthetic and Systems Biotechnology
issn 2405-805X
publishDate 2020-03-01
description 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. Keywords: Cellulosome, Dockerin, Scaffoldin, Anaerobic fungi, Thermophile, Enzyme
url http://www.sciencedirect.com/science/article/pii/S2405805X2030003X
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