Enhanced cellulose degradation by targeted integration of a cohesin-fused  -glucosidase into the Clostridium thermocellum cellulosome

ABSTRACT Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble β-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

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Draft genome sequence data of Clostridium thermocellum PAL5 possessing high cellulose-degradation ability

Data in Brief ◽

10.1016/j.dib.2019.104274 ◽

2019 ◽

Vol 25 ◽

pp. 104274 ◽

Cited By ~ 2

Author(s):

Eiko Nakazono-Nagaoka ◽

Takashi Fujikawa ◽

Ayumi Shikata ◽

Chakrit Tachaapaikoon ◽

Rattiya Waeonukul ◽

...

Keyword(s):

Genome Sequence ◽

Clostridium Thermocellum ◽

Sequence Data ◽

Cellulose Degradation ◽

Draft Genome ◽

Draft Genome Sequence ◽

Genome Sequence Data ◽

Degradation Ability ◽

High Cellulose

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fSpatial and temporal dynamics of cellulose degradation and biofilm formation by Caldicellulosiruptor obsidiansis and Clostridium thermocellum

AMB Express ◽

10.1186/2191-0855-1-30 ◽

2011 ◽

Vol 1 (1) ◽

pp. 30 ◽

Cited By ~ 24

Author(s):

Zhi-Wu Wang ◽

Seung-Hwan Lee ◽

James G Elkins ◽

Jennifer L Morrell-Falvey

Keyword(s):

Biofilm Formation ◽

Clostridium Thermocellum ◽

Temporal Dynamics ◽

Cellulose Degradation

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Hydrolysis of Cellulose by Soluble Clostridium Thermocellum and Acidothermus Cellulolyticus Cellulases

Journal of Enzymes ◽

10.14302/issn.2690-4829.jen-18-2025 ◽

2018 ◽

Vol 1 (1) ◽

pp. 5-19

Author(s):

Phillip Brumm ◽

Dan Xie ◽

Larry Allen ◽

...

Keyword(s):

Filter Paper ◽

Clostridium Thermocellum ◽

Cellulose Degradation ◽

Cellulase Activity ◽

Cellulose Hydrolysis ◽

Reducing Sugar ◽

Crystalline Cellulose ◽

Amorphous Cellulose ◽

Highly Active ◽

The Individual

The goal of this work was to clone, express, characterize and assemble a set of soluble thermostablecellulases capable of significantly degrading cellulose. We successfully cloned, expressed, and purified eleven Clostridium thermocellum (Cthe) cellulases and eight Acidothermuscellulolyticus(Acel) cellulases. The performance of the nineteen enzymes was evaluated on crystalline (filter paper) and amorphous (PASC) cellulose. Hydrolysis products generated from these two substrates were converted to glucose using beta-glucosidase and the glucose formed was determined enzymatically. Ten of the eleven Cthe enzymes were highly active on amorphous cellulose. The individual enzymes all produced <10% reducing sugar equivalents from filter paper. Combinations of Cthe cellulases gave higher conversions, with the combination of CelE, CelI, CelG, and CelK converting 34% of the crystalline cellulose. All eight Acel cellulases showed endo-cellulase activity and were highly active on PASC. Only Acel_0615 produced more than 10% reducing sugar equivalents from filter paper, and a combination of six Acel cellulases produced 32% conversion. Acel_0617, a GH48 exo-cellulase, and Acel_0619, a GH12 endo-cellulase, synergistically stimulated cellulose degradation by the combination of Cthe cellulases to almost 80%. Addition of both Acel enzymes to the Cthe enzyme mix did not further stimulate hydrolysis. Cthe CelG and CelI stimulated cellulose degradation by the combination of Acel cellulases to 66%.

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Exemplar Abstract for Clostridium thermocellum Viljoen et al. 1926 (Approved Lists 1980).

The NamesforLife Abstracts ◽

10.1601/ex.14973 ◽

2003 ◽

Author(s):

Charles Thomas Parker ◽

Dorothea Taylor ◽

George M Garrity

Keyword(s):

Clostridium Thermocellum

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Revalorizing Lignocellulose for the Production of Natural Pharmaceuticals and Other High Value Bioproducts

Current Medicinal Chemistry ◽

10.2174/0929867324666170912095755 ◽

2019 ◽

Vol 26 (14) ◽

pp. 2475-2484 ◽

Cited By ~ 4

Author(s):

Congqiang Zhang ◽

Heng-Phon Too

Keyword(s):

Clostridium Thermocellum ◽

White Rot Fungi ◽

Brown Rot ◽

Cost Effective ◽

White Rot ◽

Chemical Pretreatment ◽

Significant Challenge ◽

Drug Molecules ◽

Natural Medicine ◽

Renewable Natural Resource

Lignocellulose is the most abundant renewable natural resource on earth and has been successfully used for the production of biofuels. A significant challenge is to develop cost-effective, environmentally friendly and efficient processes for the conversion of lignocellulose materials into suitable substrates for biotransformation. A number of approaches have been explored to convert lignocellulose into sugars, e.g. combining chemical pretreatment and enzymatic hydrolysis. In nature, there are organisms that can transform the complex lignocellulose efficiently, such as wood-degrading fungi (brown rot and white rot fungi), bacteria (e.g. Clostridium thermocellum), arthropods (e.g. termite) and certain animals (e.g. ruminant). Here, we highlight recent case studies of the natural degraders and the mechanisms involved, providing new utilities in biotechnology. The sugars produced from such biotransformations can be used in metabolic engineering and synthetic biology for the complete biosynthesis of natural medicine. The unique opportunities in using lignocellulose directly to produce natural drug molecules with either using mushroom and/or ‘industrial workhorse’ organisms (Escherichia coli and Saccharomyces cerevisiae) will be discussed.

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