Title: Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins ($$ T\overline{a}pirins\ $$) from Extremely Thermophilic Caldicellulosiruptor Species

Abstract

Genomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, called $$ t\overline{a}pirins\ $$, located proximate to the type IV pilus locus. The C-terminal domain of Caldicellulosiruptor kronotskyensis $$ t\overline{a}pirin\ $$ 0844 (Calkro_0844) is structurally unique and has a cellulose binding affinity akin to that seen with family 3 carbohydrate binding modules (CBM3s). Here, full-length and C-terminal versions of $$ t\overline{a}pirins\ $$ from Caldicellulosiruptor bescii (Athe_1870), Caldicellulosiruptor hydrothermalis(Calhy_0908), Caldicellulosiruptor kristjanssonii (Calkr_0826), and Caldicellulosiruptor naganoensis (NA10_0869) were produced recombinantly in Escherichia coli and compared to Calkro_0844. All five $$ t\overline{a}pirins\ $$ bound to microcrystalline cellulose, switchgrass, poplar, and filter paper but not to xylan. Densitometry analysis of bound protein fractions visualized by SDS-PAGE revealed that Calhy_0908 and Calkr_0826 (from weakly cellulolytic species) associated with the cellulose substrates to a greater extent than Athe_1870, Calkro_0844, and NA10_0869 (from strongly cellulolytic species). Perhaps this relates to their specific needs to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Calkro_0844 and NA10_0869 share a higher degree of amino acid sequence identity (>80% identity) with each other than either does with Athe_1870 (~50%). The levels of amino acid sequence identity of Calhy_0908 and Calkr_0826 to Calkro_0844 were only 16% and 36%, respectively, although the three-dimensional structures of their C-terminal binding regions were closely related. Unlike the parent strain, C. bescii mutants lacking the $$ t\overline{a}pirin\ $$ genes did not bind to cellulose following short-term incubation, suggesting a role in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, $$ t\overline{a}pirins\ $$ likely help scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species.The mechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed. Caldicellulosiruptor species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called $$ t\overline{a}pirins\ $$, are involved in the way that Caldicellulosiruptor species interact with microcrystalline cellulose, and additional information about the diversity of these proteins across the genus, including binding affinity and three-dimensional structural comparisons, is provided here.

Authors:

Lee, Laura L. ^[1]; Hart, William S. ^[1]; Lunin, Vladimir V. ^[2]; Alahuhta, Markus ^[2]; Bomble, Yannick J. ^[2]; Himmel, Michael E. ^[2]; Blumer-Schuette, Sara E. ^[1]; Adams, Michael W. W. ^[3]; Kelly, Robert M. ^[1]

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+ Show Author Affiliations

1. North Carolina State Univ., Raleigh, NC (United States)
2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
3. Univ. of Georgia, Athens, GA (United States)

Publication Date:

2019-01-23

Research Org.:

National Renewable Energy Lab. (NREL), Golden, CO (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1494728

Report Number(s):

NREL/JA-2700-72846
Journal ID: ISSN 0099-2240

Grant/Contract Number:  

AC36-08GO28308

Resource Type:

Accepted Manuscript

Journal Name:

Applied and Environmental Microbiology

Additional Journal Information:

Journal Volume: 85; Journal Issue: 3; Journal ID: ISSN 0099-2240

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

09 BIOMASS FUELS; Caldicellulosiruptor; tapirins; lignocellulose; glycoside hydrolase; cellulase