Putative Role of Cellulosomal Protease Inhibitors in Clostridium cellulovorans Based on Gene Expression and Measurement of Activities

Meguro, Hirokazu; Morisaka, Hironobu; Kuroda, Kouichi; Miyake, Hideo; Tamaru, Yutaka; Ueda, Mitsuyoshi

doi:10.1128/jb.05022-11

Cited by 14 publications

(17 citation statements)

References 22 publications

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“…The major scaffold protein, CbpA, is comprised of 9 cohesin domains that bind to various cellulosomal proteins via cohesin-dockerin interactions (9). Interestingly, even though most of the cellulosomal proteins were glycoside hydrolases (GHs), proteins such as proteases, protease inhibitors (36), and unknown proteins were also included. These proteins, seemingly not related to saccharification, may be important in the degradation of various resources.…”

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confidence: 99%

Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources

Matsui

Bae

Esaka

et al. 2013

Appl Environ Microbiol

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The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.

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confidence: 99%

Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources

Matsui

Bae

Esaka

et al. 2013

Appl Environ Microbiol

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“…The existence of cellulosomal protease inhibitors raises questions as to how the cellulosome is maintained or modified under diverse environmental conditions (Schwarz and Zverlov, ). Similar inhibitors from C. thermocellum and C. cellulovorans exhibited inhibitory activities against bacterial and plant proteases (Kang et al ., ; Meguro et al ., ). In the present study, C. cellulolyticum Dpi was shown to be an effective inhibitor of cysteine protease.…”

Section: Discussionmentioning

confidence: 97%

Dockerin‐containing protease inhibitor protects key cellulosomal cellulases from proteolysis in Clostridium cellulolyticum

et al. 2014

Molecular Microbiology

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SummaryCellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin-containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome-localized protease inhibitors in Clostridia.

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“…As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-related clostridia, mesophilic C. acetobutylicum and C. cellulolyticum, and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome ( Table 2) and coded for not only CAZymes but also lipases, peptidases, and proteinase inhibitors [40,41]. Cellulosomal genes among clostridial genomes were identified and classified as cohesincontaining scaffolding proteins and dockerin-containing proteins.…”

Section: Lignocellulosic Biomass Utilization Toward Biorefinery Usingmentioning

confidence: 99%

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

Tamaru¹,

López-Contreras²

2013

Cellulose - Biomass Conversion

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Putative Role of Cellulosomal Protease Inhibitors in Clostridium cellulovorans Based on Gene Expression and Measurement of Activities

Cited by 14 publications

References 22 publications

Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources

Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources

Dockerin‐containing protease inhibitor protects key cellulosomal cellulases from proteolysis in Clostridium cellulolyticum

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

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