Inducing effects of cellulosic hydrolysate components of lignocellulose on cellulosome synthesis in Clostridium thermocellum

Abstract

SummaryCellulosome is a highly efficient supramolecular machine for lignocellulose degradation, and its substrate‐coupled regulation requires soluble transmembrane signals. However, the inducers for cellulosome synthesis and the inducing effect have not been clarified quantitatively. Values of cellulosome production capacity (CPC) and estimated specific activity (eSA) were calculated based on the primary scaffoldin ScaA to define the stimulating effects on the cellulosome synthesis in terms of quantity and quality respectively. The estimated cellulosome production of Clostridium thermocellum on glucose was at a low housekeeping level. Both Avicel and cellobiose increased CPCs of the cells instead of the eSAs of the cellulosome. The CPC of Avicel‐grown cells was over 20‐fold of that of glucose‐grown cells, while both Avicel‐ and glucose‐derived cellulosomes showed similar eSA. The CPC of cellobiose‐grown cells was also over three times higher than glucose‐grown cells, but the eSA of cellobiose‐derived cellulosome was 16% lower than that of the glucose‐derived cellulosome. Our results indicated that cello‐oligosaccharides played the key roles in inducing the synthesis of the cellulosome, but non‐cellulosic polysaccharides showed no inducing effects.

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

“…Also, this large amount of extracellular protein does not reflect lysis of cells. In C. thermocellum , the synthesis of the cellulosomes attached to the outside of cells is also induced by cellobiose ( 43 ). They can detach from cells, and “the cell-free cellulosome complex can be seen as a long-range cellulosome because it can diffuse away from the cell and degrade polysaccharide substrates remotely from the bacterial cell” ( 44 ).…”

Thermophilic whole‐cell degradation of polyethylene terephthalate using engineered Clostridium thermocellum

“…To analyse the secretory expression of LCC, SDS polyacrylamide gel electrophoresis (SDS‐PAGE) was carried out by loading 10 µg cell lysate or supernatant protein in each lane followed by an esterolytic activity staining using the Fast‐Red TR salt (Meilunbio, Guangzhou, China) as a dye and 1‐naphthyl acetate (Solarbio, Beijing, China) as a substrate as described previously (Billig, et al , 2010). For selected samples, the intensity of protein bands with hydrolytic activities was calculated using the Quantity One software (version 4.6.2; Bio‐Rad, Shanghai, China) as previously described (Li, et al , 2018). For comparison, polyacrylamide gels loaded by the same supernatant samples were partly also stained with Coomassie Brilliant Blue.…”

Construction of consolidated bio-saccharification biocatalyst and process optimization for highly efficient lignocellulose solubilization

“…1). The intensities of the bands referring to the primary scaffoldin ScaA, Cel48S, Cel9K and the fusion protein Cel9K–BGL were determined using the Quantity One software based on the peak intensity analysis, and the relative protein expression levels were determined by dividing their band intensities with that of the ScaA protein according to a previously described ScaA-based estimation method [33]. The Cel48S protein was with comparative expression level in Δ pyrF and ∆ pyrF ::KBm, indicating that the expression of Cel48S was not influenced in ∆ pyrF ::KBm.…”

“…An additional 150-kDa band was detected referring to the fusion protein Cel9K–BGL (red arrow). The intensities of the bands referring to Cel48S, Cel9K, and Cel9K–BGL were determined using the Quantity One software and were divided by that of the corresponding ScaA protein as previously described [33]. The obtained ratios shown below the lanes stand for the expression levels of the proteins.…”

“…The protein concentrations were determined using the Bradford method [44] immediately before further analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check protein purity and composition as previously described using protein standards ranging from 10 to 245 kDa (New England BioLabs) [33].…”