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

Abstract

Cellulosome 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.

Figure 1

Scanning electron microscopy visualization of C. thermocellum cells at early, middle and late exponential phase using glucose, cellobiose or Avicel as the sole carbon source. At the early and middle exponential phase, resting polycellulosomal protuberances are formed on the cell surface with cellobiose or Avicel as the carbon source, and the surface of cells grown on glucose appears smooth but with fibrous structures indicating protuberances in a protracted state. No or few polycellulosomal structures are observed for the cells grown at the late exponential phase because of the detachment of cellulosome from the cell. A scale bar is shown at the bottom right.

Figure 2

Cellulolytic activities of cell‐attached, cellulose‐affinity‐purified and extracellular proteins of C thermocellum with glucose, cellobiose or Avicel as the sole carbon source.A. Cell‐associated hydrolysis assay. The cells at both middle and late exponential stages were used for analysis.B. Cellulose‐affinity‐purified/extracellular protein assay. The values are shown in Table S1. The cells at late exponential stage were used for analysis. The hydrolysis assay with 5 mg Avicel at 55°C for 24 h under oxic conditions, and the amount of reduced sugars was measured using the DNS method. Control reactions containing no Avicel or cells/proteins were prepared, and the amounts of reduced sugars produced by the control reactions were subtracted from the values for the experimental samples. Average values and standard errors indicated by error bars were calculated from three independent experiments

Figure 3

The cellulosomal titre (A), yield (B), estimated production capacity (C) and estimated specific activity (D) of the C. thermocellum cells grown on glucose (Glu), cellobiose (Cb) and Avicel (Av) as the carbon sources. The total amount of carbon source was 5 g l⁻¹. For inducing experiment, 0.5 g l⁻¹ Cb or Av was added in medium containing 4.5 g l⁻¹ Glu (Glu + Cb or Glu + Av respectively). The titre and yield were calculated based on the measured protein concentration and specific activity of extracellular proteins (Table S1). The estimated specific activity (eSA_{S caA}) and cellulosomal production capacity (CPC_{S caA}) were calculated based on the proportion of ScaA (pScaA) in the extracellular proteins (Table S1). Three independent analyses were performed for statistical calculation. P‐value was calculated using Student's t‐test with Glu as the reference. *P < 0.05, **P < 0.01

Figure 4

SDS‐PAGE analysis of extracellular (E) and cellulose‐affinity‐purified (C) proteins of C. thermocellum cultivated with glucose, cellobiose or Avicel as the sole carbon source. Protein bands (red arrows) with abundance changes among different samples were selected for mass spectroscopy identification as shown to the right of the Coomassie blue‐stained gel. Besides cellulosomal proteins (ScaA, Cel48S, Cel9K, Cel8A, Xyn10C), several non‐cellulosomal proteins were detected, including S‐layer (S‐layer homology protein, Clo1313_RS15300), CbpB (sugar ABC transporter substrate‐binding protein, Clo1313_RS06075), ADH (alcohol dehydrogenase, Clo1313_RS09240), CAO (copper amine oxidase‐like protein, Clo1313_RS11630), GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase, Clo1313_RS10615) and CbpA (sugar ABC transporter substrate‐binding protein Clo1313_RS09245). M, protein standards.

Figure 5

Scanning electron microscopy visualization (A) and hydrolysis activity (B) of C. thermocellum cells grown on glucose supplemented with 0.5 g l⁻¹ various carbon sources. The total amount of carbon source is 5 g l⁻¹.A. Polycellulosomal protuberances could be observed on the cell surface when cellobiose, cellotriose, cellotetraose, cellopentaose or Avicel was supplemented, while no protuberances were observed for xylan, pectin, arabinoxylan or xylose. Scale bar is shown at the bottom right.B. The extracellular hydrolysis activities of the C. thermocellum cells. The titre and yield values were calculated based on the measured protein concentration and specific activity of extracellular proteins (Table S1). P‐values were calculated to determine using Student's t‐test with glucose as the reference. *P < 0.05, **P < 0.01.