Functional analysis of the degradation of cellulosic substrates by a Chaetomium globosum endophytic isolate

Abstract

Most photosynthetically fixed carbon is contained in cell wall polymers present in plant biomasses, the largest organic carbon source in the biosphere. The degradation of these polymers for biotechnological purposes requires the combined action of several enzymes. To identify new activities, we examined which enzymes are activated by an endophytic strain of Chaetomium globosum to degrade cellulose-containing substrates. After biochemical analyses of the secretome of the fungus grown on cellulose or woody substrates, we took advantage of the available genomic data to identify potentially involved genes. After in silico identification of putative genes encoding either proteins able to bind to cellulose or glycohydrolases (GHs) of family 7, we investigated their transcript levels by reverse transcription-quantitative PCR (RT-qPCR). Our data suggest that eight genes compose the core of the cellulose-degrading system of C. globosum. Notably, the related enzymes belong structurally to the well-described GH families 5, 6, 7, 16, and 45, which are known to be the core of the cellulose degradation systems of several ascomycetes. The high expression levels of cellobiose dehydrogenase and two GH 61 enzymes suggest the involvement of this oxidoreductive synergic system in C. globosum. Transcript analysis along with relevant coding sequence (CDS) isolation and expression of recombinant proteins proved to be a key strategy for the determination of the features of two endoglucanases used by C. globosum for the first attack of crystalline cellulose. Finally, the possible involvement of transcriptional regulators described for other ascomycetes is discussed.

Fig 1

Growth of C. globosum in terms of estimated biomass production (A) and activities measured in fungal media collected from cultures grown on glucose (B), MPW (C), and MCC (D). Glycohydrolytic activity on different substrates (CMC, MCC, ASC, and Xyl), as well as CDH activity, was measured. ASC, acid-swollen cellulose; CMC, carboxymethyl cellulose; GH, glycohydrolases; Glu, glucose; MCC, microcrystalline cellulose; MPW, milled poplar wood; TSP, total soluble proteins; Xyl, xylan.

Fig 2

Expression levels of analyzed genes compared to histone H4 (expression level = 1), measured by RT-qPCR using total RNA. Black bars report the expression levels under glucose growth conditions, and white bars report the expression levels of the fungus grown on MPW. *, genes induced >10-fold under MPW growth conditions compared to glucose growth conditions. Data are from at least two independent experiments, and error bars show standard deviations. EG, endoglucanases; GH, glycohydrolases; MPW, milled poplar wood.

Fig 3

Expression levels in C. globosum grown on MCC with respect to those on MPW, measured by RT-qPCR. Data are from at least two independent experiments, and error bars show standard deviations. *, non-GH enzyme. EG, endoglucanases; GH, glycohydrolases; MCC, microcrystalline cellulose; MPW, milled poplar wood; UD, undetectable.

Fig 4

Electrophoretic analyses of recombinant GH 5 (CHGG_01188) and GH 45 (CHGG_08509) proteins. Expression in E. coli was induced with 1 mM IPTG for 4 h at different temperatures. C, polyacrylamide gel stained with Coomassie blue; WB, immunoblot detection of His-tagged proteins.

Fig 5

Specific activities of GH 5 and GH 45 enzymes in E. coli raw extracts toward cellulose- and hemicellulose-containing substrates after 16 h of incubation at 40°C. All substrates were present at 1% in 50 mM acetate buffer, pH 5.5. “MCC+” and “MCC++” indicate MCC plus 1 and 2 μl of BG (NS22118; Novozymes), respectively, added according to the manufacturer's instructions. Data are from at least three independent experiments, and error bars show standard deviations. ASC, acid-swollen cellulose; BG, β-glucosidases; CMC, carboxymethyl cellulose; MCC, microcrystalline cellulose; TSP, total soluble proteins; Xyl, xylan.

Fig 6

Schematic map of promoter regions with putative cis regulatory elements. The positions of putative binding sites for ACEI (black) and Hap2/3/5 (white) and of nonoverlapping neighbor consensus sequences for CreA (gray) identified by the SCOPE motif finder are indicated. EG, endoglucanases; GH, glycohydrolases.

Fig 7

Venn diagram showing the distribution of differentially expressed genes of C. globosum in response to various carbon sources. MCC, microcrystalline cellulose; MPW, milled poplar wood.