Chaetomella raphigera β-glucosidase D2-BGL has intriguing structural features and a high substrate affinity that renders it an efficient cellulase supplement for lignocellulosic biomass hydrolysis

Background To produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. β-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale. Results We heterologously expressed the fungal β-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial β-glucosidase Novozyme 188 (N188; Km = 0.2 vs 2.14 mM for p-nitrophenyl β-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus β-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose. Conclusions D2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 β-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.

First Report of Chaetomella raphigera Causing Leaf Spot on Rosa chinensis in China

China rose, Rosa chinensis Jacq., is extensively cultivated as an ornamental plant in China (1). During the course of a disease survey of China rose in Henan Province, a leaf spot was observed on about 20 China roses, cultivated in a garden in Zhengzhou, Henan Province, in early October 2012. The early symptom appeared as small round, pale brown lesions on the leaves. Lesions expanded into 5 to 15-mm-diameter spots that were near round or irregular and brown. Both sporodochial and pycnidial conidiomata developed in necrotic areas of diseased leaves when placed in moist chambers. Pycnidia were elongated, reniform, with a single raphe over the top, pale to dark brown, and 260 to 350 × 150 to 210 μm. Sporodochia were pale luteous and 100 to 280 × 80 to 180 μm. Setae, conidiophores, conidiogenous cells, and conidia were the same between two types of conidioma. Setae were pale to dark brown, 0 to 2 septate, straight with rounded end, clavate to curved at apex, and 22 to 60 × 2 to 5 μm. Conidiophores were up to 120 × 1 to 2 μm, filiform, cylindric, and branched. Conidiogenous cells were enteroblastic, collar and channel minute. Conidia were nonseptate, hyaline, ellipsoid or cymbiform, smooth, guttulate, and 4 to 6.5 × 1.5 to 2.5 μm. Two pure cultures (zm12276-1 and zm12276-2) were obtained by picking spores from independent conidiomata on one leaf and then subsequently grown on potato dextrose agar (PDA), producing the same two kinds of conidiomata. The characteristics of conidial size and distinctly different conidiomata with setae are diagnostic of Chaetomella raphigera M.E. Swift (3,4). The identity of our fungus (zm12276-1) was confirmed to be C. raphigera by DNA sequencing of the ITS1-5.8S-ITS2 region. The DNA sequence was 99% identical to those of the other C. raphigera isolates (AY487076 and AY487085) (2). The ITS sequence from zm12276-1 was deposited in GenBank (KF483474). Pathogenicity was tested by inoculating 10 leaves of R. chinensis with mycelia plug from colony of zm12276-1 (0.5 cm in diameter). An equal number of fresh leaves inoculated with the plugs of non-colonized PDA medium served as the control. All leaves were incubated in clear plastic box with a dish of sterile distilled water at 25°C under ambient light. After 7 days, 90% of the inoculated leaves showed symptoms identical to those observed on R. chinensis leaves affected in the field. From each of the symptomatic leaves, C. raphigera was recovered, whereas controls remained symptom-free and no fungus was isolated from the control leaves. Koch's postulates were repeated three times with the same results using the pure culture of zm12276-1. C. raphigera has been previously reported on Rosa sp. in the United States (4). To our knowledge, this is the first report of C. raphigera infecting R. chinensis in China. The disease cycle and the control strategies in the regions are being further studied. References: (1) C. Z. Gu and K. R. Robertson. Pages 339-381 in: Flora of China, vol. 9. Science Press, Beijing and Missouri Botanical Garden, 2003. (2) A. Y. Rossman et al. Mycol. Progr. 3:275, 2004. (3) B. C. Sutton. The Coelomycetes. CAB International Publishing, New York, 1980. (4) M. E. Swift. Mycologia 22:165, 1930.

Phylloplane mycoflora of Gerbera spp. and their Pathogenic potentiality

Two types of symptoms, blight and anthracnose, were recorded on two species of Gerbera viz. Gerbera aurantiaca L. and Gerbera viridifolia L. The most common fungal species isolated from Gerbera spp. were Alternaria citrii, A. tennuisssima, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Bipolaris hawaiiensis, Chaetomella raphigera, Cladosporium cladosporoides, Colletotrichum capsici, C. coffeanum, C. dematium, C. gleosporioides, C. lindemuthianum, C. musae, C. orbiculare, Colletorichum sp.1, Colletotrichum sp.2, Colletotrichum sp.3, Colletotrichum sp.4, Curvularia clavata, C. lunata, Fusarium nivale, F. semitectum, Phomopsis sp., Penicillium sp., Pestalotia sp., Rhizopus stolonifer, Trichoderma viride, one cleistothecial ascomycetes and one sterile fungus. The frequency percentage of association of Colletotrichum spp. with Gerbera spp. was higher than any other fungi. Bipolaris hawaiiensis is a new record for Bangladesh. Among the isolated fungi Alternaria citrii, A. tennuissima, Colletotrichum capcisi, C. dematium, C. coffeanum and Curvularia clavata were found to be pathogenic to Gerbera plant. DOI: http://dx.doi.org/10.3329/jbas.v37i2.17562 Journal of Bangladesh Academy of Sciences, Vol. 37, No. 2, 211-217, 2013

Pestalozziella subsessilis. [Descriptions of Fungi and Bacteria].

A description is provided for Pestalozziella subsessilis, a colonizer of living leaves, causing leaf spot symptoms leading to leaves fading and dying. Some information on its dispersal and transmission, economic impacts, infraspecific variation and conservation status is given, along with details of its geographical distribution (USA (Florida, Kansas, Louisiana, Mississippi, Missouri, New Jersey, Wisconsin), Kazakhstan, Russia, New Zealand, Austria, Bulgaria, Czech Republic, Germany, Hungary, Italy, Poland, Ukraine, and United Kingdom), hosts (Geranium carolinianum (leaf), G. collinum (leaf), G. columbianum (leaf), G. columbinum (leaf), G. macrorrhizum (leaf), G. maculatum (leaf), G. palustre (leaf), G. pratense (leaf), G. pusillum (leaf), G. pyrenaicum (leaf), G. robertianum (leaf), G. sanguineum (leaf), G. sylvaticum (leaf), G. wlassovianum (leaf), Geranium sp., and Oxypolis rigidor [Tiedemannia rigida]) and associated fungi Chaetomella raphigera.