Defined enzyme cocktail from the anaerobic fungus Orpinomyces sp. strain C1A effectively releases sugars from pretreated corn stover and switchgrass

Abstract

The anaerobic fungus Orpinomyces strain C1A is capable of growth on various types of lignocellulosic substrates, and harbors an impressive reservoir of carbohydrate active enzymes (CAZymes). Using a minimum enzyme cocktail strategy, we constituted a four-component lignocellulolytic cocktail derived from highly transcribed C1A, and evaluated its efficacy against pretreated corn stover and switchgrass. Hydrolysis yields ranged between 65–77.4%, depending on the lignocellulosic substrate and pretreatment applied. Addition of a highly expressed anaerobic fungal swollenin improved hydrolysis yields by up to 7%. Compared to the commercial cocktail CTec2, these anaerobic fungal cocktails provided comparable or slightly lower hydrolysis yields. Further, the differences in efficacy between commercial and anaerobic cocktails were often only realized after extended (168 hr) incubations. Under certain conditions, the hydrolysis yields of the anaerobic fungal cocktail was slightly superior to that realized by CTec2. We attribute the observed high hydrolysis yields to the high specific activity and affinity of the individual enzymes of the cocktail, as well as the high level of synergy and multi-functionality observed in multiple components. Collectively, this effort provides a novel platform for constructing highly effective enzymes for biofuel production and represents the first lignocellulolytic enzyme cocktail created from anaerobic fungal enzymes.

Community structure and fibrolytic activities of anaerobic rumen fungi in dromedary camels

“…The mix of fibrolytic and proteolytic enzymes was observed in anaerobic rumen fungi such as Neocallimastix and Piromyces [47,58], which highlights rumen fungi as a promising source of feed additives [25,60]. The purified enzymes cocktail also represents a promising alternative to save the enzymes cost of production of ethanol from lignocellulosic biomass [21].…”

“…The mix of fibrolytic and proteolytic enzymes was observed in anaerobic rumen fungi such as Neocallimastix and Piromyces , which highlights rumen fungi as a promising source of feed additives . The purified enzymes cocktail also represents a promising alternative to save the enzymes cost of production of ethanol from lignocellulosic biomass . Increasing the fungal population in the rumen to enhance the fermentation poor quality forages could be performed using of fibrous diets like wheat straw or alfalfa and dietary supplement like sulfur and decreasing the population of ciliate protozoa in the rumen .…”

Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion

“…1 filter paper20 µg Swo /mg substrate , 45 °C, 48 h; SEM, photographyDeagglomeration of filter paper (reduction of particle size and count); SEM showed an increased surface roughness; no swelling was observed[37] T. asperellum / E. coli /refolding, AC/~35–50 kDaAvicel PH-1015 µg Swo /mg substrate , 50 °C, 91 h; light microscopyPartial disruption of Avicel PH-101 particles[38] T. pseudokoningii / A. niger /HIC/~75 kDaAvicel PH-101, filter paper5–20 µg Swo /mg Avicel , 0.5–2 µg Swo /mg filter paper , 40 °C, 48–72 h; light microscopy, XRDNo effects were observed by applying light microscopy; CrI was increased (88–90 %)[40] T. reesei / T. reesei /IMAC, IE/n.a.Mercerized cotton fibers10 µg Swo /mg substrate , 50 °C, overnight; CBM adsorption assay, SEMAvailable surface for CBMs was increased (~38 %); SEM showed a smoothed surface upon Tr Swo1 treatment[41] T. reesei / E. coli and N. tabacum /CS/n.a.Mercerized cotton fibers0.2–2 µg Swo /mg substrate , 37 °C or 50 °C, 8 h; phase contrast microscopyFiber expansion, inner fiber structure was altered independent of the Tr Swo1 source[28] P. oxalicum / T. reesei /precipitation, IMAC/~90 kDaAvicel PH-1014 µg Swo /mg substrate , 50 °C, 48 h; light microscopy, protein binding assayPartial disruption of Avicel PH-101 particles; B max for cellulases was increased (~20 %)[39] T. reesei / T. reesei /IMAC, IE/n.a.Dissolving pulp, various lignocellulosic pulps50 µg Swo /mg substrate , 50 °C, overnight; high-resolution fiber quality analyzerFragmentation was observed to a low extent for dissolving pulp fibers but not for lignocellulosic pulps[42] Orpinomyces sp. strain C1A/ E. coli /refolding, IMAC/~67 kDaCotton fibers0.25–5 µg Swo /mg substrate , 39 °C, 12 h; ESEM, Congo red cotton assayAverage cotton fiber width was increased (~56 %); dye adsorption was increased (CAE ~0.4 for 5 µg Swo /mg substrate )[78] aMm apparent molecular mass, CS enriched culture supernatant, AC affinity chromatography, IMAC immobilized metal adsorption chromatography (via His-tag), HIC hydrophobic interaction chromatograp...…”

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

“…The importance of rumen fungi for biomass degradation has since then been supported by in vivo studies [23][24][25] , and recently reinforced in transcriptome studies revealing that the fungi express a range of CAZymes when grown on different carbon sources 9,26 . Although enzymes of fungal origin have been regularly explored for their remarkable capacity to degrade lignocellulosic fiber 12,27,28 , their functional role in native anaerobic habitats and within the biomass-degrading enzyme repertoire of the rumen microbiome remains unclear. Thus, we lack a complete understanding of their biology and their contribution to the function and health of the rumen ecosystems.…”