Abstract
BackgroundEnzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification.ResultsTwo genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75–85 °C and pH 5.5–6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp.ConclusionsThis study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously uncharacterized CE5-CBM1 acetyl xylan esterase. This shows that sexual crossing and selection of M. heterothallica are the successful strategy to improve the composition of enzyme mixtures for efficient plant biomass degradation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0460-y) contains supplementary material, which is available to authorized users.
Highlights
Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes
Sexual crosses between M. heterothallica CBS 203.75 and CBS 663.74 combined with growth screening select for strains with improved sugar beet pulp (SBP) saccharification The ascospores of M. heterothallica isolates CBS 203.75 and CBS 663.74 were plated on agar medium with 3 % SBP
69 of the 70 progenies had the same mating type as CBS 663.74. This might be explained by interference of some germinating conidiospores of CBS 663.74, as CBS 663.74 showed better growth on SBP and higher saccharification activities compared to CBS 203.75 (15.5 ± 0.6 and 11.3 ± 0.7 μg min−1 ml−1, respectively)
Summary
Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Thermophilic Myceliophthora species have a large set of enzymes targeting different linkages in plant polysaccharides The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The filamentous fungus Myceliophthora thermophila (synonym Sporotrichum thermophile/Chrysosporium lucknowense) has shown to efficiently degrade biomass using thermostable enzymes and has more than 200 plant biomass-degrading enzymes encoded in its genome [9, 10]. These glycoside hydrolases (GH), polysaccharide lyases (PL), carbohydrate esterases (CE) and oxidases are covering most of the recognized fungal CAZy families [10]. A comparative study will be difficult for identifying the contribution of specific enzymes in releasing sugars from plant biomass
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