title: Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.
abstract: BACKGROUND
  Enzymatic 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.
  
  
  RESULTS
  Two 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands ; Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Maria Victoria
authors.initials: MV
authors.lastname: Aguilar-Pontes
authors.affiliation: Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands ; Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands ; Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Sjors
authors.initials: S
authors.lastname: van der Horst
authors.affiliation: Yeast and Basidiomycete Research, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: Theelen
authors.affiliation: Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands ; Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Ronald P
authors.initials: RP
authors.lastname: de Vries
authors.affiliation: Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands ; Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: van den Brink
keywords: Acetyl xylan esterase; CAZy enzymes; Myceliophthora heterothallica; Plant biomass; Progeny; Saccharification; Sexual crossing; Sugar beet pulp