Fungal enzyme glycosylation: Characterization of cellobiohydrolase Cel7A from Trichoderma reesei

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling

Abstract

Polysaccharides found in cells are a valuable source of starting material for the industrial production of biofuel precursors and chemicals. In biorefineries, the commonly applied material is lignocellulose from renewable plant
biomass abundant in glucose polymers, known as cellulose. The bioconversion of cellulose to fermentable sugars is challenging due to the recalcitrance of lignocellulose. Numerous microorganisms have evolved to encompass
an enzymatic apparatus to degrade cellulose. These enzymes are efficiently produced by some filamentous fungi secreting many cellulases in large amounts. Inspired by Nature, this concept has successfully been applied on an
industrial scale using selected enzymatic cocktails containing cellulose-degrading enzymes to depolymerize lignocellulosic feedstocks. Due to the recalcitrant nature of lignocellulose even after pretreatment, the application
of cellulases is costly. However, numerous engineering methods have been applied to improve the catalytic efficacy of these biocatalysts. Fungal cellulases are often decorated with short glycan chains (glycosylated) which
is a possible engineering target for improving enzyme activity. The current thesis describes the structural and functional role of glycosylation in a family glycoside hydrolase 7 (GH7) cellobiohydrolase Cel7A from Trichoderma reesei (TrCel7A) through detailed kinetic characterization. This was investigated through manipulation of the TrCel7A glycan by the site-directed mutagenesis and through application of newly developed enzymatic deglycosylation toolbox.
The influence of the glycosylation in TrCel7A was studied using steady-state kinetic approaches suitable for the characterization of cellulases acting on the interface of the insoluble cellulosic substrates. The TrCel7A architecture is comprised of a N-glycosylated catalytic domain connected to a C-terminal carbohydrate-binding module (CBM) by an O-glycosylated linker. The single and complete N-glycosylation site deficient mutants (N45Q, N270Q, N384Q) were produced in Aspergillus oryzae and Trichoderma reesei. The effect of the removal of N-glycan chains from the TrCel7A catalytic domain was investigated by comparing the stability, kinetics and binding properties of these enzymes to the TrCel7A wild-type. The results clearly demonstrated that the N-glycans
modulated binding affinity and adsorption capacity to cellulose surface without affecting the overall thermal stability or structural integrity of the enzyme. The N-glycans at site N45 were demonstrated to play a key role in the location of productive binding sites and the rate of desorption. The study of O-glycosylation in TrCel7A was approached by enzymatic trimming using a number of exoglycosidases.TrCel7A produced in either A. oryzae or T. reesei is primarily decorated with α-mannooligosaccharides and hence enzymes with α-mannosidase activity were screened resulting in the identification of a family GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92). This enzyme demonstrated activity on α-1,2-mannobiose and on α-mannan extracted from Saccharomyces cerevisiae, which enabled activity for other α-mannan degrading enzymes. NnGH92 exhibited activity on the O-glycans found in fungal glycoproteins, including TrCel7A. A crystal structure of NnGH92 was solved and the substrate-binding residues at the active site were identified. The comparison of NnGH92 to the GH92 structural homologues allowed to identify conserved catalytic and binding residues at –1 and +1 subsites, leading to a confirmation of the structural elements responsible for the activity of α-1,2-mannosidase. In the same crystal structure, four non-catalytic domains, including two family CBM32s, a β-sheet CBM-like domain and a four-helix bundle domain, were solved which together with the catalytic domain, corresponded to the full-length enzyme of NnGH92. The functions of the NnGH92 non-catalytic domains were investigated by their sequential deletion. These domains were important
for the structural integrity of the enzyme as such and no obvious function in binding to yeast α-mannan was detected.
Structural information about the type of monosaccharides and glycosidic linkages is often a prerequisite for finding a suitable glycosidase activity to deconstruct the O-glycan chains in fungal glycoproteins by the enzymatic
treatment. We performed extensive O-glycomics analysis, elucidating the glycosyl residues chemistry isolated from TrCel7A produced in A. oryzae. This allowed the identification of the most abundant O-glycan structures
present in the TrCel7A linker region. Based on these results, a number of exo-glycosidases were rationally tested, leading to the identification of a family GH2 β-galactofuranosidase from Amesia atrobrunnea and NnGH92. Both
enzymes effectively reduced the extent of O-glycosylation in TrCel7A to a single abundant glycoform consisting of nine mannose residues. A commercial jack bean α-mannosidase was found to remove the remaining mannose
residues directly attached to the protein backbone. Kinetic characterization of the TrCel7A variants with reduced degree of O-glycosylation demonstrated that the kinetic behavior did not change compared to the wild-type. This enzymatic deglycosylation toolbox can be used for the glycoengineering of other fungal glycoproteins where the glycosylation is suspected to play a role.
This work emphasizes the challenges and importance related to glycosylation in TrCel7A. The kinetic characterization allowed to expand the knowledge on how glycosylation modifies the activity and binding properties of TrCel7A. The presented methods, results and observations can be applied to other fungal cellulosedegrading enzymes or glycosylated fungal enzymes to produce enzymes with changed behavior in industrial applications.
OriginalsprogEngelsk
UdgivelsesstedRoskilde
ForlagRoskilde Universitet
Antal sider90
StatusUdgivet - mar. 2021

Citer dette