Two different aspects of natural multi-modular diversity within a xylan utilization locus to investigate catalytic synergism
Abstract
Plant biomass acting enzymes (CAZYmes) are central components of biorefinery
processes that aim to convert lignocelluloses into molecules of interest. Therefore,
efforts to discover, characterize or design novel Cazymes are particularly relevant to
valorize lignocellulosic biomass for industrial purposes.
In Nature, microorganisms deploy different strategies to degrade plant cell wall including
secretion of multi-modular enzymes, cellulosomes anchored at the surface of microbes
or genetic organizations (polysaccharide utilization loci PUL) aiming to orchestrate the
breakdown of a specific glycan. Thanks to functional metagenomics we discovered new
enzymes from the gut microbiome of the termite Pseudacanthotermes militaris (1). This
study highlighted an interesting hypothetical xylan PUL, encoding five glycoside
hydrolases ( GH) and one carbohydrate esterase (CE) annotated from Bacteroidales.
Among the GHs, a novel GH10 encoding sequence Pm25, showed details of an
unusual domain organization. It consists of one catalytic domain, which is intercalated
by two Carbohydrate Binding Modules (CBMs). This multi-modular organization was
shown to be vastly distributed in different xylan PUL found in Bacteroidetes,
demonstrating its importance in the glycan foraging for the gut microbiota (2). Therefore,36
the xylan PUL can serve as a model not only to explore the role of enzyme multimodularity but also as a basis to develop synergy between enzymes.
In this work, we will present the detailed biochemical and structural characterization of
Pm25 with or without its CBMs. The role of the CBMs was also investigated and
quantification of their interactions towards carbohydrate led to better understand the
specific role of these modules (3).
Moreover, in order to explore the molecular basis of the enzymatic synergy, a crucial
parameter for the valorization of lignocellulosic biomass, we have recently developed a
coevolution strategy using a xylanase and a xylosidase from the PUL. We will present
the development of the screening and preliminary biochemical characterization of the
best arabinoxylan-active mutants. All these results contribute to a better understanding
of the molecular determinants to design novel biotechnological tools for plant biomass
valorization.