A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin
Artikel i vetenskaplig tidskrift, 2016

Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature’s most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown. Results Here we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor–regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- and exo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm. Conclusions By combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.

recalcitrant polysaccharides

Bacteroidetes

chitin

Polysaccharide utilization locus

Författare

Johan Larsbrink

Wallenberg Wood Science Center (WWSC)

Chalmers, Biologi och bioteknik, Industriell bioteknik

Yongtao Zhu

Unknown organization

Sampada S. Kharade

Unknown organization

Kurt J. Kwiatkowski

Unknown organization

Vincent G.H. Eijsink

Unknown organization

Nicole M. Koropatkin

Unknown organization

Mark J. McBride

Unknown organization

Phillip B. Pope

Unknown organization

Biotechnology for Biofuels

1754-6834 (eISSN)

Vol. 9

Ämneskategorier

Biokemi och molekylärbiologi

Mikrobiologi

Strukturbiologi

Genetik

Styrkeområden

Energi

Livsvetenskaper och teknik

Fundament

Grundläggande vetenskaper

DOI

10.1186/s13068-016-0674-z

PubMed

27933102