Master, E. R. | Search bioRxiv

bioRxiv2022

Loosenin-like proteins from Phanerochaete carnosa impact both cellulose and chitin fiber networks

Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms, and reportedly play a role in the modification and deconstruction of cell wall polysaccharides including lignocellulose. So far, very few microbial expansin related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A; PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. Whereas all of the purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF), none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Binding of PcaLOOLs to alpha-chitin was higher than to cellulose (Avicel), and highest at pH 5.0. Notably, whereas PcaLOOL9 reduced the yield strain of chitin nanofibrils (ChNF) in a protein-dose dependent manner, the reverse pattern was observed for PcaLOOL7 despite belonging to the same LOOL subfamily. The current study reveals the potential of microbial expansin-related proteins to impact both cellulose and chitin networks, and provides further evidence pointing to a non-lytic mode of action.

microbiology↗

bioRxiv2021

PcGCE is a potent elicitor of defense responses in aspen

Using microbial enzymes in transgenesis is a powerful means to introduce new functionalities in plants. Glucuronoyl esterase (GCE) is a microbial enzyme hydrolyzing the ester bond between lignin and 4-O-methyl--D-glucuronic acid present as a side chain of glucuronoxylan. This bond mediates lignin-carbohydrate complex (LCC) formation, considered as crucial factor of lignocellulose recalcitrance to saccharification. Previous studies showed that hybrid aspen (Populus tremula L. x tremuloides Michx.) constitutively expressing Phanerochaete carnosa Burt GCE (PcGCE) had better efficiency of cellulose-to-glucose conversion but were stunned and had lower cellulose content indicating that more studies are needed to design strategy for deployment of this enzyme in planta. Here we report that the transgenic plants exhibit premature leaf senescence, increased accumulation of calcium oxalate crystals, tyloses and necrotic lesions and have strongly activated immune defense reactions as revealed by their altered profiles of transcriptomes, metabolomes and hormones in the leaves. To elucidate if these effects are triggered by damage-associated molecular patterns (DAMPs) or by PcGCE protein perceived as a pathogen-associated molecular pattern (PAMP), we ectopically expressed in aspen an enzymatically inactive PcGCES217A. The mutated PcGCE induced similar growth retardation, leaf necrosis and premature senescence as the active one, providing evidence that PcGCE protein is recognized as PAMP. Transcriptomics analysis of young expanding leaves of 35S:PcGCE plants identified several candidates for receptors of PcGCE, which were not expressed in developing wood tissues. Grafting experiments showed that PcGCE transcripts are not cell-to-cell mobile and that PcGCE expressing leaves augment systemic responses. In agreement, expressing PcGCE in developing wood by using the wood-specific promoter (WP), avoided all off-target effects. Moreover, WP:PcGCE lines grew better than control plants providing evidence that this strategy can be used in transgenic crops dedicated for biorefinery.

bioengineering↗