Flavosases

Winner of the 2024 call for projects.

Organic carbon on the Earth's surface is estimated at 550 billion tons, 82% of which is captive in plant biomass. Much of the plant carbon is stored in plant cell walls composed primarily of cellulose, the most abundant biopolymer on Earth (production: ~ 55 billion tons/year).

Cellulosic biomass thus represents a huge carbon reservoir usable for production of biofuels or commodity chemicals. Cellulose and other plant cell wall polysaccharides are woven into a matrix that is recalcitrant to degradation, requiring cellulolytic bacteria and fungi to efficiently secrete degradative enzymes (CAZymes: i.e. cellulases) to depolymerize cellulosic substrates. In aerobic biotopes, cellulolytic microorganisms generally secrete copious amounts (g/L) of CAZymes in a free diffusing state. In contrast, some anaerobic microorganisms secrete much lower amounts (mg/L) of multienzymatic complexes called cellulosomes. Partner 1 (P1)’s work showed that the remarkable efficiency of cellulosomes stems from the synergy provided by the physical proximity of the gathered enzymes and attachment of complexes to the surface of cellulose.

While harnessing microbial cellulolytic capabilities for biotechnological design and conversion of cellulose into desired compounds holds great potential, innovations are needed to realize its Flavobacteria (phylum Bacteroidota) use a highly efficient secretion apparatus called the type IX secretion system (T9SS). The T9SS is a versatile nanomachine that secretes dozens of different enzymes with various activities, notably CAZymes. After translocation into the periplasm by the Sec machinery, the enzymes, which all have in common a C-terminal secretion domain (CTD), are recognized, selected, and secreted by the T9SS across the outer membrane. It has been shown that other enzymes can be fused to a CTD to be abundantly and specifically released in the medium by the T9SS. In the

FLAVOLASES project, we seek to engineer Flavobacterium johnsoniae into a novel, efficient cellulolytic biocatalyst by exploiting the T9SS to export minicellulosomes built by P1 and free cellulases from Lachnoclostridium phytofermentans.

This work will be carried out in 3 work areas:

• Construction of recombinant strains of F. johnsoniae, a genetically tractable species, to secrete both cellulosomes and free cellulases.
• Evaluation of the cellulose degradation and secretion abilities of engineered strains.
• Most promising candidates will undergo a further adaptive laboratory evolution (ALE) using an automat available in P3’s group to improve the secretion yield of the newly integrated cellulolytic system.

Altogether, the project relies on the complementary expertise of the three partners to shape an efficient cellulolytic system necessary to plant biomass-based biorefineries design. Furthermore, ALE will also provide significant information on the T9SS mechanism of action and how to use and foster its efficiency to secrete desired proteins such as cellulolytic enzymes cocktails or minicellulosomes useful to treat plant biomass in industrial processes.

Project lifetime:
 

2025 - 2029

Scientific manager:
 

Nicolas Vita (CNRS)