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Screening, characterisation and engineering of carbohydrate harvesting machineries of microbial ecosystems

Periodic Reporting for period 1 - CaTSYS (Screening, characterisation and engineering of carbohydrate harvesting machineries of microbial ecosystems)

Reporting period: 2016-09-01 to 2018-08-31

Gut bacteria have evolved sophisticated strategies to face the complexity and the diversity of the glycans they feed on in the intestine. In order to completely metabolize these glycans, bacteria displayed multi-proteic systems involving proteins for sensing, binding, transport and degradation. These systems are from high interest and radically transformed the way we think the biotechnological processes involved in the conversion of plant biomass and agricultural by-products into valuable synthons. To expand and harness our knowledge of these enzymatic synergies naturally displayed by bacteria to internalise and breakdown complex glycans, CaTSYS was focused on the discovery and the characterisation of multi-proteic systems involving novel CAZymes and carbohydrate binders/transporters. However, because most of the gut microbes remain uncultured, metagenomics a culture-independent method developed in the last decade to analyse the pool of genomes of microbial communities (the so-called metagenomes) is required to explore their functional diversity. CaTSYS aimed at developing new ultra-high throughput functional metagenomics approaches in order to boost the discovery and engineering of bacterial pathways involved in the metabolisation of complex glycans, and to establish the molecular bases of oligosaccharide transport and degradation in mammal gut ecosystems.
Firstly, two new droplet-microfluidic-based methods were developed to mine metagenomic and directed evolution libraries for glycan metabolization, in collaboration with the F. Hollfelder’s group at the University of Cambridge and with A. Dagkesamanskaya at INSAT. One is dedicated to the screening of functional carbohydrate transporters by positive selection in water-in-oil droplets. The second one is specific to the screening of glycan degradation pathways. These new developments allowed us to blow the locks of metagenomic DNA recovery from droplets, and to screen natural and artificial sequence diversity 1000 times cheaper and faster than with conventional approaches. Thanks to these technological breakthroughs, and to the metagenome screening campaigns previously performed at INSAT, we discovered new pathways involved in the food-microbiota-host crosstalk in the human gut. After analysis of their abundance and prevalence in the human gut microbiome, the most original catalysts and carbohydrate transporters and binders were further biochemically characterised.
Part of these results have been published or are about to be in the next few months. In addition, some results were presented at several conferences worldwide such as 13th Carbohydrate Bioengineering Meeting (Vienna, 23-26 April 2017), Gordon Conference Cellulases and others carbohydrate-active enzymes (Andover, 23-28 July 2017), and Enzyme Engineering XXIV (Toulouse, 24-28 September 2017). The interest of biodiversity mining to find biotechnological tools that are so useful in our daily life was also presented to general public at the Science in the City festival of ESOF (Toulouse, July 9-12 July), together with members of the H2020 Carbazymes and Metafluidics projects.
To conclude, new methods have been developed to boost the discovery and engineering of carbohydrate-active enzymes and transporters issued from complex microbiomes. The speed of screening has been increased by thousand fold by droplet-based microfluidics and several technological bottlenecks have been circumvented, enabling to explore, extremely quickly and precisely, the vast world of still unknown bacterial functions Furthermore, the discovery and characterization of key glycan metabolization pathways in the human gut microbiome led to a better understanding of the relationships between host, food and intestinal microbes. These strategies could now be extended to rapidly explore a series of microbial ecosystems which represent a goldmine for the discovery of new polysaccharide degrading/transporting proteins to boost the development of bioeconomy.
Ultra high-throughput metagenomics for the discovery of glycan metabolizing pathways