Development of a droplet-based, “Vectorless” system for in vitro, ultrahigh-throughput functional metagenomics

Resumen

Enzymes of prokaryotic microorganisms are of particular interest due to their massive metabolic diversity and their widespread application as biocatalysts for industrial transformations, contributing to the development of more sustainable and environmentally friendly practices. However, only a small fraction of bacteria is successfully cultivable by current techniques and their enzymes become only accessible by the application of strategies such as functional metagenomics. Classic functional metagenomic workflows require the construction of metagenomic libraries in vectors (plasmids, fosmids…) and subsequent transformation in host-organisms for the expression of possible hits. This process is time-consuming and is limited by low efficiency. Furthermore, the use of host-organisms may entail toxicity of the expressed proteins, recombination events and low transformation frequencies. In this doctoral thesis, an alternative, more straightforward, vector independent and host-independent workflow (dubbed “Vectorless”) for the in vitro functional screening of metagenomic libraries has been developed. The Vectorless workflow is based on a specifically designed new class of metagenomic libraries generated completely in vitro and constituted by dumbbell-DNA (dbDNA) molecules instead of vectors. In the developed Vectorless workflow, the application of droplet-based microfluidics enables the in vitro compartmentalization for the amplification, in vitro transcription and translation (IVTT) and subsequent screening by of fluorescence-assisted droplet sorting (FADS) of the dbDNA libraries. Three dbDNA metagenomic libraries have been generated from three diverse natural environments and have been employed for the screening of esterases resulting in the identification of 16 diverse enzymes showing esterase activity, including weak events of catalytic promiscuity. The sequences of the identified proteins in which the esterase activity resides have been cloned for their subsequent overexpression, purification and partial biochemical characterization. After completing this process, the esterase specific activity of three esterases (ISL_02, ISL_08 and CAB_20) was determined and a Thal0063 showed very low esterase activity, which is congruent with the phenomenon of catalytic promiscuity