Metagenomics approaches to discover new industrially-relevant enzymes
- PhD, Metagenomics, Esterase, Methyltransferase, Enrichment
Biocatalysis has become an indispensable part of the industrial production of organic compounds due to the superior functional properties of enzymes over chemical synthesis and due to the need of mitigating the environmental impact of chemical processes. The demand for new biocatalysts is constantly growing and besides the numerous characterised enzymes, only few candidates meet the requirements dictated by the industrial sector. Metagenomics has made possible the exploration of the incredible microbial biochemical diversity, significantly increasing the number of novel enzymes identified thanks to naïve screening and direct sequencing of environmental DNA. By applying a metagenomics-based analysis, we investigated the rhizosphere of Sorghum plant for the identification of novel biocatalysts of industrial interest. In detail, we aimed at finding a methylation reaction for the enzymatic production of sorgoleone, an allelopathic metabolite naturally released by sorghum roots that acts as a potent weed control compound, and other hydrolytic activities with enhanced properties of common use in biotechnological applications. After enrichment with Cashew Nut Shell Liquid (CNSL), a waste product from cashew industry rich in phenolic lipids resembling our target substrate/product, de novo sequencing of the total metagenomic DNA and fosmid expression libraries were produced. The changes in microbial community composition and its biodegradation potential was evaluated and resulted in a reconstruction of catabolic network, which suggested the significant enrichment in bacteria known for their aromatic hydrocarbons degradation capabilities in response to CNSL addition. Metagenome analysis suggested a number of enzyme candidates that were cloned and expressed. One of them was an efficient SAM-O-methyltransferase from uncultured microorganism distantly related to Mycobacterium, able to react with the aromatic moiety of the target substrate molecule cardol triene. Preliminary experiments suggested this enzyme as a basis for the establishment and further improvement of the two-stage process for semisynthetic sorgoleone production. Furthermore, functional screening of fosmid libraries identified two highly promiscuous esterases, capable of hydrolysing a broader range of ester substrates than the most-active industrial prototypes. The work has a number of potential environmental and economic implications, namely, in moving towards the establishing of an environmentally-friendly (greener) process for production of a natural herbicide for efficient weed control. Furthermore, a notable contribution was delivered to the functional annotation of poorly described enzyme families in databases, as well as to the definition of structural determinants of the enzyme promiscuity phenomenon, feature highly valued in industrial enzymes.
|Award date||17 Apr 2019|