A New High-Throughput Screening Method to Detect Antimicrobial Volatiles from Metagenomic Clone Libraries.

The ever-growing spread of resistance in medicine and agriculture highlights the need to identify new antimicrobials. Microbial volatile organic compounds (VOCs) are one of the most promising groups of chemicals to meet this need. These rarely exploited molecules exhibit antimicrobial activity and their high vapour pressure makes them ideal for application in surface sterilisation, and in particular, in biofumigation. Therefore, we adapted the previously developed Two Clamp VOCs Assay (TCVA) to a new high-throughput screening for the detection of novel antifungal VOCs from metagenomic clone libraries. As a proof of concept, we tested the new high-throughput TCVA (htTCVA) by sourcing a moss metagenomic library against Fusarium culmorum. This led to the identification of five clones that inhibited the growth of mycelium and spores in vitro by up to 8% and 30% and subsequently, to the identification of VOCs that are potentially, and in part responsible for the clones' antifungal activity. For these VOCs, the in vitro effect of the pure compounds was as high as 100%. These results demonstrate the robustness and feasibility of the htTCVA, which provides access to completely new and unexplored biosynthetic pathways and their secondary metabolites.

P450Jα : A New, Robust and α-Selective Fatty Acid Hydroxylase Displaying Unexpected 1-Alkene Formation.

Oxyfunctionalization of fatty acids (FAs) is a key step in the design of novel synthetic pathways for biobased/biodegradable polymers, surfactants and fuels. Here, we show the isolation and characterization of a robust FA α-hydroxylase (P450Jα ) which catalyses the selective conversion of a broad range of FAs (C6:0-C16:0) and oleic acid (C18:1) with H2 O2 as oxidant. Under optimized reaction conditions P450Jα yields α-hydroxy acids all with >95 % regioselectivity, high specific activity (up to 15.2 U mg-1 ) and efficient coupling of oxidant to product (up to 85 %). Lauric acid (C12:0) turned out to be an excellent substrate with respect to productivity (TON=394 min-1 ). On preparative scale, conversion of C12:0 reached 83 % (0.9 g L-1 ) when supplementing H2 O2 in fed-batch mode. Under similar conditions P450Jα allowed further the first biocatalytic α-hydroxylation of oleic acid (88 % conversion on 100 mL scale) at high selectivity and in good yields (1.1 g L-1 ; 79 % isolated yield). Unexpectedly, P450Jα displayed also 1-alkene formation from shorter chain FAs (≤C10:0) showing that oxidative decarboxylation is more widely distributed across this enzyme family than reported previously.