The expanding antibiotic resistance crisis calls for a more in depth understanding of the importance of antimicrobial resistance genes (ARGs) in pristine environments. We, therefore, studied the microbiome associated with Sphagnum moss forming the main vegetation in undomesticated, evolutionary old bog ecosystems. In our complementary analysis of culture collections, metagenomic data and a fosmid library from different geographic sites in Europe, we identified a low abundant but highly diverse pool of resistance determinants, which targets an unexpectedly broad range of 29 antibiotics including natural and synthetic compounds. This derives both, from the extraordinarily high abundance of efflux pumps (up to 96%), and the unexpectedly versatile set of ARGs underlying all major resistance mechanisms. Multi-resistance was frequently observed among bacterial isolates, e.g. in Serratia, Rouxiella, Pandoraea, Paraburkholderia and Pseudomonas. In a search for novel ARGs, we identified the new class A β-lactamase Mm3. The native Sphagnum resistome comprising a highly diversified and partially novel set of ARGs contributes to the bog ecosystem´s plasticity. Our results reinforce the ecological link between natural and clinically relevant resistomes and thereby shed light onto this link from the aspect of pristine plants. Moreover, they underline that diverse resistomes are an intrinsic characteristic of plant-associated microbial communities, they naturally harbour many resistances including genes with potential clinical relevance.

Oxyfunctionalization of fatty acids (FAs) is ak ey step in the design of novel synthetic pathways for biobased/ biodegradable polymers, surfactants and fuels. Here, we show the isolation and characterizationo farobust FA a-hydroxylase (P450 Ja)w hich catalyses the selective conversion of ab road range of FAs(C6:0-C16:0) and oleic acid (C18:1) with H 2 O 2 as oxidant. Under optimized reaction conditions P450 Ja yields a-hydroxy acids all with > 95 %r egioselectivity, high specific activity (up to 15.2 Umg À1)a nd efficient coupling of oxidant to product (up to 85 %). Lauric acid (C12:0) turned out to be an excellent substrate with respectt op ro-ductivity (TON = 394 min À1). On preparative scale, conversion of C12:0r eached 83 %(0.9 gL À1)w hen supplementing H 2 O 2 in fed-batch mode. Under similarc onditions P450 Ja allowed further the first biocatalytic a-hydroxylation of oleic acid (88 %c onversion on 100 mL scale) at high selectivity and in good yields (1.1 gL À1 ;7 9% isolatedy ield). Unexpectedly, P450 Ja displayed also 1-alkenef ormationf rom shorter chain FAs(C10:0) showingt hat oxidative decarboxylation is more widely distributed across this enzyme family than reportedp reviously.

The usage of natural products, especially in the treatment of diseases, has a long history. While natural products used to be administered directly, they today serve as lead compounds and structural scaffolds for the development of new drugs and other market products. The success of combinatorial approaches to develop new products strongly depends on natural product-likeness. This exemplifies the importance of natural products as structural leads during product development and demonstrates natural product discovery to be as important as ever. This chapter highlights endophytes as a rich bio-resource for the identification of novel natural compounds and emphasizes functional metagenomics as a promising method to source the endophytic potential. With the majority of microorganisms not readily cultivable under laboratory conditions, a vast amount of natural products synthesized by endophytes remains inaccessible. Functional metagenomics circumvents current cultivation limitations by direct cloning of bacterial community DNA. This procedure is, however, rarely performed exclusively on endophytes. This chapter outlines the procedures underlying this methodology with focus on its application to endophytes.

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.