Combining OSMAC, metabolomic and genomic methods for the production and annotation of halogenated azaphilones and ilicicolins in termite symbiotic fungi

Abstract

We gathered a collection of termite mutualistic strains from French Guiana to explore the metabolites of symbiotic microorganisms. Molecular networks reconstructed from a metabolomic analysis using LC–ESI–MS/MS methodology led us to identify two families of chlorinated polyketides, i.e., azaphilones from Penicillium sclerotiorum and ilicicolins from Neonectria discophora. To define the biosynthetic pathways related to these two types of scaffolds, we used a whole genome sequencing approach followed by hybrid assembly from short and long reads. We found two biosynthetic gene clusters, including two FAD-dependent halogenases. To exploit the enzymatic promiscuity of the two identified FAD halogenases, we sought to biosynthesize novel halogenated metabolites. An OSMAC strategy was used and resulted in the production of brominated analogs of ilicicolins and azaphilones as well as iodinated analogs of azaphilones.

Enhancing chemical and biological diversity by co-cultivation

“…These approaches mostly focus on the untargeted regulation of the metabolome, unbiasedly increasing chemical diversity without direct manipulation of the genome. Several of these strategies have been described in the literature and comprise mainly the use of substrate feeding ( De Boer and Schmidt-Dannert, 2003 ), small molecules elicitors ( Pettit, 2011a ; Seyedsayamdost, 2014 ; Xu et al, 2017 ; Mao et al, 2018 ; Moon et al, 2019b ; Xu et al, 2019 ), co-cultivation ( Pettit, 2009 ; Shank and Kolter, 2009 ) and the variation of nutrient sources and availability by One Strain, Many Compounds (OSMAC) ( Bode et al, 2002 ; Wei et al, 2010 ; Rateb et al, 2011 ; Pan et al, 2019 ; Selegato et al, 2019 ; Hebra et al, 2022 ). These have been shown to provide a broader expression of the metabolic pathways than their respective monocultures, thus increasing chemodiversity in a more holistic and unsupervised manner.…”

Nitrogen Enriched Solid-State Cultivation for the Overproduction of Azaphilone Red Pigments by Penicillium sclerotiorum SNB-CN111

“…The advantage of t-SNE molecular networks over regular molecular network is that datasets are represented as a whole, preserving the structural relationship between distinct clusters. This methodology has already been successfully applied to extracts of Penicillium sclerotiorum SNB-CN111 on solid PD conditions to decipher the chemical diversity of produced azaphilones [ 24 , 25 ]. Cultivation on solid CZK medium, PD medium, and solid YE medium generated 255, 620 and 1069 distinct metabolic features, respectively, indicating that the chemical diversity is increasing with the complexity and concentration of the organic nitrogen sources ( Figure 1 b).…”

“…In the two last years, processes of liquid submerged cultivation to obtain red azaphilone pigments with specific amino acids incorporation have been reported [ 21 , 22 , 23 ]. Nevertheless, in some particular cases, such as Penicillium sclerotiorum SNB-CN111 isolated from Nasutitermes similis termite aerial nests [ 24 , 25 ], the selected strain did not produce azaphilones in liquid potato dextrose media but in a solid one. For that purpose, we developed of a unique two-step nitrogen enriched solid-state cultivation method able to overproduce red azaphilone pigments, and their chemical diversity related to the nitrogen sources was characterized using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and molecular network by MetGem software [ 26 , 27 , 28 ] offering a new pipeline for large-scale red azaphilone production [ 25 ].…”

A Survey of Recently Discovered Naturally Occurring Organohalogen Compounds

“…69 Two studies of Penicillium sclerotiorum E23Y-1A cultivated from an unidentified sponge in one case and from the sponge Holoxea sp. in the second study, both from Hainan, China, yielded penicilazaphilones F (163) and G (164), 70 philone H (166), and 5-bromosclerotiorin (167). The latter two are obtained when KBr is present in the fermentation broth.…”