The full potential of fungal secondary metabolism has until recently been impeded by the lack of universal genetic tools for most species. However, the emergence of several CRISPR-Cas9-based genome editing systems adapted for several genera of filamentous fungi have now opened the doors for future efforts in discovery of novel natural products and elucidation and engineering of their biosynthetic pathways in fungi where no genetic tools are in place. So far, most studies have focused on demonstrating the performance of CRISPR-Cas9 in various fungal model species, and recently we presented a versatile CRISPR-Cas9 system that can be successfully applied in several diverse Aspergillus species. Here we take it one step further and show that our system can be used also in a phylogenetically distinct and largely unexplored species from the genus of Talaromyces. Specifically, we exploit CRISPR-Cas9-based genome editing to identify a new gene in T. atroroseus responsible for production of polyketide-nonribosomal peptide hybrid products, hence, linking fungal secondary metabolites to their genetic origin in a species where no genetic engineering has previously been performed.

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry.

No abstract

No abstract

Whilst the effects of antibiotics on microorganisms are widely studied, it remains less well-understood how antibiotics affect the physiology of the native producing organisms. Here, using a marine bacterium Photobacterium galatheae S2753 that produces the antibiotic holomycin, we generated a holomycin deficient strain by in-frame deletion of hlmE, the core gene responsible for holomycin production. Mass spectrometry analysis of cell extracts confirmed that ΔhlmE did not produce holomycin and that the mutant was devoid of antibacterial activity. Biofilm formation of ΔhlmE was significantly reduced compared to that of the wild-type S2753 and was restored in an hlmE complementary mutant. Consistently, exogenous holomycin, but not its dimethylated and less antibacterial derivative, S,S’-dimethyl holomycin, restored the biofilm formation of ΔhlmE. Furthermore, zinc starvation was found essential for both holomycin production and biofilm formation of S2753, although the molecular mechanism remains elusive. Collectively, these data suggest that holomycin promotes biofilm formation of S2753 via its ene-disulfide group. Lastly, the addition of holomycin in sub-inhibitory concentrations also enhanced the biofilm of four other Vibrionaceae strains. P. galatheae likely gains an ecological advantage from producing holomycin as both an antibiotic and a biofilm stimulator, which facilitates the nutrition acquisition and protects P. galatheae from environmental stresses. Studying the function of antibiotic compounds in the native producer will shed light on their role in nature and could potentially point to novel bioprospecting strategies. Importance Despite the societal impact of antibiotics, their ecological functions remain elusive and have mostly been studied by exposing non-producing bacteria to sub-inhibitory concentrations. Here, we studied the effects of the antibiotic holomycin on its native producer, Photobacterium galatheae S2753, a Vibrionaceae bacterium. Holomycin provides a distinct advantage to S2753 both as an antibiotic and by enhancing biofilm formation in the producer. Vibrionaceae species successfully thrive in global marine ecosystems, where they play critical ecological roles as free-living, symbiotic, or pathogenic bacteria. Genome mining has demonstrated that many have the potential to produce several bioactive compounds, including P. galaltheae. To unravel the contribution of the microbial metabolites to the development of marine microbial ecosystems, better insight into the function of these compounds in the producing organisms is needed. Our finding provides a model to pursue this and highlights the ecological importance of antibiotics to the fitness of the producing organisms.