A candidate gene cluster for the bioactive natural product gyrophoric acid in lichen-forming fungi

Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Yet they a have limited implementation in industry as for most of the natural products, the corresponding genes remain unknown. Here we implement a long-read sequencing and bioinformatic approach to identify the biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus Umbilicaria, we identify the most likely GA cluster and investigate cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria with only three genes that are conserved across species, including the PKS gene. In addition, our results suggest that the same cluster codes for different but structurally similar NPs, i.e., GA, umbilicaric acid and hiascic acid, bringing new evidence that lichen metabolite diversity is also generated through regulatory mechanisms at the molecular level. Ours is the first study to identify the most likely GA cluster. This information is essential for opening up avenues for biotechnological approaches to producing and modifying GA, and possibly other lichen compounds. We show that bioinformatics approaches are useful in linking genes and potentially associated natural products. Genome analyses help unlocking the pharmaceutical potential of organisms such as lichens, which are biosynthetically diverse, but slow growing, and usually uncultivable due to their symbiotic nature.

Exploring the transportome of the biosurfactant producing yeast Starmerella bombicola

Starmerella bombicola is a non-conventional yeast mainly known for its capacity to produce high amounts of the glycolipids ‘sophorolipids’. Although its product has been used as biological detergent for a couple of decades, the genetics of S. bombicola are still largely unknown. Computational analysis of the yeast’s genome enabled us to identify 254 putative transporter genes that make up the entire transportome. For each of them, a potential substrate was predicted using homology analysis, subcellular localization prediction and RNA sequencing in different stages of growth. One transporter family is of exceptional importance to this yeast: the ATP Binding Cassette (ABC) transporter Superfamily, because it harbors the main driver behind the highly efficient sophorolipid export. Furthermore, members of this superfamily translocate a variety of compounds ranging from antibiotics to hydrophobic molecules. We conducted an analysis of this family by creating deletion mutants to understand their role in the export of hydrophobic compounds, antibiotics and sophorolipids. Doing this, we could experimentally confirm the transporters participating in the efflux of medium chain fatty alcohols, particularly decanol and undecanol, and identify a second sophorolipid transporter that is located outside the sophorolipid biosynthetic gene cluster.

The crystal structure of DynF from the dynemicin-biosynthesis pathway of Micromonospora chersina

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded β-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.

Genome Sequence-Guided Finding of Lucensomycin Production by Streptomyces achromogenes Subsp. streptozoticus NBRC14001

Information on microbial genome sequences is a powerful resource for accessing natural products with significant activities. We herein report the unveiling of lucensomycin production by Streptomyces achromogenes subsp. streptozoticus NBRC14001 based on the genome sequence of the strain. The genome sequence of strain NBRC14001 revealed the presence of a type I polyketide synthase gene cluster with similarities to a biosynthetic gene cluster for natamycin, which is a polyene macrolide antibiotic that exhibits antifungal activity. Therefore, we investigated whether strain NBRC14001 produces antifungal compound(s) and revealed that an extract from the strain inhibited the growth of Candida albicans. A HPLC analysis of a purified compound exhibiting antifungal activity against C. albicans showed that the compound differed from natamycin. Based on HR-ESI-MS spectrometry and a PubChem database search, the compound was predicted to be lucensomycin, which is a tetraene macrolide antibiotic, and this prediction was supported by the results of a MS/MS analysis. Furthermore, the type I polyketide synthase gene cluster in strain NBRC14001 corresponded well to lucesomycin biosynthetic gene cluster (lcm) in S. cyanogenus, which was very recently reported. Therefore, we concluded that the antifungal compound produced by strain NBRC14001 is lucensomycin.

Bioinformatic and Mechanistic Analysis of the Palmerolide PKS-NRPS Biosynthetic Pathway From the Microbiome of an Antarctic Ascidian

Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the ∼75 kbp trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicihabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

Genome-based characterization of a plasmid-associated micrococcin P1 biosynthetic gene cluster and virulence factors in Mammaliicoccus sciuri IMDO-S72

Analysis of the de novo assembled genome of Mammaliicoccus sciuri IMDO-S72 revealed the genetically encoded machinery behind its earlier reported antibacterial phenotype and gave further insight into the repertoire of putative virulence factors of this recently reclassified species. A plasmid-encoded biosynthetic gene cluster was held responsible for the antimicrobial activity of M. sciuri IMDO-S72, comprising genes involved in thiopeptide production. The compound encoded by this gene cluster was structurally identified as micrococcin P1. Further examination of its genome highlighted the ubiquitous presence of innate virulence factors mainly involved in surface colonization. Determinants contributing to aggressive virulence were generally absent, with exception of a plasmid-associated ica cluster. The native antibiotic resistance genes sal (A) and mecA were detected within the genome, amongst others, but were not consistently linked with a resistant phenotype. While mobile genetic elements were identified within the genome, such as an untypeable SCC element, they proved to be generally free of virulence- and antibiotic-related genes. These results further suggest a commensal lifestyle of M. sciuri and indicate the association of antibiotic resistance determinants with mobile genetic elements, as an important factor in conferring antibiotic resistance, in addition to their unilateral annotation. Importance Mammaliicoccus sciuri has been put forward as an important carrier of virulence and antibiotic resistance genes, which can be transmitted to clinically important staphylococcal species such as Staphylococcus aureus . As a common inhabitant of mammal skin, this species is believed to have a predominant commensal lifestyle although it has been reported as an opportunistic pathogen in some cases. This study provides an extensive genome-wide description of its putative virulence potential taking into consideration the genomic context in which these genes appear, an aspect that is often overlooked during virulence analysis. Additional genome and biochemical analysis linked M. sciuri with the production of micrococcin P1, gaining further insight to which extent these biosynthetic gene cluster are distributed amongst different related species. The frequent plasmid-associated character hints that these traits can be horizontally transferred and might confer a competitive advantage to its recipient within its ecological niche.

Genomic Insights into the Distribution and Phylogeny of Glycopeptide Resistance Determinants within the Actinobacteria Phylum

The spread of antimicrobial resistance (AMR) creates a challenge for global health security, rendering many previously successful classes of antibiotics useless. Unfortunately, this also includes glycopeptide antibiotics (GPAs), such as vancomycin and teicoplanin, which are currently being considered last-resort drugs. Emerging resistance towards GPAs risks limiting the clinical use of this class of antibiotics—our ultimate line of defense against multidrug-resistant (MDR) Gram-positive pathogens. But where does this resistance come from? It is widely recognized that the GPA resistance determinants—van genes—might have originated from GPA producers, such as soil-dwelling Gram-positive actinobacteria, that use them for self-protection. In the current work, we present a comprehensive bioinformatics study on the distribution and phylogeny of GPA resistance determinants within the Actinobacteria phylum. Interestingly, van-like genes (vlgs) were found distributed in different arrangements not only among GPA-producing actinobacteria but also in the non-producers: more than 10% of the screened actinobacterial genomes contained one or multiple vlgs, while less than 1% encoded for a biosynthetic gene cluster (BGC). By phylogenetic reconstructions, our results highlight the co-evolution of the different vlgs, indicating that the most diffused are the ones coding for putative VanY carboxypeptidases, which can be found alone in the genomes or associated with a vanS/R regulatory pair.

Horizontal Transfer and Evolution of the Biosynthetic Gene Cluster for Benzoxazinoid

Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites, first identified in maize (Zea mays) and subsequently shown to be encoded by a biosynthetic gene cluster (BGC), the Bx cluster. Data mining of mining 40 high-quality grass genomes identified complete Bx clusters (containing genes Bx1 to Bx5 and Bx8) in three genera (Zea, Echinochloa and Dichanthelium) in the Panicoideae and partial clusters in the Triticeae. The Bx cluster originated from gene duplication of native analogues of Bx genes and chromosomal translocation. An ancient Bx cluster including additional Bx genes (e.g., Bx6) is found in ancestral Panicoideae. The ancient Bx cluster was gained by the Triticeae ancestor via a horizontal transfer (HT) event from the ancestral Panicoideae and later separated into three parts on different chromosomes. Bx6 appears to have been under less constrained selection during evolution of the Panicoideae as evidenced by the fact that was translocated ∼1.31-Mb away from the Bx cluster in Z. mays, moved to other chromosomes in Echinochloa, and even lost in Dichanthelium. Further investigation indicated that intense selection and polyploidization shaped the evolutionary trajectory of the Bx cluster in the grass family. This study provides the first case of HT of BGCs among plants and sheds new insights on the evolution of BGCs.SignificanceBiosynthetic gene clustering and horizontal gene transfer are two evolutionary inventions for rapid adaption by organisms. Horizontal transfer of a gene cluster has been reported in fungi and bacteria, but not in plants up to now. By mining the genomes of 40 monocot species, we deciphered the organization of Bx gene cluster, a biosynthetic gene cluster for benzoxazinoids in grasses. We found that the Bx cluster was formed by gene duplication of native analogues of individual Bx genes and directional translocation. More importantly, the Bx cluster in Triticeae was inherited from the Panicoideae via horizontal transfer. Compared with the native analogues, Bx clusters in grasses show constrained purifying selection underscoring their significance in environmental adaption.