Biosynthetic gene cluster synteny: Orthologous polyketide synthases in Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus), which together generate a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here, we provide a comparative view of the biosynthetic gene clusters of three lichen mycobionts derived from Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata. In addition, we present a high-quality PacBio metagenome of Parmelia sulcata, from which we extracted the mycobiont bin containing 214 biosynthetic gene clusters. Most biosynthetic gene clusters in these genomes were associated with T1PKSs, followed by NRPSs and terpenes. This study focused on biosynthetic gene clusters related to polyketide synthesis. Based on ketosynthase homology, we identified nine highly syntenic clusters present in all three species. Among the four clusters belonging to nonreducing PKSs, two are putatively linked to lichen substances derived from orsellinic acid (orcinol depsides and depsidones, e.g., lecanoric acid, physodic acid, lobaric acid), one to compounds derived from methylated forms of orsellinic acid (beta orcinol depsides, e.g., atranorin), and one to melanins. Five clusters with orthologs in all three species are linked to reducing PKSs. Our study contributes to sorting and dereplicating the vast PKS diversity found in lichenized fungi. High-quality sequences of biosynthetic gene clusters of these three common species provide a foundation for further exploration into biotechnological applications and the molecular evolution of lichen substances.

Differential RNA-Seq Analysis Predicts Genes Related to Terpene Tailoring in Caryopteris × clandonensis.

Enzymatic terpene functionalization is an essential part of plant secondary metabolite diversity. Within this, multiple terpene-modifying enzymes are required to enable the chemical diversity of volatile compounds essential in plant communication and defense. This work sheds light on the differentially transcribed genes within Caryopteris × clandonensis that are capable of functionalizing cyclic terpene scaffolds, which are the product of terpene cyclase action. The available genomic reference was subjected to further improvements to provide a comprehensive basis, where the number of contigs was minimized. RNA-Seq data of six cultivars, Dark Knight, Grand Bleu, Good as Gold, Hint of Gold, Pink Perfection, and Sunny Blue, were mapped on the reference, and their distinct transcription profile investigated. Within this data resource, we detected interesting variations and additionally genes with high and low transcript abundancies in leaves of Caryopteris × clandonensis related to terpene functionalization. As previously described, different cultivars vary in their modification of monoterpenes, especially limonene, resulting in different limonene-derived molecules. This study focuses on predicting the cytochrome p450 enzymes underlying this varied transcription pattern between investigated samples. Thus, making them a reasonable explanation for terpenoid differences between these plants. Furthermore, these data provide the basis for functional assays and the verification of putative enzyme activities.

Biosynthetic Potential of Hypogymnia Holobionts: Insights into Secondary Metabolite Pathways.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus). They are known to produce a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here we provide a comprehensive view of the biosynthetic gene clusters of all organisms comprising a lichen thallus: fungi, green algae, and bacteria. We present two high-quality PacBio metagenomes, in which we identified a total of 460 biosynthetic gene clusters. Lichen mycobionts yielded 73-114 clusters, other lichen associated ascomycetes 8-40, green algae of the genus Trebouxia 14-19, and lichen-associated bacteria 101-105 clusters. The mycobionts contained mainly T1PKSs, followed by NRPSs, and terpenes; Trebouxia reads harbored mainly clusters linked to terpenes, followed by NRPSs and T3PKSs. Other lichen-associated ascomycetes and bacteria contained a mix of diverse biosynthetic gene clusters. In this study, we identified for the first time the biosynthetic gene clusters of entire lichen holobionts. The yet untapped biosynthetic potential of two species of the genus Hypogymnia is made accessible for further research.

Comparative Genome-Wide Analysis of Two Caryopteris x Clandonensis Cultivars: Insights on the Biosynthesis of Volatile Terpenoids.

Caryopteris x Clandonensis, also known as bluebeard, is an ornamental plant containing a large variety of terpenes and terpene-like compounds. Four different cultivars were subjected to a principal component analysis to elucidate variations in terpenoid-biosynthesis and consequently, two representative cultivars were sequenced on a genomic level. Functional annotation of genes as well as comparative genome analysis on long read datasets enabled the identification of cultivar-specific terpene synthase and cytochrome p450 enzyme sequences. This enables new insights, especially since terpenoids in research and industry are gaining increasing interest due to their importance in areas such as food preservation, fragrances, or as active ingredients in pharmaceutical formulations. According to BUSCO assessments, the presented genomes have an average size of 355 Mb and about 96.8% completeness. An average of 52,090 genes could be annotated as putative proteins, whereas about 42 were associated with terpene synthases and about 1340 with cytochrome p450 enzymes.

Selective ene-reductase immobilization to magnetic nanoparticles through a novel affinity tag.

BACKGROUND: Magnetic nanoparticles (MNPs) are becoming more important as carriers, because of their large specific surface area and easy separability. They are increasingly used in enzyme technology, diagnostics, and drug delivery. MAJOR RESULTS: For the directed and almost irreversible immobilization of proteins on MNPs, we have developed a new selective (His-Arg)4 peptide-tag, that binds fusion proteins directly from an E. coli cell lysate to non-functionalized, low-cost MNPs. Using the immobilization of an ene-reductase as an example, we could demonstrate that the fusion with this tag increases thermostability without reducing overall activity (ER w/o tag: t1/2  = 3.7 h, (HR)4 -ER: t1/2  = 9.9 h). Immobilization by adsorption in Tris buffer resulted in very high enzyme loads with approx. 380 mg g-1 and 67% residual activity. The immobilization on the MNPs allowed a fast concentration, buffer exchange, and reuse. While about 50% of the activity was lost after the first reuse, we were able to show that the activity did not decrease further and was stable for another nine cycles. CONCLUSION: According to our studies, our tag highly works for any kind of immobilization on MNPs and holds the potential for enzyme immobilizations as well as for drug delivery and sensors.

Knowledge, attitude and practice for breast cancer risk factors and screening modalities in staff nurses of Ayub Teaching Hospital Abbottabad.

BACKGROUND: Breast cancer is the commonest cancer modality in female worldwide. Avoiding the risk factors can reduce its incidence and adhering to screening and early detection can reduce its mortality. A sufficient knowledge regarding the risk factors and screening modalities is therefore essential. We assessed the knowledge level about these parameters in our staff nurses. METHODS: A self-administered questionnaire survey was performed. Knowledge regarding the risk factors and screening modalities were categorised into good, fair, poor and very poor categories. RESULTS: Knowledge regarding most of the factors was found to be fair. A few things were termed as good knowledge like role of breast-feeding in protecting against breast cancer. Practice regarding the screening modalities was not satisfactory. Only a few nurses had good knowledge of the risk factors and screening modalities. Practice of the Screening modalities was also poor. CONCLUSION: There is a need to improve the nursing curriculum, training at the workplace and motivate them for screening practices. They should be encouraged to talk to their patients and their female attendants about prevention and early detection of breast cancer.