Comparative Genomics Reveals Insights into Induction of Violacein Biosynthesis and Adaptive Evolution in Janthinobacterium.

Violacein has different bioactive properties conferring distinct selective advantages, such as defense from predation and interspecific competition. Adaptation of Janthinobacterium to diverse habitats likely leads to variation in violacein production among phylogenetically closely related species inhabiting different environments, yet genomic mechanisms and the influence of adaptive evolution underpinning violacein biosynthesis in Janthinobacterium are not clear. In this study, we performed genome sequencing, comparative genomic analysis, and phenotypic characterization to investigate genomic factors regulating violacein production in nine Janthinobacterium strains, including a type strain from soil and eight strains we isolated from terrestrial subsurface sediment and groundwater. Results show that although all nine Janthinobacterium strains are phylogenetically closely related and contain genes essential for violacein biosynthesis, they vary in carbon usage and violacein production. Sediment and groundwater strains are weak violacein producers and possess far fewer secondary metabolite biosynthesis genes, indicating genome adaptation compared to soil strains. Further examination suggests that quorum sensing (QS) may play an important role in regulating violacein in Janthinobacterium: the strains exhibiting strong potential in violacein production possess both N-acyl-homoserine lactone (AHL) QS and Janthinobacterium QS (JQS) systems in their genomes, while weaker violacein-producing strains harbor only the JQS system. Preliminary tests of spent media of two Janthinobacterium strains possessing both AHL QS and JQS systems support the potential role of AHLs in inducing violacein production in Janthinobacterium. Overall, results from this study reveal potential genomic mechanisms involved in violacein biosynthesis in Janthinobacterium and provide insights into evolution of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment. IMPORTANCE Phylogenetically closely related bacteria can thrive in diverse environmental habitats due to adaptive evolution. Genomic changes resulting from adaptive evolution lead to variations in cellular function, metabolism, and secondary metabolite biosynthesis. The most well-known secondary metabolite produced by Janthinobacterium is the purple-violet pigment violacein. To date, the mechanisms of induction of violacein biosynthesis in Janthinobacterium is not clear. Comparative genome analysis of closely related Janthinobacterium strains isolated from different environmental habitats not only reveals potential mechanisms involved in induction of violacein production by Janthinobacterium but also provides insights into the survival strategy of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment.

Hunting Bioactive Molecules from the Agave Genus: An Update on Extraction and Biological Potential.

Agaves are plants used in the production of alcoholic beverages and fibers. Ever since ancient times, pre-Hispanic cultures in Mexico have used them in traditional medicine to cure different ailments. Over the years, studies of the active principles responsible for the therapeutic benefits of agaves have increased. Leaves and fibers are the main agro-wastes generated in tequila and mezcal production, while fibers are the main waste product in the textile sector. Different investigations have referred to the agro-waste from agave processing as a source of bioactive molecules called secondary metabolites (SM). Among them, phenols, flavonoids, phytosterols, and saponins have been extracted, identified, and isolated from these plants. The role of these molecules in pest control and the prospect of metabolites with the biological potential to develop novel drugs for chronic and acute diseases represent new opportunities to add value to these agro-wastes. This review aims to update the biological activities and recent applications of the secondary metabolites of the genus Agave.

Metabolite analysis in Nymphaea 'Blue Bird' petals reveal the roles of flavonoids in color formation, stress amelioration, and bee orientation.

In this study, metabolome of open petals (OP) and closed petals (CP) from Nymphaea 'Blue Bird' was firstly investigated. A total of 455 metabolites was identified in Nymphaea 'Blue Bird' petals, which was mainly composed of 100 flavonoids, 83 phenolic acids, 64 amino acids and derivatives, 60 lipids, 32 alkaloids, 32 organic acids, 24 nucleotides and derivatives, and 12 lignans and coumarins. By differential analysis, 192 metabolites were identified with variable importance in project ≥ 1, among which 83 and 109 metabolites were up- and down-regulated in OP group, respectively. Further analysis (Log2 fold change ≥ 1) identified 26 and 7 metabolites exhibited significantly lower and higher contents in CP group, relative to OP group. Importantly, KEGG analysis indicated that flavonoid biosynthesis exhibited the most significant enrichment. qRT-PCR analysis indicated that the PAL, CHS, and HCDBR genes showed a significantly higher expression in OP group than in CP group. These data explain the increase of naringenin chalcone and phloretin in OP. However, there was no significant difference of total flavonoids between OP and CP groups. Considering the increase of H2O2 content and ultraviolet (UV) absorption peak in OP, our results implied that diurnal stressful conditions induced the degradation of flavonoids, which contributed to environmental stress amelioration. Moreover, a higher absorption peak of 360-380 nm UV was observed in the extract liquor of OP. The sensitivity maximum of the UV-photoreceptor of bees is situated around 340-380 nm UV. This suggested, as noted for the maximum absorption of dihydrokaempferol in 340-370 nm, rhythmic accumulation and loss of these differential flavonoids in Nymphaea 'Blue Bird' petals might enhance UV pattern to some degree, influencing pollinator attraction.

Transcriptome repository of North-Western Himalayan endangered medicinal herbs: a paramount approach illuminating molecular perspective of phytoactive molecules and secondary metabolism.

Medicinal plants of the North-Western Himalayan region are known for their unprecedented biodiversity and valuable secondary metabolites that are unique to this dynamic geo-climatic region. From ancient times these medicinal herbs have been used traditionally for their therapeutic potentials. But from the last 2 decades increasing pharmaceutical demand, illegal and unorganized trade of these medicinal plants have accelerated the rate of over-exploitation in a non-scientific manner. In addition, climate change and anthropogenic activities also affected their natural habitat and driving most of these endemic plant species to critically endangered that foresee peril of mass extinction from this eco-region. Hence there is an urgent need for developing alternative sustainable approaches and policies to utilize this natural bioresource ensuring simultaneous conservation. Hither, arise the advent of sequencing-based transcriptomic studies significantly contributes to better understand the background of important metabolic pathways and related genes/enzymes of high-value medicinal herbs, in the absence of genomic information. The use of comparative transcriptomics in conjunction with biochemical techniques in North-Western Himalayan medicinal plants has resulted in significant advances in the identification of the molecular players involved in the production of secondary metabolic pathways over the last decade. This information could be used to further engineer metabolic pathways and breeding programs, ultimately leading to the development of in vitro systems dedicated to the production of pharmaceutically important secondary metabolites at the industrial level. Collectively, successful adoption of these approaches can certainly ensure the sustainable utilization of Himalayan bioresource by reducing the pressure on the wild population of these critically endangered medicinal herbs. This review provides novel insight as a transcriptome-based bioresource repository for the understanding of important secondary metabolic pathways genes/enzymes and metabolism of endangered high-value North-Western Himalayan medicinal herbs, so that researchers across the globe can effectively utilize this information for devising effective strategies for the production of pharmaceutically important compounds and their scale-up for sustainable usage and take a step forward in omics-based conservation genetics.

Natural Products from Tongan Marine Organisms.

The islands of the South Pacific Ocean have been in the limelight for natural product biodiscovery, due to their unique and pristine tropical waters and environment. The Kingdom of Tonga is an archipelago in the central Indo-Pacific Ocean, consisting of 176 islands, 36 of which are inhabited, flourishing with a rich diversity of flora and fauna. Many unique natural products with interesting bioactivities have been reported from Indo-Pacific marine sponges and other invertebrate phyla; however, there have not been any reviews published to date specifically regarding natural products from Tongan marine organisms. This review covers both known and new/novel Marine Natural Products (MNPs) and their biological activities reported from organisms collected within Tongan territorial waters up to December 2020, and includes 109 MNPs in total, the majority from the phylum Porifera. The significant biological activity of these metabolites was dominated by cytotoxicity and, by reviewing these natural products, it is apparent that the bulk of the new and interesting biologically active compounds were from organisms collected from one particular island, emphasizing the geographic variability in the chemistry between these organisms collected at different locations.

Meroterpenoids: A Comprehensive Update Insight on Structural Diversity and Biology.

Meroterpenoids are secondary metabolites formed due to mixed biosynthetic pathways which are produced in part from a terpenoid co-substrate. These mixed biosynthetically hybrid compounds are widely produced by bacteria, algae, plants, and animals. Notably amazing chemical diversity is generated among meroterpenoids via a combination of terpenoid scaffolds with polyketides, alkaloids, phenols, and amino acids. This review deals with the isolation, chemical diversity, and biological effects of 452 new meroterpenoids reported from natural sources from January 2016 to December 2020. Most of the meroterpenoids possess antimicrobial, cytotoxic, antioxidant, anti-inflammatory, antiviral, enzyme inhibitory, and immunosupressive effects.

Pleiotropic effects of ActVI-ORFA as an unusual regulatory factor identified in the biosynthetic pathway of actinorhodin in Streptomyces coelicolor.

Regulatory networks play critical roles in controlling the the biosynthesis of natural products in Streptomyces. ActVI-ORFA, a regulatory factor encoded by the actinorhodin biosynthetic gene cluster (act cluster), positively controls the production of actinorhodin (ACT) in Streptomyces coelicolor, although its regulatory mechanism remains obscure. This study aimed to identify the regulatory targets of ActVI-ORFA. Deletion of ActVI-ORFA caused the differential expression of hundreds of proteins, as determined by two-dimensional electrophoresis and peptide mass fingerprinting analysis. qRT-PCR analysis of some genes encoding these differentially expressed proteins, including act genes and non-act genes, confirmed that ActVI-ORFA could control their transcriptional levels. In an electrophoretic mobility shift assay with a promoter region of a target gene located in the act cluster, no binding was detected, consistent with the lack of a recognizable DNA-binding domain in ActVI-ORFA. Overall, our findings suggest that ActVI-ORFA is a pleiotropic regulatory factor that controls multiple physiological pathways, including secondary metabolite production, probably via an indirect mode.

Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations.

The bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing dsDNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54 kb genomic region in Enterobacteriaceae. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in Enterobacteriaceae. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella species and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the Escherichia coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

Penicillium chrysogenum Fermentation and Analysis of Benzylpenicillin by Bioassay and HPLC.

Penicillium chrysogenum, recently re-identified as Penicillium rubens, is the microorganism used for the industrial production of penicillin. This filamentous fungus (mold) probably represents the best example of adaptation of a microorganism to industrial production conditions and therefore, it can be considered as a model organism for the study of primary and secondary metabolism under a highly stressful environment. In this regard, biosynthesis and production of benzylpenicillin can be used as an interesting phenotypic trait for those studies. In this chapter, we describe P. chrysogenum culture procedures for the production of benzylpenicillin and the process of antibiotic quantitation either by bioassay or by high-performance liquid chromatography (HPLC).

Screening Fermentation and Extract Generation.

This chapter describes the process of fermenting actinomycetes in flask and the generation of extracts from these broths. A medium for secondary metabolite production and a general procedure for flask fermentation are specified. Directions are given to reproduce aeration when using different flasks. The generation of extracts is based on a solvent mixture that could be varied in order to improve the extraction of products with different polarity. These extracts are then stored in a 96-well microtube format to facilitate their usage for the screening of bioactive compounds.

In Silico Analysis of P450s and Their Role in Secondary Metabolism in the Bacterial Class Gammaproteobacteria.

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.

Chemical Diversity and Biological Activity of Secondary Metabolites Isolated from Indonesian Marine Invertebrates.

Marine invertebrates have been reported to be an excellent resource of many novel bioactive compounds. Studies reported that Indonesia has remarkable yet underexplored marine natural products, with a high chemical diversity and a broad spectrum of biological activities. This review discusses recent updates on the exploration of marine natural products from Indonesian marine invertebrates (i.e., sponges, tunicates, and soft corals) throughout 2007-2020. This paper summarizes the structural diversity and biological function of the bioactive compounds isolated from Indonesian marine invertebrates as antimicrobial, antifungal, anticancer, and antiviral, while also presenting the opportunity for further investigation of novel compounds derived from Indonesian marine invertebrates.

The effects of different potato dextrose agar media on secondary metabolite production in Fusarium.

Potatoes contain several nutrients essential for fungal growth, making them an excellent component of media such as the popular Potato Dextrose Agar (PDA) medium. Commercially, PDA is available from multiple retailers offering virtually the same product. These media, however, could contain small differences in composition of nutrients affecting the expression of secondary metabolites. This study aims to investigate the use of four PDA media from different manufacturers (Fluka, Oxoid, Sigma, and VWR) and their effect on the metabolite profile of four species of Fusarium (F. fujikuroi, F. graminearum, F. pseudograminearum and F. avenaceum). Secondary metabolites were analysed using HPLC-HRMS, from which statistically significant differences in intensities were observed for 9 out of 10 metabolites.

A second riboflavin import system is present in flavinogenic Streptomyces davaonensis and supports roseoflavin biosynthesis.

The antibiotic roseoflavin is produced by Streptomyces davaonensis in the stationary phase of growth. To support biosynthesis of the secondary metabolite roseoflavin, S. davaonensis underwent several genetic adaptations with regard to metabolism of the roseoflavin precursor and primary metabolite riboflavin. In addition to 17 riboflavin biosynthesis genes at different chromosomal locations, S. davaonensis contains the riboflavin transporter gene ribM being part of the riboflavin biosynthetic operon ribE1MAB5H. Deletion of this operon generated riboflavin auxotrophic S. davaonensis strains. The finding that S. davaonensis ΔribE1MAB5H was able to grow in a culture medium containing low levels of riboflavin indicated that in addition to RibM, a second riboflavin transporter is present in this bacterium. The S. davaonensis genes ribXY (former rosXY) represented candidate genes for such a second riboflavin transport system and the results of our experiments now show that RibXY from S. davaonensis is a highly efficient riboflavin importer but not a roseoflavin importer.

The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase.

Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.

Competition Sensing Changes Antibiotic Production in Streptomyces.

One of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require "elicitors" to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors, and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing strains of streptomycetes, we show that strains commonly inhibit each other's growth and that this occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreases in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competition. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes.IMPORTANCE Bacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces cells are more likely to produce antibiotics when grown with competitors that are closely related or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor's toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces cells also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

Metabolons, enzyme-enzyme assemblies that mediate substrate channeling, and their roles in plant metabolism.

Metabolons are transient multi-protein complexes of sequential enzymes that mediate substrate channeling. They differ from multi-enzyme complexes in that they are dynamic, rather than permanent, and as such have considerably lower dissociation constants. Despite the fact that a huge number of metabolons have been suggested to exist in plants, most of these claims are erroneous as only a handful of these have been proven to channel metabolites. We believe that physical protein-protein interactions between consecutive enzymes of a pathway should rather be called enzyme-enzyme assemblies. In this review, we describe how metabolons are generally assembled by transient interactions and held together by both structural elements and non-covalent interactions. Experimental evidence for their existence comes from protein-protein interaction studies, which indicate that the enzymes physically interact, and direct substrate channeling measurements, which indicate that they functionally interact. Unfortunately, advances in cell biology and proteomics have far outstripped those in classical enzymology and flux measurements, rendering most reports reliant purely on interactome studies. Recent developments in co-fractionation mass spectrometry will likely further exacerbate this bias. Given this, only dynamic enzyme-enzyme assemblies in which both physical and functional interactions have been demonstrated should be termed metabolons. We discuss the level of evidence for the manifold plant pathways that have been postulated to contain metabolons and then list examples in both primary and secondary metabolism for which strong evidence has been provided to support these claims. In doing so, we pay particular attention to experimental and mathematical approaches to study metabolons as well as complexities that arise in attempting to follow them. Finally, we discuss perspectives for improving our understanding of these fascinating but enigmatic interactions.

Natural Products in Polyclad Flatworms.

Marine invertebrates are promising sources of novel bioactive secondary metabolites, and organisms like sponges, ascidians and nudibranchs are characterised by possessing potent defensive chemicals. Animals that possess chemical defences often advertise this fact with aposematic colouration that potential predators learn to avoid. One seemingly defenceless group that can present bright colouration patterns are flatworms of the order Polycladida. Although members of this group have typically been overlooked due to their solitary and benthic nature, recent studies have isolated the neurotoxin tetrodotoxin from these mesopredators. This review considers the potential of polyclads as potential sources of natural products and reviews what is known of the activity of the molecules found in these animals. Considering the ecology and diversity of polyclads, only a small number of species from both suborders of Polycladida, Acotylea and Cotylea have been investigated for natural products. As such, confirming assumptions as to which species are in any sense toxic or if the compounds they use are biosynthesised, accumulated from food or the product of symbiotic bacteria is difficult. However, further research into the group is suggested as these animals often display aposematic colouration and are known to prey on invertebrates rich in bioactive secondary metabolites.

Recent progress in biodiversity research on the Xylariales and their secondary metabolism.

The families Xylariaceae and Hypoxylaceae (Xylariales, Ascomycota) represent one of the most prolific lineages of secondary metabolite producers. Like many other fungal taxa, they exhibit their highest diversity in the tropics. The stromata as well as the mycelial cultures of these fungi (the latter of which are frequently being isolated as endophytes of seed plants) have given rise to the discovery of many unprecedented secondary metabolites. Some of those served as lead compounds for development of pharmaceuticals and agrochemicals. Recently, the endophytic Xylariales have also come in the focus of biological control, since some of their species show strong antagonistic effects against fungal and other pathogens. New compounds, including volatiles as well as nonvolatiles, are steadily being discovered from these ascomycetes, and polythetic taxonomy now allows for elucidation of the life cycle of the endophytes for the first time. Moreover, recently high-quality genome sequences of some strains have become available, which facilitates phylogenomic studies as well as the elucidation of the biosynthetic gene clusters (BGC) as a starting point for synthetic biotechnology approaches. In this review, we summarize recent findings, focusing on the publications of the past 3 years.

Links among extracellular enzymes, lignin degradation and cell growth establish the models to identify marine lignin-utilizing bacteria.

A major conundrum in the isolation of prokaryotes from open environments is stochasticity. It is especially difficult to study low abundance groups where very little biological information exists, although single-cell genomics and metagenomics have alleviated some of this bottleneck. Here, we report an approach to capture lignin-utilizing bacteria by linking a physical model to actual organisms. Extracellular enzymes, lignin degradation and cell growth are crucial phenotypes of lignin-utilizing bacteria, but their interrelationships remain poorly understood. In this study, the phenotypes of bacteria isolated from in situ lignocellulose enrichment samples in coastal waters were traced and statistically analysed. It suggested cell growth, dye-decolorizing peroxidase (DyP) and reactive oxygen species (ROS) were significantly correlated with lignin degradation, exhibiting a genus-specific property. The established models enabled us to efficiently capture lignin-utilizing bacteria and rapidly evaluate lignin degradation for Bacillus and Vibrio strains. Through the model, we identified several previously unrecognized marine bacterial lignin degraders. Moreover, it demonstrated that the isolated marine lignin-utilizing bacteria employ a DyP-based system and ROS for lignin depolymerization, providing insights into the mechanism of marine bacterial lignin degradation. Our findings should have implications beyond the capture of lignin-utilizing bacteria, in the isolation of other microorganisms with as-yet-unknown molecular biomarkers.