Characterization of Bioactivities and Biosynthesis of Angucycline/Angucyclinone Derivatives Derived from Gephyromycinifex aptenodytis gen. nov., sp. nov.

Strain NJES-13T is the type strain and currently the only species of the newly established actinobacteria genera Aptenodytes in the family Dermatophilaceae isolated from the gut microbiota of the Antarctic emperor penguin. This strain demonstrated excellent bioflocculation activity with bacteria-derived exopolysaccharides (EPSs). Moreover, it produced bioactive angucycline/angucyclinone derivatives (ADs) and contained one type III polyketide synthase (T3PKS), thus demonstrating great potential to produce novel bioactive compounds. However, the low productivity of the potential new AD metabolite was the main obstacle for its chemical structure elucidation. In this study, to increase the concentration of targeted metabolites, the influence of cellular morphology on AD metabolism in strain NJES-13T was determined using glass bead-enhanced fermentation. Based on the cellular ultra-structural observation driven by bacterial EPSs, and quantitative analysis of the targeted metabolites, the successful increasing of the productivity of three AD metabolites was achieved. Afterward, a new frigocyclinone analogue was isolated and then identified as 2-hydroxy-frigocyclinone, as well as two other known ADs named 2-hydroxy-tetrangomycin (2-HT) and gephyromycin (GPM). Three AD metabolites were found to demonstrate different bioactivities. Both C-2 hydroxyl substitutes, 2-hydroxy-tetrangomycin and 2-hydroxy-frigocyclinone, exhibited variable inhibitory activities against Staphylococcus aureus, Bacillus subtilis and Candida albicans. Moreover, the newly identified 2-hydroxy-frigocyclinone also showed significant cytotoxicity against three tested human-derived cancerous cell lines (HL-60, Bel-7402 and A549), with all obtained IC50 values less than 10 µM. Based on the genetic analysis after genomic mining, the plausible biogenetic pathway of the three bioactive ADs in strain NJES-13T was also proposed.

Identification and Heterologous Expression of the Kendomycin B Biosynthetic Gene Cluster from Verrucosispora sp. SCSIO 07399

Verrucosispora sp. SCSIO 07399, a rare marine-derived actinomycete, produces a set of ansamycin-like polyketides kendomycin B–D (1–3) which possess potent antibacterial activities and moderate tumor cytotoxicity. Structurally, kendomycin B–D contain a unique aliphatic macrocyclic ansa scaffold in which the highly substituted pyran ring is connected to the quinone moiety. In this work, a type I/type III polyketide synthase (PKS) hybrid biosynthetic gene cluster coding for assembly of kendomycin B (kmy), and covering 33 open reading frames, was identified from Verrucosispora sp. SCSIO 07399. The kmy cluster was found to be essential for kendomycin B biosynthesis as verified by gene disruption and heterologous expression. Correspondingly, a biosynthetic pathway was proposed based on bioinformatics, cluster alignments, and previous research. Additionally, the role of type III PKS for generating the precursor unit 3,5-dihydroxybenzoic acid (3,5-DHBA) was demonstrated by chemical complementation, and type I PKS executed the polyketide chain elongation. The kmy cluster was found to contain a positive regulatory gene kmy4 whose regulatory effect was identified using real-time quantitative PCR (RT-qPCR). These advances shed important new insights into kendomycin B biosynthesis and help to set the foundation for further research aimed at understanding and exploiting the carbacylic ansa scaffold.

CRISPR/Cas9-mediated disruption of the PYRROLIDINE KETIDE SYNTHASE gene reduces the accumulation of tropane alkaloids in Atropa belladonna hairy roots

Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A non-canonical type III polyketide synthase (PKS), pyrrolidine ketide synthase (PYKS), catalyzes a two-step decarboxylative reaction, which involves imine-ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.

Probiotic and Antifungal Attributes of Levilactobacillus brevis MYSN105, Isolated From an Indian Traditional Fermented Food Pozha

The use of probiotics and antifungal capabilities of the lactic acid bacteria (LAB) isolated from different niches is a strategy to prepare functional cultures and biopreservatives for food/feed industries. In the present study, LAB strains isolated from an Indian traditional fermented food, Pozha, were evaluated for their probiotic properties and biocontrol potential. A total of 20 LAB isolates were selected from Pozha samples collected aseptically and screened for their antagonistic activity against Fusarium verticillioides. Among the bioactive isolates, Lacticaseibacillus brevis MYSN105 showed the highest antifungal activity in vitro, causing some morphological alterations such as damaged mycelia and deformed conidia. Cell-free supernatant (CFS) from L. brevis MYSN105 at 16% concentration effectively reduced the mycelial biomass to 0.369 g compared to 1.938 g in control. Likewise, the conidial germination was inhibited to 20.12%, and the seed treatment using CFS induced a reduction of spore count to 4.1 × 106 spores/ml compared to 1.1 × 109 spores/ml for untreated seeds. The internal transcribed spacer (ITS) copy number of F. verticillioides decreased to 5.73 × 107 and 9.026 × 107 by L. brevis MYSN105 and CFS treatment, respectively, compared to 8.94 × 1010 in control. The L. brevis MYSN105 showed high tolerance to in vitro gastrointestinal conditions and exhibited high adhesive abilities to intestinal epithelial cell lines. The comparative genome analysis demonstrated specific secondary metabolite region coding for bacteriocin and T3PKS (type III polyketide synthase) possibly related to survival and antimicrobial activity in the gut environment. Our results suggest that L. brevis MYSN105 has promising probiotic features and could be potentially used for developing biological control formulations to minimize F. verticillioides contamination and improve food safety measures.

A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis

Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer’s disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate–dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.

Comparative genomics and physiological investigation supported safety, cold adaptation, efficient hydrolytic and plant growth-promoting potential of psychrotrophic Glutamicibacter arilaitensis LJH19, isolated from night-soil compost

Background Night-soil compost (NSC) has traditionally been conserving water and a source of organic manure in northwestern Himalaya. Lately, this traditional method is declining due to modernization, its unhygienic conditions, and social apprehensions. Reduction in the age-old traditional practice has led to excessive chemical fertilizers and water shortage in the eco-sensitive region. In the current study, a bacterium has been analyzed for its safety, cold-adaptation, efficient degradation, and plant growth-promoting (PGP) attributes for its possible application as a safe bioinoculant in psychrotrophic bacterial consortia for improved night-soil composting. Results Glutamicibacter arilaitensis LJH19, a psychrotrophic bacterium, was isolated from the NSC of Lahaul valley in northwestern Himalaya. The strain exhibited amylase (186.76 ± 19.28 U/mg), cellulase (21.85 ± 0.7 U/mg), and xylanase (11.31 ± 0.51 U/mg) activities at 10 °C. Possessing efficient hydrolytic activities at low-temperature garners the capability of efficient composting to LJH19. Additionally, the strain possessed multiple PGP traits such as indole acetic acid production (166.11 ± 5.7 μg/ml), siderophore production (85.72 ± 1.06% psu), and phosphate solubilization (44.76 ± 1.5 μg/ml). Enhanced germination index and germination rate of pea seeds under the LJH19 inoculation further supported the bacterium’s PGP potential. Whole-genome sequencing (3,602,821 bps) and genome mining endorsed the cold adaptation, degradation of polysaccharides, and PGP traits of LJH19. Biosynthetic gene clusters for type III polyketide synthase (PKS), terpene, and siderophore supplemented the endorsement of LJH19 as a potential PGP bacterium. Comparative genomics within the genus revealed 217 unique genes specific to hydrolytic and PGP activity. Conclusion The physiological and genomic evidence promotes LJH19 as a potentially safe bio-inoculant to formulate psychrotrophic bacterial consortia for accelerated degradation and improved night-soil compost.

Quantitative trait locus mapping combined with variant and transcriptome analyses identifies a cluster of gene candidates underlying the variation in leaf wax between upland and lowland switchgrass ecotypes

Key message Mapping combined with expression and variant analyses in switchgrass, a crop with complex genetics, identified a cluster of candidate genes for leaf wax in a fast-evolving region of chromosome 7K. Abstract Switchgrass (Panicum virgatum L.) is a promising warm-season candidate energy crop. It occurs in two ecotypes, upland and lowland, which vary in a number of phenotypic traits, including leaf glaucousness. To initiate trait mapping, two F2 mapping populations were developed by crossing two different F1 sibs derived from a cross between the tetraploid lowland genotype AP13 and the tetraploid upland genotype VS16, and high-density linkage maps were generated. Quantitative trait locus (QTL) analyses of visually scored leaf glaucousness and of hydrophobicity of the abaxial leaf surface measured using a drop shape analyzer identified highly significant colocalizing QTL on chromosome 7K (Chr07K). Using a multipronged approach, we identified a cluster of genes including Pavir.7KG077009, which encodes a Type III polyketide synthase-like protein, and Pavir.7KG013754 and Pavir.7KG030500, two highly similar genes that encode putative acyl-acyl carrier protein (ACP) thioesterases, as strong candidates underlying the QTL. The lack of homoeologs for any of the three genes on Chr07N, the relatively low level of identity with other switchgrass KCS proteins and thioesterases, as well as the organization of the surrounding region suggest that Pavir.7KG077009 and Pavir.7KG013754/Pavir.7KG030500 were duplicated into a fast-evolving chromosome region, which led to their neofunctionalization. Furthermore, sequence analyses showed all three genes to be absent in the two upland compared to the two lowland accessions analyzed. This study provides an example of and practical guide for trait mapping and candidate gene identification in a complex genetic system by combining QTL mapping, transcriptomics and variant analysis.

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.