Ribosomally derived lipopeptides containing distinct fatty acyl moieties

Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non–gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.

BDSF is a degradation-prone quorum-sensing signal detected by the histidine kinase RpfC of Xanthomonas campestris pv. campestris

Diffusible signal factors (DSFs) are medium-chain fatty acids that induce bacterial quorum sensing. Among these compounds, BDSF is a structural analog of DSF that is commonly detected in bacterial species (e.g., Xanthomonas, Pseudomonas, and Burkholderia). Additionally, BDSF contributes to the interkingdom communication regulating fungal life stage transitions. How BDSF is sensed in Xanthomonas spp. and the functional diversity between BDSF and DSF remain unclear. In this study, we generated genetic and biochemical evidence that BDSF is a low-active regulator of X. campestris pv. campestris quorum sensing, whereas trans-BDSF seems not a signaling compound. BDSF is detected by the sensor histidine kinase RpfC. Although BDSF has relatively low physiological activities, it binds to the RpfC sensor with a high affinity and activates RpfC autophosphorylation to a level that is similar to that induced by DSF in vitro. The inconsistency in the physiological and biochemical activities of BDSF is not due to RpfC–RpfG phosphorylation or RpfG hydrolase. Neither BDSF nor DSF controls the phosphotransferase and phosphatase activities of RpfC or the ability of RpfG hydrolase to degrade the bacterial second messenger cyclic di-GMP. We demonstrated that BDSF is prone to degradation by RpfB, a critical fatty acyl-CoA ligase involved in the turnover of DSF-family signals. rpfB mutations lead to substantial increases in BDSF-induced quorum sensing. Although DSF and BDSF are similarly detected by RpfC, our data suggest that their differential degradation in cells is the major factor responsible for the diversity in their physiological effects.

Characterization of a Pentacyclic Triterpene Acetyltransferase Involved in the Biosynthesis of Taraxasterol and ψ-Taraxasterol Acetates in Lettuce

Triterpenoids exist in a free state and/or in conjugated states, such as triterpene glycosides (saponins) or triterpene esters. There is no information on the enzyme participating in the production of triterpene esters from free triterpenes. Lettuce (Lactuca sativa) contains various pentacyclic triterpene acetates (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, β-amyrin acetates, and germanicol acetate). In this study, we report a novel triterpene acetyltransferase (LsTAT1) in lettuce involved in the biosynthesis of pentacyclic triterpene acetates from free triterpenes. The deduced amino acid sequences of LsTAT1 showed a phylogenetic relationship (43% identity) with those of sterol O-acyltransferase (AtSAT1) of Arabidopsis thaliana and had catalytic amino acid residues (Asn and His) that are typically conserved in membrane-bound O-acyltransferase (MBOAT) family proteins. An analysis of LsTAT1 enzyme activity in a cell-free system revealed that the enzyme exhibited activity for the acetylation of taraxasterol, ψ-taraxasterol, β-amyrin, α-amyrin, lupeol, and taraxerol using acetyl-CoA as an acyl donor but no activity for triterpene acylation using a fatty acyl donor. Lettuce oxidosqualene cyclase (LsOSC1) is a triterpene synthase that produces ψ-taraxasterol, taraxasterol, β-amyrin and α-amyrin. The ectopic expression of both the LsOSC1 and LsTAT1 genes in yeast and tobacco could produce taraxasterol acetate, ψ-taraxasterol acetate, β-amyrin acetate, and α-amyrin acetate. However, expression of the LsTAT1 gene in tobacco was unable to induce the conversion of intrinsic sterols (campesterol, stigmasterol, and β-sitosterol) to sterol acetates. The results demonstrate that the LsTAT1 enzyme is a new class of acetyltransferase belong to the MBOAT family that have a particular role in the acetylation of pentacyclic triterpenes and are thus functionally different from sterol acyltransferase conjugating fatty acyl esters.

Dioctanoyl Ultrashort Tetrabasic β-Peptides Sensitize Multidrug-Resistant Gram-Negative Bacteria to Novobiocin and Rifampicin

Recently reported peptidomimetics with increased resistance to trypsin were shown to sensitize priority multidrug-resistant (MDR) Gram-negative bacteria to novobiocin and rifampicin. To further optimize proteolytic stability, β-amino acid-containing derivatives of these compounds were prepared, resulting in three dioctanoyl ultrashort tetrabasic β-peptides (dUSTBβPs). The nonhemolytic dUSTBβP 3, comprised of three β3-homoarginine residues and two fatty acyl tails eight carbons long, enhanced the antibacterial activity of various antibiotics from different classes. Notably, compound 3 retained the ability to potentiate novobiocin and rifampicin in wild-type Gram-negative bacteria against MDR clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. dUSTBβP 3 reduced the minimum inhibitory concentration of novobiocin and rifampicin below their interpretative susceptibility breakpoints. Furthermore, compound 3 exhibited improved in vitro stability (86.8 ± 3.7% remaining) relative to its α-amino acid-based counterpart (39.5 ± 7.4% remaining) after a 2 h incubation in human plasma.

An Untargeted Lipidomic Analysis Reveals Depletion of Several Phospholipid Classes in Patients with Familial Hypercholesterolemia on Treatment with Evolocumab

Rationale: Familial hypercholesterolemia (FH) is caused by mutations in genes involved in low-density lipoprotein cholesterol (LDL-C) metabolism, including those for pro-protein convertase subtilisin/kexin type 9 (PCSK-9). The effect of PCSK-9 inhibition on the plasma lipidome has been poorly explored. Objective: Using an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, the plasma lipidome of FH subjects before and at different time intervals during treatment with the PCSK-9 inhibitor Evolocumab was explored. Methods and Results: In 25 FH subjects, heterozygotes or compound heterozygotes for different LDL receptor mutations, untargeted lipidomic revealed significant reductions in 26 lipid classes belonging to phosphatidylcholine (PC), sphingomyelin (SM), ceramide (CER), cholesteryl ester (CE), triacylglycerol (TG) and phosphatidylinositol (PI). Lipid changes were graded between baseline and 4- and 12-week treatment. At 12-week treatment, five polyunsaturated diacyl PC, accounting for 38.6 to 49.2% of total PC at baseline; two ether/vinyl ether forms; seven SM; five CER and glucosyl/galactosyl-ceramide (HEX-CER) were reduced, as was the unsaturation index of HEX-CER and lactosyl—CER (LAC-CER). Although non quantitative modifications were observed in phosphatidylethanolamine (PE) during treatment with Evolocumab, shorter and more saturated fatty acyl chains were documented. Conclusions: Depletion of several phospholipid classes occurs in plasma of FH patients during treatment with the PCSK-9 inhibitor Evolocumab. The mechanism underlying these changes likely involves the de novo synthesis of SM and CER through the activation of the key enzyme sphingomyelin synthase by oxidized LDL and argues for a multifaceted system leading to vascular improvement in users of PCSK-9 inhibitors.

Transcriptome analysis identifies candidate genes in the biosynthetic pathway of sex pheromones from a zygaenid moth, Achelura yunnanensis (Lepidoptera: Zygaenidae)

In most moth species, sex pheromones responsible for mating and communication of both sexes are primarily produced by the pheromone glands (PGs) of female moths. Although the PG transcriptomes and pheromone production related genes from 24 moth species have been characterized, studies on the related information remain unknown in the Zygaenidae family. Here, we sequenced the PG transcriptome of a zygaenid moth, Achelura yunnanensis. Such the sequencing resulted in the yields of 47,632,610 clean reads that were assembled into 54,297 unigenes, coupled with RNA sequencing data from 12 other tissues. Based on the transcriptome, a total of 191 genes encoding pheromone biosynthesis and degradation enzymes were identified, 161 of which were predicted to have full-length sequences. A comparative analysis among 24 moth species of nine families indicated that the numbers of the genes were variable, ranging from 14 in two Grapholita species to 191 in A. yunnanensis. Phylogenetic analysis in parallel with the expression data highlighted some key genes, including three △9 and four △11 desaturases, four fatty acyl-CoA reductases (FARs) clustering in the pgFAR clade, and three significantly antennae-enriched aldehyde oxidases. An extensive tissue- and sex- expression profile revealed a broad distribution of the genes, in which 128 relatives were detected in the PGs and 127 in the antennae. This study reports, for the first time, the gene repertoires associated with the pheromone production in Zygaenidae, and provides a valuable resource for exploring putative roles of the PG-enriched genes in A. yunnanensis.

Integrated Analysis of Fatty Acid Metabolism and Transcriptome Involved in Olive Fruit Development to Improve Oil Composition

Olea europaea L. is an important oil crop with excellent nutritional properties. In this study, a full-length transcriptome combined with fatty acid composition was used to investigate the molecular mechanism of fatty acid (FA) metabolism of olive fruits at various stages of development (S1–S5). A total of 34 fatty acids (FAs) were measured using gas chromatography-mass spectrometry (GC-MS). All transcripts of FA metabolism during olive fruit development were studied, including glycolysis, fatty acid synthesis, triacylglycerol synthesis, and FA degradation. A total of 100 transcripts of 11 gene families, 68 transcripts of 12 gene families, 55 transcripts of 7 gene families, and 28 transcripts of 7 gene families were identified as encoding for enzymes involved in FA metabolism. Furthermore, one of the critical reactions in TAG metabolism is the activation of fatty acyl chains to fatty acyl CoA, which is catalyzed by long-chain acyl CoA synthetases (LACS). Phylogenetic analysis showed that 13 putative LACS-encoding genes clustered into five groups, of which two putative transcripts encoding LACS6/7 may participate in FA degradation. The aim of this study was to evaluate the fatty acid from synthesis to degradation pathways during olive fruit development to provide a better understanding of the molecular mechanism of FA metabolism during olive fruit maturation and provide information to improve the synthesis of oil components that are beneficial to human health.