Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides.

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.

Characteristics of Bacterial Strains with Desirable Flavor Compounds from Korean Traditional Fermented Soybean Paste (Doenjang).

To identify and analyze the characteristics of the microorganisms involved in the formation of the desirable flavor of Doenjang, a total of 179 strains were isolated from ninety-four Doenjang collected from six regions in South Korea, and fourteen strains were selected through a sensory evaluation of the aroma of each culture. The enzyme activities of amylase, protease and lipase was shown in the various strains. Bacillus sp.-K3, Bacillus sp.-K4 and Bacillus amyloliquefaciens-J2 showed relatively high protease activity, at 317.1 U, 317.3 U and 319.5 U, respectively. The Bacillus sp.-K1 showed the highest lipase activity at 2453.6 U. In the case of amylase, Bacillus subtilis-H6 showed the highest activity at 4105.5 U. The results of the PCA showed that Bacillus subtilis-H2, Bacillus subtilis-H3, and Bacillus sp.-K2 were closely related to the production of 3-hydroxy-2-butanone (23.51%~43.37%), and that Bacillus subtilis-H5 and Bacillus amyloliquefaciens-J2 were significantly associated with the production of phenethyl alcohol (0.39% and 0.37%). The production of peptides was observed to vary among the Bacillus cultures such as Val-Val-Pro-Pro-Phe-Leu and Pro-Ala-Glu-Val-Leu-Asp-Ile. These peptides are precursors of related volatile flavor compounds created in Doenjang via the enzymatic or non-enzymatic route; it is expected that these strains could be used to enhance the flavor of Doenjang.

Metabolic Shift of an Isogenic Strain of Enterococcus faecalis 14, Deficient in Its Own Bacteriocin Synthesis, as Revealed by a Transcriptomic Analysis.

The production of antimicrobial molecules often involves complex biological pathways. This study aimed at understanding the metabolic and physiological networks of enterocin EntDD14-associated function, in the bacteriocinogenic strain, Enterococcus faecalis 14. A global and comparative transcriptomic study was carried out on E. faecalis 14 and its isogenic mutant Δbac, inactivated in genes coding for EntDD14. The in vitro ability to form biofilm on polystyrene plates was assessed by the crystal violet method, while the cytotoxicity on human colorectal adenocarcinoma Caco-2 cells was determined by the Cell Counting Kit-8. Transcriptomic data revealed that 71 genes were differentially expressed in both strains. As expected, genes coding for EntDD14 were downregulated in the Δbac mutant, whereas the other 69 genes were upregulated. Upregulated genes were associated with phage-related chromosomal islands, biofilm formation capability, resistance to environmental stresses, and metabolic reprogramming. Interestingly, the Δbac mutant showed an improved bacterial growth, a high capacity to form biofilm on inanimate surfaces and a very weak cytotoxicity level. These multiple metabolic rearrangements delineate a new line of defense to counterbalance the loss of EntDD14.

MiniBacillus PG10 as a Convenient and Effective Production Host for Lantibiotics.

Efficient bacterial cell factories are important for the screening and characterization of potent antimicrobial peptides such as lantibiotics. Although lantibiotic production systems have been established in Lactococcus lactis and Escherichia coli, the industrial workhorse Bacillus subtilis has been left relatively unexplored as a lantibiotic production host. Therefore, we tested different B. subtilis strains for their ability to produce lantibiotic peptides by using the subtilin modification and transport enzymes derived from the natural subtilin producer B. subtilis ATCC 6633. Our study shows that although B. subtilis ATCC 6633 and 168 are able to produce various processed lantibiotic peptides, an evident advantage of using either the 8-fold protease-deficient strain WB800 or the genome-minimized B. subtilis 168 strain PG10 is the lack of extracellular serine protease activity. Consequently, leader processing of lantibiotic precursor peptides is circumvented and thus potential toxicity toward the production host is prevented. Furthermore, PG10 provides a clean secondary metabolic background and therefore appears to be the most promising B. subtilis lantibiotic production host. We demonstrate the production of various lantibiotic precursor peptides by PG10 and show different options for their in vitro activation. Our study thus provides a convenient B. subtilis-based lantibiotic production system, which facilitates the search for novel antimicrobial peptides.

Artificial Splitting of a Non-Ribosomal Peptide Synthetase by Inserting Natural Docking Domains.

The interaction in multisubunit non-ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit-to-subunit interaction. We introduced natural docking domains into the three-module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC-ESI-MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS-PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split-module cases compared to the full-length wild-type enzyme.

Recombinant peptide fusion construction for protein-templated catalytic palladium nanoparticles.

Although peptide-enabled synthesis of nanostructures has garnered considerable interest for use in catalytic applications, it has so far been achieved mostly via Fmoc based solid phase peptide synthesis. Consequently, the potential of longer peptides in nanoparticle synthesis have not been explored largely due to the complexities and economic constraints of this chemical synthesis route. This study examines the potential of a 45-amino acid long peptide expressed as fusion to green fluorescence protein (GFPuv) in Escherichia coli for use in palladium nanoparticle synthesis. Fed-batch fermentation with E. coli harboring an arabinose-inducible plasmid produced a product containing three copies of Pd4 peptide fused to N-terminus of GFPuv ((Pd4)3 -GFPuv). Using the intrinsic fluorescence of GFPuv, expression and enrichment of the fusion product was easily monitored. Crude lysate, desalted lysate, and an ion-exchange enriched fraction containing (Pd4)3 -GFPuv were used to test the hypothesis that high purity of the biologic material used as the nanoparticle synthesis template may not be necessary. Nanoparticles were characterized using a variety of material science techniques and used to catalyze a model Suzuki-Miyaura coupling reaction. Results demonstrated that palladium nanoparticles can be synthesized using the soluble cell extract containing (Pd4)3 -GFPuv without extensive purification or cleavage steps, and as a catalyst the crude mixture is functional.

Discovery of the Biosynthetic Machinery for Stravidins, Biotin Antimetabolites.

Stravidins are peptide antibiotics produced by Streptomyces spp. Their antibacterial activity derives from an unusual amiclenomycin monomer, the warhead that inhibits biotin biosynthesis. Despite being discovered over five decades ago, stravidin biosynthesis has remained a mystery. Using our "metabologenomics" platform, we discover new stravidin analogues and identify the novel biosynthetic machinery responsible for their production. Analysis of the newly identified biosynthetic gene cluster (BGC) indicates the unusual amiclenomycin warhead is derived from chorismic acid, with initial steps similar to those involved in p-amino phenylalanine biosynthesis. However, a distinctive decarboxylation retains the nonaromatic character of a key ring and precedes a one-carbon extension to afford the warhead in its bioactive, untriggered state. Strikingly, we also identified two streptavidin genes flanking the new stravidin BGC reported here. This aligns with the known synergistic activity between the biotin-binding activity of streptavidin and the stravidins to antagonize both biotin biogenesis and bacterial growth.

DeepRiPP integrates multiomics data to automate discovery of novel ribosomally synthesized natural products.

Microbial natural products represent a rich resource of evolved chemistry that forms the basis for the majority of pharmacotherapeutics. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a particularly interesting class of natural products noted for their unique mode of biosynthesis and biological activities. Analyses of sequenced microbial genomes have revealed an enormous number of biosynthetic loci encoding RiPPs but whose products remain cryptic. In parallel, analyses of bacterial metabolomes typically assign chemical structures to only a minority of detected metabolites. Aligning these 2 disparate sources of data could provide a comprehensive strategy for natural product discovery. Here we present DeepRiPP, an integrated genomic and metabolomic platform that employs machine learning to automate the selective discovery and isolation of novel RiPPs. DeepRiPP includes 3 modules. The first, NLPPrecursor, identifies RiPPs independent of genomic context and neighboring biosynthetic genes. The second module, BARLEY, prioritizes loci that encode novel compounds, while the third, CLAMS, automates the isolation of their corresponding products from complex bacterial extracts. DeepRiPP pinpoints target metabolites using large-scale comparative metabolomics analysis across a database of 10,498 extracts generated from 463 strains. We apply the DeepRiPP platform to expand the landscape of novel RiPPs encoded within sequenced genomes and to discover 3 novel RiPPs, whose structures are exactly as predicted by our platform. By building on advances in machine learning technologies, DeepRiPP integrates genomic and metabolomic data to guide the isolation of novel RiPPs in an automated manner.

Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.

Ruminococcin C, an anti-clostridial sactipeptide produced by a prominent member of the human microbiota Ruminococcus gnavus.

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to-α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool.

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, which are proposed to be introduced by YcaO-TfuA proteins. Using RiPPER, we show that previously undescribed RiPP gene clusters encoding YcaO and TfuA proteins are widespread in Actinobacteria and encode a highly diverse landscape of precursor peptides that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new structural class of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

Trimetaphosphate Activates Prebiotic Peptide Synthesis across a Wide Range of Temperature and pH.

The biochemical activation of amino acids by adenosine triphosphate (ATP) drives the synthesis of proteins that are essential for all life. On the early Earth, before the emergence of cellular life, the chemical condensation of amino acids to form prebiotic peptides or proteins may have been activated by inorganic polyphosphates, such as tri metaphosphate (TP). Plausible volcanic and other potential sources of TP are known, and TP readily activates amino acids for peptide synthesis. But de novo peptide synthesis also depends on pH, temperature, and processes of solvent drying, which together define a varied range of potential activating conditions. Although we cannot replay the tape of life on Earth, we can examine how activator, temperature, acidity and other conditions may have collectively shaped its prebiotic evolution. Here, reactions of two simple amino acids, glycine and alanine, were tested, with or without TP, over a wide range of temperature (0-100 °C) and acidity (pH 1-12), while open to the atmosphere. After 24 h, products were analyzed by HPLC and mass spectrometry. In the absence of TP, glycine and alanine readily formed peptides under harsh near-boiling temperatures, extremes of pH, and within dry solid residues. In the presence of TP, however, peptides arose over a much wider range of conditions, including ambient temperature, neutral pH, and in water. These results show how polyphosphates such as TP may have enabled the transition of peptide synthesis from harsh to mild early Earth environments, setting the stage for the emergence of more complex prebiotic chemistries.

ADPr-Peptide Synthesis.

Synthetic mono-ADPr-peptides are useful for structural, biochemical, and proteomics studies. We describe here a protocol for the preparation of mono-ADPr-peptides based on a fairly standard Fmoc-based solid-phase synthesis. Phosphoribosylated precursor building blocks are introduced into the peptide chain on solid-phase and subsequently converted to ADPr-sites by chemical phosphorylation with adenosine phosphoramidite. Suitably protected phosphoribosylated glutamine, asparagine, and citrulline building blocks described in this protocol allow introduction of ADP-Gln, ADPr-Asn, and ADPr-Cit into peptide chains as demonstrated for three peptides. Trifunctional amino acids, for which base-sensitive side-chain protection is available, can be accommodated in the sequences flanking the ADPr-cites.

A prebiotic template-directed peptide synthesis based on amyloids.

The prebiotic replication of information-coding molecules is a central problem concerning life's origins. Here, we report that amyloids composed of short peptides can direct the sequence-selective, regioselective and stereoselective condensation of amino acids. The addition of activated DL-arginine and DL-phenylalanine to the peptide RFRFR-NH2 in the presence of the complementary template peptide Ac-FEFEFEFE-NH2 yields the isotactic product FRFRFRFR-NH2, 1 of 64 possible triple addition products, under conditions in which the absence of template yields only single and double additions of mixed stereochemistry. The templating mechanism appears to be general in that a different amyloid formed by (Orn)V(Orn)V(Orn)V(Orn)V-NH2 and Ac-VDVDVDVDV-NH2 is regioselective and stereoselective for N-terminal, L-amino-acid addition while the ornithine-valine peptide alone yields predominantly sidechain condensation products with little stereoselectivity. Furthermore, the templating reaction is stable over a wide range of pH (5.6-8.6), salt concentration (0-4 M NaCl), and temperature (25-90 °C), making the amyloid an attractive model for a prebiotic peptide replicating system.

The cell free protein synthesis system from the model filamentous fungus Neurospora crassa.

Cell-free protein synthesis (CFPS) can be used in many applications to produce polypeptides and to analyze mechanisms of mRNA translation. Here we describe how to make and use a CPFS system from the model filamentous fungus Neurospora crassa. The extensive genetic resources available in this system provide capacities to exploit robust CFPS for understanding translational control. Included are procedures for the growth and harvesting of cells, the preparation of cell-free extracts that serve as the source of the translational machinery in the CFPS and the preparation of synthetic mRNA to program the CFPS. Methods to accomplish cell-free translation and analyze protein synthesis, and to map positions of ribosomes on mRNAs by toeprinting, are described.

A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis.

BACKGROUND: Lactic acid bacteria are a family of "generally regarded as safe" organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields. RESULTS: To expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4-2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding. CONCLUSIONS: Overall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells.

A short upstream open reading frame (uORF) was recently identified in the 5' untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle.

Aminoacyl-tRNA synthetases and the evolution of coded peptide synthesis: the Thioester World.

Coded peptide synthesis must have been preceded by a prebiotic stage, in which thioesters played key roles. Fossils of the Thioester World are found in extant aminoacyl-tRNA synthetases (AARSs). Indeed, studies of the editing function reveal that AARSs have a thiol-binding site in their catalytic modules. The thiol-binding site confers the ability to catalyze aminoacyl~coenzyme A thioester synthesis and peptide bond formation. Genomic comparisons show that AARSs are structurally related to proteins involved in sulfur and coenzyme A metabolisms and peptide bond synthesis. These findings point to the origin of the amino acid activation and peptide bond synthesis functions in the Thioester World and suggest that the present-day AARSs had originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis.

Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis.

Improving the productivity of secondary metabolites is highly beneficial for the utilization of natural products. Here, we found that gene duplication of the goadsporin biosynthetic gene locus resulted in hyper-production. Goadsporin is a linear azole containing peptide that is biosynthesized via a ribosome-mediated pathway in Streptomyces sp. TP-A0584. Recombinant strains containing duplicated or triplicated goadsporin biosynthetic gene clusters produced 1.46- and 2.25-fold more goadsporin than the wild-type strain. In a surrogate host, Streptomyces lividans, chromosomal integration of one or two copies of the gene cluster led to 342.7 and 593.5 mg/L of goadsporin production. Expression of godI, a self-resistance gene, and of godR, a pathway-specific transcriptional regulator, under a constitutive promoter gave 0.79- and 2.12-fold higher goadsporin production than the wild-type strain. Our experiments indicated that a proportional relationship exists between goadsporin production per culture volume and the copy number of the biosynthetic gene cluster.

Proteome-wide epitope mapping of antibodies using ultra-dense peptide arrays.

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.