Standard for Synthesis of Customized Peptides by Non-Ribosomal Peptide Synthetases

The purpose of this RFC is to introduce a standardized framework for the engineering of customizable non-ribosomal peptide synthetases (NRPS) and their application for in vivo and in vitro synthesis of short non-ribosomal peptides (NRPs) of user-defined sequence and structure

Isolation and crystallization of PvdJp2, a non-ribosomal peptide synthetase domain in Pseudomonas aeruginosa

This article was published in the Spring 2014 issue of the Journal of Undergraduate Researc

Unusual Building Blocks and Domain Organization of Non-Ribosomal Peptide Synthetases

The diverse class of non-ribosomal peptides consists of manifold pharmacologically important natural products. They are clinically used in antibiotic, antiviral and antitumor therapy, furthermore some are known immunosuppresants. The biological activity is based on their structural diversity, as they contain various non-proteinogenic building blocks and amino acids of which many are beta-modified. It was shown that the latter are important for biological activity, but little is known about their biosynthetic origin. In particular, these building blocks are key determinants of the class of acidic lipopeptide antibiotics and kutznerides, which are in the focus of this thesis. To determine the mechanism underlying the biosynthetic origin of the synthetically challenging beta-hydroxylated asparagine (hAsn) moieties, found in the acidic lipopeptides CDA and A54145, the corresponding recombinant non-heme iron (II)/alpha-ketoglutarate dependent hydroxylases AsnO and LptL have been examined in vitro. Direct hydroxylation of the free amino acid was observed in both cases, clearly indicating a precursor synthesis pathway. The crystal structure of one of the two hydroxylases (AsnO) was determined at high resolution and revealed a substrate induced fit mechanism of the enzyme. Upon addition of asparagine, a lid-like region seals the active site and shields it from sterically demanding substrates, which explains the observed specificity for free asparagine. Furthermore, the AsnO structure could be seen as an archetype enzyme for non-heme iron hydroxylases acting on free amino acids. It was possible to predict amino acid binding residues for homologous enzymes by 3D modeling. In order to fully understand the mechanisms of beta-hydroxylated building blocks synthesis, the hydroxylases KtzO and KtzP, predicted to be responsible for the generation of the two 3-hydroxyglutamic acid isomers found in the mixture of antifungal and antimicrobial kutznerides, were produced recombinantly and analyzed in vitro. Notably, they were found to work in trans to the assembly line on PCP-tethered glutamic acid rather than on the free amino acid. Unexpectedly, as the two isomers are found in approximately equal amounts in mature kutznerides, KtzO was shown to stereospecifically generate threo-hydroxyglutamate, while KtzP catalyzed the formation of the erythro isomer by co-elution HPLC experiments with synthetic dabsylated standards. A powerful method that employs non-hydrolyzable coenzyme A analogs was developed, which allowed the determination of the kinetic parameters of enzymes working on PCP-bound substrates for the first time. Furthermore, a hitherto unknown mechanism of NRPS assembly line restoration was observed. The corresponding adenylation (A) domain for glutamic acid activation in the kutzneride NRPS was found to be corrupted. Herein, it is shown that this lack of a functional A domain is compensated in trans by a stand-alone A domain. These findings elucidated the mechanism for the in trans compensation and the stereospecific hydroxyglutamate generation in detail and may guide the usage of in trans hydroxylation/compensation enzymes in biocombinatorial engineering approaches. In the third part of this work, the acquired knowledge about the mechanisms underlying enzymatic beta-hydroxylation of amino acids was exploited for the synthesis of the pharmaceutically relevant beta-hydroxyaspartate. Primarily, this was facilitated by the structure elucidation of AsnO in which the substrate binding residues were identified. By site directed mutagenesis, an AsnO variant was generated, which notably did not hydroxylate the original substrate asparagine, instead it was found to stereospecifically catalyze the formation of L-threo-hydroxyaspartic acid, even in commercially interesting amounts. Therefore, the AsnO variant is an excellent example for the application of basic research in order to generate pharmacologically relevant non-proteinogenic amino acids

A Novel Broad-spectrum Lipopeptide Antimicrobial Agent, Paenibacterin, against Drug-resistant Bacteria: Structural Elucidation, Biosynthesis, and mechanisms of action

Food, Agricultural, and Environmental Sciences (FAES): 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)The ongoing explosion of infections caused by antibiotic-resistant bacteria continues to challenge the global public health. However, the discovery and development of novel antibacterial drugs are on the decline in the past decades. Therefore, it is urgent to develop potent and safe antimicrobial agents. Here we report the chemical structure, biosynthesis and modes of action of a novel antimicrobial agent, paenibacterin, from a strain of Paenibacillus thiaminolyticus OSY-SE. The producer microorganism was isolated from a soil sample collected in Columbus, OH in 2011. The potent antimicrobial agent was extracted by acetonitrile from the producer cells and purified using HPLC. The core peptide structure of paenibacterin was elucidated by mass-spectrometry (MS) and nuclear magnetic resonance (NMR) while the lipid tail of compound was determined by GC-MS. In order to identify the biosynthetic pathway of the compound, the whole genome of the producer strain OSY-SE was sequenced using the high throughput Illumina sequencing technology. The paenibacterin gene cluster (pbt) was further identified by bioinformatic analyses and confirmed by in vitro protein functional analyses. Furthermore, the mechanisms of action of paenibacterin were studied by fluorescence microscopy, membrane integrity assays, and hydroxyl radical production assays. Paenibacterin is a broad-spectrum antimicrobial agent with potent activity against foodborne pathogens and clinical drug-resistant isolates. Paenibacterin generally yielded a minimum inhibitory concentration (MIC) at 2-8 μg/ml against Gram-negative strains, and 8-64 μg/ml against Gram-positive strains. Paenibacterin is a cyclic lipopeptide, consisting of 13 amino acids and a C15 fatty acid side chain. Among the amino acids, some are D-amino acids and non-proteinogenic amino acid (ornithine). Paenibacterin is biosynthesized by the producer strain through non-ribosomal peptide synthetases (NRPS). The biosynthetic gene cluster was identified within 52-kb region, encoding three NRPSs (PbtA, PbtB and PbtC) and two ABC-transporters (PbtD and PbtE). Paenibacterin damages bacterial cell membrane, resulting in cytoplasmic membrane deporlarization and potassium ions release. Furthermore, paenibacterin triggers radical production via Fenton reaction and subsequently leads to cell death. This study reported a unique and potent antimicrobial agent with activity against drug-resistant bacteria. The structure of the compound could serve a scaffold for designing even more potent compounds by chemical synthesis. In addition, the elucidation of the biosynthetic pathway expedites the effort to produce paenibacterin derivatives by genetic engineering. This new and potent compound is a very promising candidate for agricultural and clinical application.A five-year embargo was granted for this item

Antibacterial Activity of Sponge Associated-bacteria Isolated From North Java Sea

A total of 90 bacterial isolates were obtained from association with sponges collected from Bandengan water, Jepara and Karimun island of Karimunjawa islands in the north java sea. Antibacterial screening by using a pathogenic Escherichia coli indicated that two isolates BSP.12 and MKSP.5 inhihited the growth of tested strain. Molecular identification based on 16S rDNA approach revealed that isolate BSP.12 was closely related to Vibrio harveyi with a homology of 100% and isolate MKSP.5 showed highest similarity to Brachybacterium rhamnosum (99%), respectively.A PCR-based approach by using specific primers targeting the occurrence of gene fragments necessary for the biosynthesis of major natural products, namely Non-ribosomal peptide synthetases (NRPS) and Polyketide synthases (PKS) was applied to estimate the genetic potential of these active strains. Both isolates were capable of amplifying the NRPS gene fragments but not the PKS gene fragments

Antibacterial Property of Marine Bacterium Pseudomonas SP. Associated with a Soft Coral Against Pathogenic Streptococcus Equi Subsp. Zooepidemicus

A marine bacterium associated with a soft coral Sinularia polydactyla collected from Bandengan water, Jepara, North Java Sea, Indonesia, was successfully screened for antibacterial activity against pathogenic bacterium Streptococcus equi subsp. zooepidemicus K6.72 isolated from infected monkey of the island of Bali and identified based on morphological, biochemical and molecular methods. Marine bacterium was identified as Pseudomonas sp. based on its 16S rDNA and was found to amplify gene fragments of Non-ribosomal peptide synthetase (NRPS). Cloning and subsequent sequencing, a 360 bp long DNA fragment was obtained and the deduced amino acid sequence showed conserved signature regions for peptide synthetases and revealed a high similarity of 61.1% to genes peptide synthetase of Bacillus subtilis

A trans-acting cyclase off-loading strategy for non-ribosomal peptide synthetases

The terminal step in the biosynthesis of nonribosomal peptides is the hydrolytic release and, frequently, macrocyclization of an aminoacyl-S-thioester by an embedded thioesterase. The surugamide biosynthetic pathway is composed of two nonribosomal peptide synthetase (NRPS) assembly lines in which one produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded thioesterase, which led us to question how the peptides are released from the assembly line (and cyclized in the case of surugamide A). We characterized a cyclase belonging to the β-lactamase superfamily in vivo, established that it is a trans-acting release factor for both compounds, and verified this functionality in vitro with a thioester mimic of linear surugamide A. Using bioinformatics, we estimate that ∼11% of filamentous Actinobacteria harbor an NRPS system lacking an embedded thioesterase and instead employ a trans-acting cyclase. This study improves the paradigmatic understanding of how nonribosomal peptides are released from the terminal peptidyl carrier protein and adds a new dimension to the synthetic biology toolkit

Anti-Staphylococcal Activity of Variovorax paradoxus EPS

Variovorax paradoxus EPS is a gram-negative rod isolated from the sunflower rhizosphere at CSUSB. Preliminary research has shown that Variovorax paradoxus EPS has anti-staphylococcal activity in liquid and solid co-culture. Anti-staphylococcal activity of Wild type and V. paradoxus EPS 4519 on 0.5% YE agar with embedded S. aureus AH1710 supports the idea that a soluble molecule is responsible for this activity, as the agar acted as a physical barrier between V. paradoxus EPS and S. aureus colonies. Preliminary genetic analysis of V. paradoxus EPS identified three loci that suitable candidates for the synthesis of a potential anti-staphylococcal small molecule. Preliminary data failed to detect expression at two of the three identified loci and a strain with a mutation at the third locus continues to produce anti-staphylococcal activity. We hypothesize that the microbial agent is expressed at a different locus or loci that have not yet been identified. These gene products are responsible for the synthesis of the microbial agent and are controlled by exposure to Staphylococcus aureus. Optimal growth conditions were identified for V. paradoxus EPS and S. aureus to demonstrate the formation of a zone of inhibition on Tryptic Soy Agar. The use of a V. paradoxus EPS Δ 4519 transposon library at optimal growth conditions allowed us candidate mutants with altered antimicrobial activity phenotypes. We identified 28 insertion sites that resulted in altered antimicrobial activities, which will allow us to identify the genes involved in this biosynthetic pathway