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Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l . This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.

In the postgenomic era it has become increasingly apparent that the vast number of predicted biosynthesis genes of microorganisms is not reflected by the metabolic profile observed under standard fermentation conditions. In the absence of a particular (in most cases unknown) trigger these gene loci remain silent. Because these cryptic gene clusters may code for the biosynthesis of important virulence factors, toxins, or even drug candidates, new strategies for their activation are urgently needed to make use of this largely untapped reservoir of potentially bioactive compounds. The discovery of new microbial metabolites through genome mining has proven to be a very promising approach. Even so, the investigation of silent gene clusters is still a substantial challenge, particularly in fungi. Here we report a new strategy for the successful induction of a silent metabolic pathway in the important model organism Aspergillus nidulans, which led to the discovery of novel PKS-NRPS hybrid metabolites.

Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.

Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry.

Nonribosomal peptides (NRPs) are of great pharmacological importance, but there is currently no technology for high-throughput NRP 'dereplication' and sequencing. We used multistage mass spectrometry followed by spectral alignment algorithms for sequencing of cyclic NRPs. We also developed an algorithm for comparative NRP dereplication that establishes similarities between newly isolated and previously identified similar but nonidentical NRPs, substantially reducing dereplication efforts.

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds. Type I thioesterases (TEI) catalyse the release step from the assembly line of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.

Purpose The enzymes gamma-glutamyl hydrolase (GGH) and folylpolyglutamate synthetase (FPGS) regulate intracellular folate concentrations needed for cell proliferation, DNA synthesis, and repair. High GGH expression affects 5-FU thymidylate synthase (TS) inhibition and is a risk factor for various malignancies. Here, the clinical significance of GGH and FPGS expression was investigated in Stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1. Methods Surgical specimens of cancer tissue and adjacent normal mucosa, obtained from 253 patients with previously untreated gastric cancer, were examined. GGH and FPGS mRNA expression was measured by qPCR to evaluate their clinicopathological significance in gastric cancer patients after curative resection. Results While FPGS expression showed no significant differences between the cancerous and normal samples, GGH expression was higher in cancer tissue than in adjacent normal mucosa. High GGH expression was correlated with age, histological type, and vascular invasion. Overall survival (OS) of patients with high GGH mRNA expression was significantly poorer than of patients with low GGH expression. Multivariate analysis showed that high GGH expression was an independent prognostic factor of OS (HR: 2.58, 95% CI 1.29–5.16). Patients who received S-1 adjuvant treatment showed a significantly poor OS between high GGH/ low FPGS and low GGH /high FPGS . Patients without adjuvant treatment showed no significant difference. Conclusion GGH expression was significantly higher in gastric cancer tissue than in adjacent normal mucosa. High GGH and low FPGS expression is a useful independent predictor of poor outcomes in stage II/III gastric cancer patients undergoing postoperative adjuvant chemotherapy with S-1.

Hybrid multienzyme systems composed of polyketide synthase (PKS) and nonribosomal polypeptide synthetase (NRPS) modules direct the biosynthesis of clinically valuable natural products in bacteria. The fidelity of this process depends on specific recognition between successive polypeptides in each assembly line-interactions that are mediated by terminal 'docking domains'. We have identified a new family of N-terminal docking domains, exemplified by TubCdd from the tubulysin system of Angiococcus disciformis An d48. TubCdd is homodimeric, which suggests that NRPS subunits in mixed systems self-associate to interact with partner PKS homodimers. The NMR structure of TubCdd reveals a new fold featuring an exposed beta-hairpin that serves as the binding site for the C-terminal docking domain of the partner polypeptide. The pattern of charged residues on the contact surface of the beta-hairpin is a key determinant of the interaction and seems to constitute a 'docking code' that can be used to alter binding affinity.