Evolutionary Imprint of Catalytic Domains in Fungal PKS–NRPS Hybrids

Fungal hybrid enzymes consisting of a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) module are involved in the biosynthesis of a vast array of ecologically and medicinally relevant natural products. Whereas a dozen gene clusters could be assigned to the requisite PKS–NRPS pathways, the programming of the multifunctional enzymes is still enigmatic. Through engineering and heterologously expressing a chimera of PKS (lovastatin synthase, LovB) and NRPS (cytochalasin synthase, CheA) in Aspergillus terreus, we noted the potential incompatibility of a fungal highly reducing PKS (hrPKS) with the NRPS component of fungal PKS–NRPS hybrids. To rationalize the unexpected outcome of the gene fusion experiments, we conducted extensive bioinformatic analyses of fungal PKS–NRPS hybrids and LovB‐type PKS. From motif studies and the function of the engineered chimeras, a noncanonical function of C‐terminal condensation (C) domains in truncated PKS–NRPS homologues was inferred. More importantly, sequence alignments and phylogenetic trees revealed an evolutionary imprint of the PKS–NRPS domains, which reflect the evolutionary history of the entire megasynthase. Furthermore, a detailed investigation of C and adenylation (A) domains provides support for a scenario in which not only the A domain but also the C domain participates in amino acid selection. These findings shed new light on the complex code of this emerging class of multifunctional enzymes and will greatly facilitate future combinatorial biosynthesis and pathway engineering approaches towards natural product analogues.     

Expanding Substrate Promiscuity by Engineering a Novel Adenylating‐Methylating NRPS Bifunctional Enzyme

Nonribosomal peptides synthetases (NRPSs), which are multifunctional mega‐enzymes producing many biologically active metabolites, are ideal targets for enzyme engineering. NRPS adenylation domains play a critical role in selecting/activating the amino acids to be transferred to downstream NRPS domains in the biosynthesis of natural products. Both monofunctional and bifunctional A domains interrupted with an auxiliary domain are found in nature. Here, we show that a bifunctional interrupted A domain can be uninterrupted by deleting its methyltransferase auxiliary domain portion to make an active monofunctional enzyme. We also demonstrate that a portion of an auxiliary domain with almost no sequence identity to the original auxiliary domain can be insert into naturally interrupted A domain to develop a new active bifunctional A domain with increased substrate profile. This work shows promise for the creation of new interrupted A domains in engineered NRPS enzymes.     

Targeted Discovery of Tetrapeptides and Cyclic Polyketide-Peptide Hybrids from a Fungal Antagonist of Farming Termites

Herein, we report the targeted isolation and characterization of four linear nonribosomally synthesized tetrapeptides (pseudoxylaramide A–D) and two cyclic nonribosomal peptide synthetase-polyketide synthase-derived natural products (xylacremolide A and B) from the termite-associated stowaway fungus Pseudoxylaria sp. X187. The fungal strain was prioritized for further metabolic analysis based on its taxonomical position and morphological and bioassay data. Metabolic data were dereplicated based on high-resolution tandem mass spectrometry data and global molecular networking analysis. The structure of all six new natural products was elucidated based on a combination of 1D and 2D NMR analysis, Marfey's analysis and X-ray crystallography.     

Exploring Chemical Diversity of α‐Pyrone Antibiotics: Molecular Basis of Myxopyronin Biosynthesis

Myxopyronins and corallopyronins are structurally related α‐pyrone antibiotics from myxobacteria. They are thought to represent a highly promising compound class for the development of broad‐spectrum antibacterial therapeutic agents, because of their ability to inhibit RNA polymerase through interaction with the “switch region”, a recently identified novel drug target. Here we describe the identification and characterization of the myxopyronin biosynthetic pathway from Myxococcus fulvus Mx f50. A detailed comparison with the recently identified corallopyronin biosynthetic pathway revealed the genetic and biochemical basis, thus explaining the observed structural differences between the two natural product families. Directed mutagenesis procedures for M. fulvus Mx f50 were developed to enable functional studies and pathway modifications. Our work provided new insights into myxopyronin biosynthesis and led to the production of a novel and unexpected myxopyronin derivative.     

Alkyl Formate Ester Synthesis by a Fungal Baeyer–Villiger Monooxygenase

We investigated Baeyer–Villiger monooxygenase (BVMO)‐mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ～80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space‐time yield (STY) of 4.2 g L^?1 d^?1 (3.4 g L^?1 d^?1 extracted product).     

Galectin‐1–Asialofetuin Interaction Is Inhibited by Peptides Containing the Tyr‐Xxx‐Tyr Motif Acting on the Glycoprotein

Galectin‐1 (Gal‐1), a ubiquitous β‐galactoside‐binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal‐1 depend on its affinity for β‐galactoside‐containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr‐Xxx‐Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal‐1–asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr‐Xxx‐Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr‐Xxx‐Tyr peptides studied do not bind to Gal‐1, whereas their binding to ASF is clearly detected. ¹⁵N,¹H HSQC titrations with ¹⁵N‐labeled Gal‐1 confirm the absence of any peptide–Gal‐1 interaction. These data indicate that the Tyr‐Xxx‐Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.     

Controlling Amyloid‐β Peptide(1–42) Oligomerization and Toxicity by Fluorinated Nanoparticles

The amyloid‐β peptide (Aβ) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. Soluble oligomers that precede fibril formation have been proposed as the main neurotoxic species that contributes to neurodegeneration and dementia. We hypothesize that oligomerization and cytotoxicity can be repressed by nanoparticles (NPs) that induce conformational changes in Aβ42. We show here that fluorinated and hydrogenated NPs with different abilities to change Aβ42 conformation influence oligomerization as assessed by atomic force microscopy, immunoblot and SDS‐PAGE. Fluorinated NPs, which promote an increase in α‐helical content, exert an antioligomeric effect, whereas hydrogenated analogues do not and lead to aggregation. Cytotoxicity assays confirmed our hypothesis by indicating that the conformational conversion of Aβ42 into an α‐helical‐enriched secondary structure also has antiapoptotic activity, thereby increasing the viability of cells treated with oligomeric species.     

An Ensemble of Rapidly Interconverting Orientations in Electrostatic Protein–Peptide Complexes Characterized by NMR Spectroscopy

Protein complex formation involves an encounter state in which the proteins are associated in a nonspecific manner and often stabilized by interactions between charged surface patches. Such patches are thought to bind in many different orientations with similar affinity. To obtain experimental evidence for the dynamics in encounter complexes, a model was created using the electron transfer protein plastocyanin and short charged peptides. Three plastocyanins with distinct surface charge distributions were studied. The experimental results from chemical shift perturbations, paramagnetic relaxation enhancement (PRE) NMR, and theoretical results from Monte Carlo simulations indicate the presence of multiple binding orientations that interconvert quickly and are dominated by long‐range charge interactions. The PRE data also suggest the presence of highly transient orientations stabilized by short‐range interactions.     

Structural defects created on natural graphite surface by slight treatment of oxygen plasma: – STM observations –

The creation of structural defects on natural graphite surfaces by slight treatment of oxygen plasma was studied by scanning tunneling microscopy (STM) at nanoscopic scale. Most of the defects were vacancies with the depth of one or two layers, while the mean number of defects per μm² of graphite surface and the mean area of defects increased with the increase in input power, time and temperature of the irradiation. After very slight treatment at room temperature, single carbon atom vacancies were obtained on graphite surfaces with a percentage more than 50%, though some large vacancies were formed. After slight irradiation at a high temperature (400°C), an agglomeration of defects occurred (the mean area increases whereas the defect density decreases), while the defects once formed were difficult to be annealed by heating at high temperatures up to 400°C in vacuum. The present work showed the possibility to control the modification of natural graphite surfaces for further applications by changing the condition of oxygen plasma treatment.     

Grain growth in TiO2-added ZnO–Bi2O3–CoO–MnO ceramics prepared by chemical processing

The effect of TiO₂ on the grain growth of the ZnO–Bi₂O₃–CoO–MnO ceramic system prepared by chemical coprecipitation, was studied between 1150 and 1300 °C in air. Bi₂O₃ melts during firing, and then TiO₂ dissolves into Bi₂O₃-rich liquid. TiO₂ initially reacts with Bi₂O₃ to form Bi₄Ti₃O₁₂. Above ≈1050 °C, Bi₄Ti₃O₁₂ reacts with ZnO to form Zn₂TiO₄ spinel phase. The kinetic study of grain growth carried out using the expression Gⁿ–G_oⁿ=K_o·t·exp(−Q/RT) gave grain exponent (n) value as 6 and the apparent activation energy (Q) as 226.46 kJ/mol. 1.00 mol% TiO₂ addition increased the grain growth exponent value from 6 to 7 and apparent activation energy with 1.00 mol% TiO₂ addition was found to be 197.10 kJ/mol. The ZnO grain size gradually increases with increasing TiO₂ content. Addition of TiO₂ may increase the reactivity of the Bi₂O₃-rich liquid towards the ZnO grain, thus affecting the ZnO grain growth.