Cryptic Isomerization in Diterpene Biosynthesis and the Restoration of an Evolutionarily Defunct P450.

Biosynthetic modifications of the 6/10-bicyclic hydrocarbon skeletons of the eunicellane family of diterpenoids are unknown. We explored the biosynthesis of a bacterial trans-eunicellane natural product, albireticulone A (3), and identified a novel isomerase that catalyzes cryptic isomerization in the biosynthetic pathway. We also assigned functions of two cytochromes P450 that oxidize the eunicellane skeleton, one of which was a naturally evolved non-functional P450 that, when genetically repaired, catalyzes allylic oxidation. Finally, we described the chemical susceptibility of the trans-eunicellane skeleton to undergo Cope rearrangement to yield inseparable atropisomers.

First trans-eunicellane terpene synthase in bacteria.

Terpenoids are the largest family of natural products, but prokaryotes are vastly underrepresented in this chemical space. However, genomics supports vast untapped biosynthetic potential for terpenoids in bacteria. We discovered the first trans-eunicellane terpene synthase (TS), AlbS from Streptomyces albireticuli NRRL B-1670, in nature. Mutagenesis, deuterium labeling studies, and quantum chemical calculations provided extensive support for its cyclization mechanism. In addition, parallel stereospecific labeling studies with Bnd4, a cis-eunicellane TS, revealed a key mechanistic distinction between these two enzymes. AlbS highlights bacteria as a valuable source of novel terpenoids, expands our understanding of the eunicellane family of natural products and the enzymes that biosynthesize them, and provides a model system to address fundamental questions about the chemistry of 6,10-bicyclic ring systems.

Mutation of the eunicellane synthase Bnd4 alters its product profile and expands its prenylation ability.

Bnd4 catalyzes the first committed step in the biosynthesis of the bacterial diterpenoid benditerpenoic acid and was the first eunicellane synthase identified from nature. We investigated the catalytic roles of the aromatic residues in the active site of Bnd4 through a series of mutation studies. These experiments revealed that large hydrophobic or aromatic side chains are required at F162 and Y197 for eunicellane formation and that selected mutations at W316 converted Bnd4 into a cembrane synthase. In addition, the Bnd4Y197A variant expanded the native prenylation ability of Bnd4 from accepting C5 and C10 prenyl donors to C15. This study supports the mechanism of eunicellane formation by Bnd4 and encourages further engineering of terpene synthases into practical and efficient prenyltransferases.

Production and characteristics of a novel chicken egg yolk antibody (IgY) against periodontitis-associated pathogens.

Periodontitis is a bacterial biofilm-induced oral disease, mostly caused by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) and Porphyromonas gingivalis (P. gingivalis). Oral administration of chicken egg yolk antibody (IgY) is a promising nutritional strategy to control pathogen infections. The objective of this study was to produce an A. actinomycetemcomitans- and P. gingivalis-specific IgY and evaluate its effects on bacterial agglutination and biofilm formation. Thirty laying hens were immunized with a complex of lysate containing typical molecular weights of membrane proteins of A. actinomycetemcomitans and P. gingivalis. IgY was isolated by polyethylene glycol 6000 and ammonium sulfate and purified by dialysis. The results of enzyme-linked immunosorbent assay showed that the obtained IgY were specific to both A. actinomycetemcomitans and P. gingivalis. In addition, immunoelectron microscopy scanning and crystal violet staining showed that the IgY could bind to cell wall of the pathogens and efficiently accelerate agglutination and inhibit biofilm formation. Furthermore, the activity of the IgY remained stable at different temperature, pH, and storage period. This is the first report that a novel two-in-one IgY was produced to modulate the agglutination and biofilm formation of A. actinomycetemcomitans and P. gingivalis, suggesting the potential of IgY to control periodontitis caused by oral pathogens.

Fabrication of Self-Cleaning Superhydrophobic Surfaces with Improved Corrosion Resistance on 6061 Aluminum Alloys.

Aluminum alloys are widely used, but they are prone to contamination or damage under harsh working environments. In this paper, a self-cleaning superhydrophobic aluminum alloy surface with good corrosion resistance was successfully fabricated via the combination of sand peening and electrochemical oxidation, and it was subsequently covered with a fluoroalkylsilane (FAS) film. The surface morphology, surface wettability, and corrosion resistance were investigated using a scanning electron microscope (SEM), an optical contact angle measurement, and an electrochemical workstation. The results show that binary rough structures and an FAS film with a low surface energy on the Al alloy surfaces confer good superhydrophobicity with a water contact angle of 167.5 ± 1.1° and a sliding angle of 2.5 ± 0.7°. Meanwhile, the potentiodynamic polarization curve shows that the corrosion potential has a positively shifted trend, and the corrosion current density decreases by three orders of magnitude compared with that of the original aluminum alloy sample. In addition, the chemical stability of the as-prepared superhydrophobic surface was evaluated by dripping test using solutions with different pH values for different immersion time. It indicates that the superhydrophobic surface could provide long-term corrosion protection for aluminum alloys. Consequently, the as-prepared superhydrophobic surface has excellent contamination resistance and self-cleaning efficacy, which are important for practical applications.

Experimental Investigation on Ultrasonic Atomization Assisted Turning of Titanium Alloy.

There are high cutting temperatures, large tool wear, and poor tool life in conventional machining, owing to the superior strength and low thermal conductivity of titanium alloy. In this work, ultrasonic atomization assisted turning (UAAT) of Ti6Al4V was performed with a mixed water-soluble oil-based cutting fluid, dispersed into tiny droplets by the high frequency vibration of a piezoelectric crystal. Different cutting speeds and two machining environments, dry and ultrasonic atomization assisted machining, were considered in the investigation of tool life, tool wear morphology, surface roughness, and chip morphology. In comparison with dry machining, UAAT shows lower tool wear and longer tool life due to the advantages of cooling and lubrication. Furthermore, better surface roughness, smoother chip edges, and shorter tool-chip contact length were obtained with UAAT.