Engineering a coenzyme-independent dioxygenase for one-step production of vanillin from ferulic acid.

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. IMPORTANCE: The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Modulation by neonicotinoids of honeybee α1/chicken ß2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

Neonicotinoids targeting insect nicotinic acetylcholine (ACh) receptors (insect nAChRs) are used for crop protection, but there is a concern about adverse effects on pollinators such as honeybees (Apis mellifera). Thus, we investigated the agonist actions of neonicotinoids (imidacloprid, thiacloprid and clothianidin) on A. mellifera α1 (Amα1)/chicken ß2 hybrid nAChRs in Xenopus laevis oocytes according to the subunit stoichiometry of (Amα1)3(ß2)2 and (Amα1)2(ß2)3 using voltage-clamp electrophysiology. ACh activated (Amα1)3(ß2)2 and (Amα1)2(ß2)3 nAChRs with similar current amplitude. We investigated the agonist activity of imidacloprid, thiacloprid and clothianidin for the two hybrid nAChRs and found that: 1) imidacloprid showed higher affinity than clothianidin, whereas clothianidin showed higher efficacy than imidacloprid for the nAChRs; 2) Thiacloprid showed the highest agonist affinity and the lowest efficacy for the nAChRs. The Amα1/ß2 subunit ratio influenced the efficacy of imidacloprid and thiacloprid, but hardly affected that of clothianidin. Hydrogen bond formation by the NH group in clothianidin with the main chain carbonyl of the loop B may account, at least in part, for the unique agonist actions of clothianidin on the hybrid nAChRs tested.

Combined effects of mutations in loop C and the loop D-E-G triangle on neonicotinoid interactions with Drosophila Dα1/chicken ß2 hybrid nAChRs.

Neonicotinoid insecticides interact with the orthosteric sites of nicotinic acetylcholine receptors (nAChRs) formed at the interfaces of (a) two adjacent α subunits and (b) α and non-α subunits. However, little is known of the detailed contributions of these two orthosteric sites to neonicotinoid actions. We therefore applied voltage-clamp electrophysiology to the Dα1/chicken ß2 hybrid nAChR expressed in Xenopus laevis oocytes to explore the agonist actions of imidacloprid and thiacloprid on wild type receptors and following binding site mutations. First, we studied the S221E mutation in loop C of the ACh binding site of the Dα1 subunit. Secondly, we explored the impact of combining this mutation in loop C with others in the loop D-E-G triangle (R57S; E78K; K140T; S221E). The S221E loop C mutation alone reduced the affinity of the neonicotinoids tested, while hardly affecting the concentration-response curve for acetylcholine. Addition of the three R57S; E78K; K140T mutations in the loop D-E-G triangle led to a further reduction in neonicotinoid sensitivity, suggesting that all four binding site loops (C, D, E, G) in the Dα1 subunit, which are located upstream of loop B in the N-terminal, extracellular domain, contribute to the selective actions of neonicotinoid insecticides.

The mechanism of loop C-neonicotinoid interactions at insect nicotinic acetylcholine receptor α1 subunit predicts resistance emergence in pests.

Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, we have investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken ß2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of ß2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the ß2 subunit lacking basic residues critical for binding neonicotinoids. Hence, we predict the possibility of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the ß1 subunit.

Multi-functional glycoside hydrolase: Blon_0625 from Bifidobacterium longum subsp. infantis ATCC 15697.

We here describe a unique ß-D-glucosidase (BGL; Blon_0625) derived from Bifidobacterium longum subsp. infantis ATCC 15697. The Blon_0625 gene was expressed by recombinant Escherichia coli. Purified recombinant Blon_0625 retains hydrolyzing activity against both p-nitrophenyl-ß-D-glucopyranoside (pNPG; 17.3±0.24Umg(-1)) and p-nitrophenyl-ß-D-xylopyranoside (pNPX; 16.7±0.32Umg(-1)) at pH 6.0, 30°C. To best of our knowledge, no previously described BGL retains the same level of both pNPGase and pNPXase activity. Furthermore, Blon_0625 also retains the activity against 4-nitrophenyl-α-l-arabinofranoside (pNPAf; 5.6±0.09Umg(-1)). In addition, the results of the degradation of phosphoric acid swollen cellulose (PASC) or xylan using endoglucanase from Thermobifida fusca YX (Tfu_0901) or xylanase from Kitasatospora setae KM-6054 (KSE_59480) show that Blon_0625 acts as a BGL and as a ß-D-xylosidase (XYL) for hydrolyzing oligosaccharides. These results clearly indicate that Blon_0625 is a multi-functional glycoside hydrolase which retains the activity of BGL, XYL, and also α-l-arabinofuranosidase. Therefore, the utilization of multi-functional Blon_0625 may contribute to facilitating the efficient degradation of lignocellulosic materials and help enhance bioconversion processes.

Starchy biomass-powered enzymatic biofuel cell based on amylases and glucose oxidase multi-immobilized bioanode.

The present study reports the design of a novel bioanode to directly utilize starch as a fuel in an enzymatic biofuel cell. The enzymatic fuel cell is based on three enzymes (alpha-amylase, glucoamylase and glucose oxidase). The carbon paste electrode containing these three enzymes and tetrathiafulvalene can both saccharize and oxidize starchy biomass. In cyclic voltammetry, catalytic currents were successfully observed with both glucose and starchy white rice used as a substrate. Finally, a membrane-less white rice/O2 biofuel cell was assembled and the electrochemical performance was evaluated. The three enzyme based electrode was used as a bioanode and an immobilized bilirubin oxidase (derived from Myrothecium verrucaria) electrode was used as a biocathode. The biofuel cell delivered an open circuit voltage of 0.522V and power density of up to 99.0 µWcm(-2). Our results show that a readily available fuel can be used for enzymatic fuel cells, and will lead to new designs.