Rieske non-heme iron-dependent oxygenases catalyse diverse reactions in natural product biosynthesis.

Covering: up to the end of 2017 The roles played by Rieske non-heme iron-dependent oxygenases in natural product biosynthesis are reviewed, with particular focus on experimentally characterised examples. Enzymes belonging to this class are known to catalyse a range of transformations, including oxidative carbocyclisation, N-oxygenation, C-hydroxylation and C-C desaturation. Examples of such enzymes that have yet to be experimentally investigated are also briefly described and their likely functions are discussed.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Effects of pesticides on the bacterial production of pyrrolnitrin.

Pyrrolnitrin is a halogenated bacterial metabolite with antifungal and antibacterial activities which served as a lead structure of synthetic fungicides. Several pyrrolnitrin-producing bacteria are considered to be promising biopesticides. However, the application of these microorganisms is not straightforward since many synthetic pesticides usually coexist in agricultural fields and inevitably affect the efficacy of biocontrol agents. In this regard, effects of 25 xenobiotics, including 18 pesticides, were investigated for pyrrolnitrin biosynthesis by Burkholderia sp. O33 and Pseudomonas fluorescens Pf-5. Strong inhibition of pyrrolnitrin synthesis was observed in 9 chemicals, including 6 pesticides, while glyphosate and validamycin enhance biosynthesis. Fenpiclonil and fludioxonil strongly inhibit the oxidative transformation of aminopyrrolnitrin to pyrrolnitrin. Halogenation reaction to aminopyrrolnitrin was reduced by methimazole, a well-known flavin-dependent monooxygenase inhibitor. Most pesticides gave moderate growth inhibitory effects. The results suggested that synthetic chemicals can modulate the efficacy of pyrrolnitrin producing bacteria, through the inhibition of cell growth or pyrrolnitrin biosynthesis. Pathway specific inhibition by fenpiclonil, fludioxonil, and methimazole will give structural insights of corresponding enzymes.

Preliminary crystallographic characterization of PrnB, the second enzyme in the pyrrolnitrin biosynthetic pathway.

Pyrrolnitrin is the active ingredient of drugs for the treatment of superficial fungal infections and was used as a lead structure for the development of fludioxonil. It is an effective agent for plant diseases caused by the fungal pathogen Rhizoctonia solani. Pyrrolnitrin is made in four steps, the second of which, catalyzed by PrnB, is a novel chemical rearrangement of 7-chlorotryptophan. PrnB was overproduced in Pseudomonas fluorescens (BL915) and well diffracting crystals were obtained of a triple cysteine-to-serine mutant by sitting-drop vapour diffusion. Crystals grown in the presence of L-7-chlorotryptophan, D-tryptophan and L-tryptophan are reported. Data sets for each are reported with high-resolution limits of 2.0, 1.75 and 1.75 A, respectively. Two crystals (PrnB in the presence of D-tryptophan and L-7-chlorotryptophan) belong to space group C2 with similar unit-cell parameters (a = 68.6, b = 79.5, c = 92.7 A, alpha = gamma = 90.0, beta = 103.8 degrees). Crystals grown in the presence of L-tryptophan belong to space group C222(1) and have unit-cell parameters a = 67.7, b = 80.1, c = 129.5 A. All crystals contain a monomer in the asymmetric unit.