Jason Leigh Vincent

Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis

Abstract Fungal maleidrides are an important family of bioactive secondary metabolites that consist of 7, 8, or 9‐membered carbocycles with one or two fused maleic anhydride moieties. The biosynthesis of byssochlamic acid (a nonadride) and agnestadride A (a heptadride) was investigated through gene disruption and heterologous expression experiments. The results reveal that the precursors for cyclization are formed by an iterative highly reducing fungal polyketide synthase supported by a hydrolase, together with two citrate‐processing enzymes. The enigmatic ring formation is catalyzed by two proteins with homology to ketosteroid isomerases, and assisted by two proteins with homology to phosphatidylethanolamine‐binding proteins.

Anti-mesotrione single chain antibody fragments derived using either phage display or via hybridoma technology: Selection, characterization and stabilization

Single-chain antibody fragments (scAb), specific for the triketone herbicide mesotrione, have been expressed and purified from the bacterium Escherichia coli. ScAbs were identified following the cloning of variable heavy and light chains from anti-mesotrione monoclonal antibodies (mAbs) and from panning an immunized phage display library. Both the monoclonal and phage derived scAbs were produced from the same immunized mouse spleen. In competition ELISA, the mAb derived scAbs (35 and 36) showed similar specificity for mesotrione compared with the parental mAbs. MAb 35 was the most sensitive with a limit of detection (IC20 value) within EC legislative limits for drinking water at 0.04 μg l−1. The best phage scAb (H6) had sensitivities approximately 20× less than scAb 35. In competition ELISA, scAb 35 showed reduced sensitivity to sulcotrione, a structurally related triketone, with IC50 values in the μM range. The introduction of an interdomain disulphide bond into scAb 35 resulted in greater stability in non-physiological environments. Immunoassay may provide a simple, cheap and rapid test for detection of mesotrione in a range of environments including soil and food.