Beauvericin, a cyclohexadepsipeptide ionophore from the entomopathogen Beauveria bassiana, shows antibiotic, antifungal, insecticidal, and cancer cell antiproliferative and antihaptotactic (cell motility inhibitory) activity in vitro. The bbBeas gene encoding the BbBEAS nonribosomal peptide synthetase was isolated from B. bassiana and confirmed to be responsible for beauvericin biosynthesis by targeted disruption. BbBEAS utilizes D-2-hydroxyisovalerate (D-Hiv) and L-phenylalanine (Phe) for the iterative synthesis of a predicted N-methyl-dipeptidol intermediate, and forms the cyclic trimeric ester beauvericin from this intermediate in an unusual recursive process. Heterologous expression of the bbBeas gene in Escherichia coli to produce the 3189 amino acid, 351.9 kDa BbBEAS enzyme provided a strain proficient in beauvericin biosynthesis. Comparative infection assays with a BbBEAS knockout B. bassiana strain against three insect hosts revealed that beauvericin plays a highly significant but not indispensable role in virulence. © 2008 Elsevier Ltd. All rights reserved.
Fungal cyclooligomer depsipeptides such as beauvericin, bassianolide, and enniatins display antibiotic, antifungal, insecticidal, broad-spectrum cancer cell antiproliferative, and cell migration inhibitory activities. We have identified a gene encoding a novel enzyme, ketoisovalerate reductase (KIVR), which is the sole provider of D-hydroxyisovalerate (D-Hiv), a common precursor for cyclooligomer depsipeptide biosynthesis in Beauveria bassiana. KIVR and related hypothetical oxidoreductases encoded in fungal genomes are similar to ketopantoate reductases but not to D-hydroxycarboxylate dehydrogenases. We demonstrate that a KIVR knockout B. bassiana strain can be used for the efficient mutasynthesis of unnatural beauvericin congeners. Simultaneous feeding of precursor analogues enabled the combinatorial mutasynthesis of scrambled beauvericins, some assembled entirely from unnatural precursors. The effects of the introduced structural changes on the antiproliferative and cell migration inhibitory ACHTUNGTRENNUNGactivities of these analogues were evaluated. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Precursor-directed biosynthesis was used to produce analogues of the cyclic depsipeptide mycotoxin beauvericin (1) using the filamentous fungus Beauveria bassiana ATCC 7159. Feeding 30 analogues of D-2-hydroxyisovalerate and L-phenylalanine, the natural 2-hydroxycarboxylic acid and amino acid precursors of beauvericin, led to the biosynthesis of novel beauvericins. Six of these were isolated and characterized, and their cytotoxicity and directional cell migration (haptotaxis) inhibitory activity against the metastatic prostate cancer cell line PC-3M were evaluated. Replacement of one, two, or all three of the D-2-hydroxyisovalerate constituents in beauvericin (1) with 2-hydroxybutyrate moieties (beauvericins G1-3, compounds 2-4) caused a parallel decline of cell migration inhibitory activity and cytotoxicity, suggesting a requirement for a branched side chain for both of these biological activities at the corresponding positions of beauvericins. Replacement of one, two, or all three N-methyl-L-phenylalanine residues of beauvericin with N-methyl-L-3-fluorophenylalanine moieties (beauvericins H1-3, compounds 5-7) increased cytotoxicity without affecting antihaptotactic activity. © 2007 American Chemical Society and American Society of Pharmacognosy.
Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.