Chemical and Genetic Studies on the Formation of Pyrrolones During the Biosynthesis of Cytochalasans.

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. In this work, we investigate the role of the highly conserved α,ß-hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite, respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3-dihydro-2H-pyrrol-2-one tautomer, rather than the required 1,5-dihydro-2H-pyrrol-2-one tautomer. Taken together our results suggest that the α,ß-hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined.

Early Oxidative Transformations During the Biosynthesis of Terrein and Related Natural Products.

The mycotoxin terrein is derived from the C10 -precursor 6-hydroxymellein (6-HM) via an oxidative ring contraction. Although the corresponding biosynthetic gene cluster (BGC) has been identified, details of the enzymatic oxidative transformations are lacking. Combining heterologous expression and in vitro studies we show that the flavin-dependent monooxygenase (FMO) TerC catalyzes the initial oxidative decarboxylation of 6-HM. The reactive intermediate is further hydroxylated by the second FMO TerD to yield a highly oxygenated aromatic species, but further reconstitution of the pathway was hampered. A related BGC was identified in the marine-derived Roussoella sp. DLM33 and confirmed by heterologous expression. These studies demonstrate that the biosynthetic pathways of terrein and related (polychlorinated) congeners diverge after oxidative decarboxylation of the lactone precursor that is catalyzed by a conserved FMO and further indicate that early dehydration of the side chain is an essential step.

Biosynthesis of 6-Hydroxymellein Requires a Collaborating Polyketide Synthase-like Enzyme.

The polyketide synthase (PKS)-like protein TerB, consisting of inactive dehydratase, inactive C-methyltransferase, and functional ketoreductase domains collaborates with the iterative non reducing PKS TerA to produce 6-hydroxymellein, a key pathway intermediate during the biosynthesis of various fungal natural products. The catalytically inactive dehydratase domain of TerB appears to mediate productive interactions with TerA, demonstrating a new mode of trans-interaction between iterative PKS components.

Diversely Functionalised Cytochalasins through Mutasynthesis and Semi-Synthesis.

Mutasynthesis of pyrichalasin H from Magnaporthe grisea NI980 yielded a series of unprecedented 4'-substituted cytochalasin analogues in titres as high as the wild-type system (≈60 mg L-1 ). Halogenated, O-alkyl, O-allyl and O-propargyl examples were formed, as well as a 4'-azido analogue. 4'-O-Propargyl and 4'-azido analogues reacted smoothly in Huisgen cycloaddition reactions, whereas p-Br and p-I compounds reacted in Pd-catalysed cross-coupling reactions. A series of examples of biotin-linked, dye-linked and dimeric cytochalasins was rapidly created. In vitro and in vivo bioassays of these compounds showed that the 4'-halogenated and azido derivatives retained their cytotoxicity and antifungal activities; but a unique 4'-amino analogue was inactive. Attachment of larger substituents attenuated the bioactivities. In vivo actin-binding studies with adherent mammalian cells showed that actin remains the likely intracellular target. Dye-linked compounds revealed visualisation of intracellular actin structures even in the absence of phalloidin, thus constituting a potential new class of actin-visualisation tools with filament-barbed end-binding specificity.

Diels-Alder Reactions During the Biosynthesis of Sorbicillinoids.

The sorbicillinoids are a class of biologically active and structurally diverse fungal polyketides arising from sorbicillin. Through co-expression of sorA, sorB, sorC, and sorD from Trichoderma reesei QM6a, the biosynthetic pathway to epoxysorbicillinol and dimeric sorbicillinoids, which resemble Diels-Alder-like and Michael-addition-like products, was reconstituted in Aspergillus oryzae NSAR1. Expression and feeding experiments demonstrated the crucial requirement of the flavin-dependent monooxygenase SorD for the formation of dimeric sorbicillinoids, hybrid sorbicillinoids, and epoxysorbicillinol in vivo. In contrast to prior reports, SorD catalyses neither the oxidation of 2',3'-dihydrosorbicillin to sorbicillin nor the oxidation of sorbicillinol to oxosorbicillinol. This is the first report that both the intermolecular Diels-Alder and Michael dimerization reactions, as well as the epoxidation of sorbicillinol are catalysed in vivo by SorD.

Evidence for enzyme catalysed intramolecular [4+2] Diels-Alder cyclization during the biosynthesis of pyrichalasin H.

Cytochalasans are highly complex fungal metabolites which exhibit diverse biological activities. Little is known of the chemical steps involved in the construction of the tricyclic core, which consists of an octahydro-isoindole skeleton fused to a macrocyclic ring. Here, using a directed gene knockout and complementation strategy, we show that PyiF is implicated as the proposed intramolecular [4+2] Diels-Alderase required for construction of the tricyclic core of pyrichalasin H 1.

Function of pathway specific regulators in the ACE1 and pyrichalasin H biosynthetic gene clusters.

Ectopic expression of BC1 which encodes a putative pathway specific transcription factor from the ACE1 biosynthetic gene cluster of the rice pathogen Pyricularia oryzae Guy11 did not lead to the production of ACE1-related compounds. However the known compound hinnulin A was formed. A putative partial gene cluster potentially involved in the biosynthesis of hinnulin A and DHN melanin was validated by RT-PCR and a possible biosynthetic pathway is proposed. Ectopic expression of pyiR which encodes a pathway specific transcription factor from the pyrichalasin H biosynthetic gene cluster in Magnaporthe grisea NI980 led to the apparent up-regulation of the pyi cluster and a 3-fold increase in pyrichalasin production under standard fermentation conditions, but did not lead to the formation of new compounds.

Mutation of key residues in the C-methyltransferase domain of a fungal highly reducing polyketide synthase.

Site directed mutations of the C-methyltransferase domain of squalestatin tetraketide synthase were made in an attempt to alter the methylation pattern of the synthase expressed in vivo: mutation resulted in either no effect or in complete abrogation of polyketide production.

Highly selective synthesis of oxabicycloalkanes by indium tribromide-mediated cyclization reactions of epoxyalkenes.

The cyclization of epoxyalkenes to oxabicycloalkanes is catalyzed by stoichiometric quantities of indium tribromide which exhibits excellent selectivity giving the oxabicyclic product in high yield in preference to other cyclized or rearrangement products.