Molecular Breeding of a Fungus Producing a Precursor Diterpene Suitable for Semi-Synthesis by Dissection of the Biosynthetic Machinery

Abstract

Many clinically useful pharmaceuticals are semi-synthesized from natural products produced by actinobacteria and fungi. The synthetic protocols usually contain many complicated reaction steps and thereby result in low yields and high costs. It is therefore important to breed microorganisms that produce a compound most suitable for chemical synthesis. For a long time, desirable mutants have been obtained by random mutagenesis and mass screening. However, these mutants sometimes show unfavorable phenotypes such as low viability and low productivity of the desired compound. Fusicoccin (FC) A is a diterpene glucoside produced by the fungus Phomopsis amygdali. Both FC and the structurally-related cotylenin A (CN) have phytohormone-like activity. However, only CN exhibits anti-cancer activity. Since the CN producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out, and elimination of the hydroxyl group at position 12 of FC was essential to mimic the CN-like activity. Based on detailed dissection of the biosynthetic machinery, we constructed a mutant producing a compound without a hydroxyl group at position 12 by gene-disruption. The mutant produced this compound as a sole metabolite, which can be easily and efficiently converted into an anti-cancer drug, and its productivity was equivalent to the sum of FC-related compounds produced by the parental strain. Our strategy would be applicable to development of pharmaceuticals that are semi-synthesized from fungal metabolites.

A Point Mutation inftmDBlocks the Fumitremorgin Biosynthetic Pathway inAspergillus fumigatusStrain Af293

“…27,28) Blocked mutants as well as gene deletion mutants provide not only clues to the identification of key steps in the biosynthetic pathway but also biosynthetic intermediates and shunt metabolites, which normally are present only in very small amounts in wild-type cultures. [28][29][30] In fact, the metabolite profile of Af293 gave us a hint as to previously unknown metabolite 4.…”

Biosynthesis of Terpenoid Natural Products in Fungi

“…Enzymes responsible for oxidative modification of the hydrocarbon scaffold were subsequently biochemically characterized [188]. Later, a second gene cluster was identified that encodes nine additional genes needed for the biosynthesis of fusicoccin A [189] (Fig. 8).…”

“…This cluster was identified in the draft genome sequence of P. amygdali based on a previously identified ABBA-type prenyltransferases that catalyzed in vitro the reverse O-prenylation of glucose with DMAPP [70], which is a prenylation that is also present in fusiccocin A. A combination of in vitro assays with recombinant enzymes and gene disruption allowed Dairi's group to then establish the complete fusicoccin A pathway [189]. His group also identified and functionally characterized the brassicicene C biosynthetic pathway from A. brassicola, which includes, in addition to a fusicoccadiene synthase (AbFs), a dioxygenase and oxidoreductase as well as five P450s [188,190,191].…”

A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline

“…3) to supply more IPP for indole diterpene biosynthesis was constructed because P. amygdali produced large amounts (>1 g/L) of fusicoccin-related compounds. 34) That strain, however, produced no specific compounds. Then, we focused on a transcriptional regulator, since the activation of transcriptional regulators sometimes results in high production of secondary metabolites in fungi, 35,36) but no candidate genes were located at the flanking regions of the cluster.…”

“…1(A)), from the strain we cloned the biosynthetic genes of fusicoccin A. 34) We attempted to identify a compound governed by the cluster, but all attempts failed. The following results, however, suggest that the cluster might be responsible for the biosynthesis of a lolilline/lolitrem type of compound.…”