Engineering of new pneumocandin side-chain analogues by mutasynthesis and evaluation of their antifungal activity

Pneumocandins are lipohexapeptides of the echinocandin family that block fungal cell wall formation through the inhibition of 1,3-β-glucan synthase. Pneumocandin B₀ is the precursor for semisynthesis of the antifungal drug caspofungin. Most of the biosynthetic steps have been functionally characterized in the producing fungus Glarea lozoyensis. However, the lipoinitiation reaction has not yet been experimentally verified. We have investigated the lipoinitiation step of pneumocandin biosynthesis and demonstrated that the gene product, GLligase, an acyl AMP ligase, shuttles the polyketide side chain to the nonribosomal peptide synthase, while GLHYD, a putative type II thioesterase, may assist in offloading the polyketide side chain product from the PKS acyl carrier protein domain. Inactivation of GLligase totally abolishes pneumocandin production. Inactivation of GLHYD significantly impaired the production of pneumocandins. Mutasynthesis experiments with a disruption mutant of the side-chain PKS, GLPKS4, afforded us an opportunity to test the substrate specificity of GLligase in the absence of its native chain and to diversify pneumocandins with substituted side chains. Feeding fermentations alternative side chain precursors yielded acrophiarin and four new pneumocandin congeners with straight C14, C15, and C16 side chains. A comprehensive biological evaluation showed that one compound, pneumocandin I, has elevated antifungal activity and similar hemolytic activity compared to pneumocandin B₀, the caspofungin precursor. This study provides new insights into the lipoinitiation mechanism in pneumocandin biosynthesis, a comparison of the SAR among pneumocandins with different-length acyl side chains, and the first example of the potential for using GLligase for engineering of new echinocandin analogues.