Investigating and engineering fungal tropolone meroterpenoid biosynthesis

Abstract

The main focus of the presented work concentrated on understanding the biosynthetic pathways of tropolone sesquiterpenoids (TS) and scytolide in fungi. Tropolone sesquiterpenoids, such as pycnidione 22 and eupenifeldin 23, share the structural motif of a core 11-membered macrocycle derived from humulene, which is connected to one or two polyketide-derived tropolones via bridging dihydropyran rings. The whole genome of fungus CF236968 was sequenced, which allowed in silico prediction of the pyc BGC as a good candidate for pycnidione-type TS. In parallel work from our group the eup2 BGC was predicted as a promising candidate for eupenifeldin-type TS in the fungus CF150626. The eup2 BGC was connected to eupenifeldin 23 biosynthesis based on reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. Ten homologous genes between the pyc BGC and the eup2 BGC were detected and five among them were shown to encode proteins homologous to the xenovulene A 24 biosynthetic pathway in Acremonium strictum. In vitro assays with Eup L4, a short chain dehydrogenase, indicated that it catalyzes the reduction of stipitaldehyde 102 and is necessary for eupenifeldin 23 production.

Scytolide 158 is a promising natural herbicide that is proposed to be derived biogenetically via lactonization of the enol pyruvate of methyl 3-epishikimate 205. Feeding [2-13C]-glycerol to Phyllosticta cirsii confirmed that scytolide 158 is derived from the shikimate pathway. Looking for the homologous genes of pentafunctional aromatic polypeptide (AROM) in P. cirsii. and T. reesei Δtmus53 resulted in a promising BGC candidate for scytolide 158 and a novel biosynthetic pathway partly related to the shikimate pathway was proposed based on this BGC.