Synthetic biology based construction of biological activity-related library of fungal decalin-containing diterpenoid pyrones

Kento Tsukada,Emil Salim,Takayuki Kuraishi,Teigo Asai,Hironori Hayashi,Naoya Hirata,Aki Hori,Kumi Kawaji,Shono Shinki,Akiho Kaneko,Kazuma Murakami,Masatoshi Murai,Shingo Dan,Yasunari Kanda,Kazuhiro Irie,Hideto Miyoshi,Eiichi N Kodama

doi:10.1038/s41467-020-15664-4

Abstract

A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host, Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.

Highlights

A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis
We demonstrate the advantage of the synthetic biology approach based on heterologous and combinatorial biosynthesis coupled with genome mining for constructing a biologically relevant diterpenoid pyrones (DDPs)-focused library composed of a variety of DDPs
Five candidate gene clusters with subA–E orthologous genes that may encode non-reducing polyketide synthase (NR-PKS), geranylgeranyl diphosphate synthase (GGPPS), PT, flavin adenine dinucleotide (FAD)-dependent epoxidase (FMOep) and terpene cyclase (TC) were found in five fungal genomes, Fusarium graminearum PH-1, Macrophomina phaseolina MS6 Colletotrichum higginsianum IMI349063, Metarhizium anisopliae E6, and Arthrinium sacchari, which we previously isolated from a spider (Fig. 3b and Supplementary Table 1)

Summary

Introduction

A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach to translate enormous amounts of biosynthetic gene information to richly diverse natural products. We demonstrate the advantage of the synthetic biology approach based on heterologous and combinatorial biosynthesis coupled with genome mining for constructing a biologically relevant DDP-focused library composed of a variety of DDPs. biosynthetic studies on DDPs are limited, a putative biosynthetic gene cluster and pathway for subglutinols (subA–F) in an entomopathogenic fungus, Metarhizium robertsii, has been estimated (Fig. 3a). We perform genome mining based on this information and find putative DDP gene clusters distributed in five fungal genera Arthrinium, OO OH

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Conclusion