An Aspergillus nidulans Platform for the Complete Cluster Refactoring and Total Biosynthesis of Fungal Natural Products

Abstract

Fungal natural products (NPs) comprise a vast number of bioactive molecules with diverse activities, and among them are many important drugs. However, the yields of fungal NPs from native producers are usually low, and total synthesis of structurally complex NPs is challenging. As such, downstream derivatization and optimization of lead fungal NPs can be impeded by the high cost of obtaining sufficient starting material. In recent years, reconstitution of NP biosynthetic pathways in heterologous hosts has become an attractive alternative approach to produce complex NPs. Here, we present an efficient, cloning-free strategy for the cluster refactoring and total biosynthesis of fungal NPs in Aspergillus nidulans. Our platform places our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted activation upon induction. We demonstrated the utility of our system by creating strains that can synthesize high-value NPs, citreoviridin (1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can be used not only for producing NPs of interests (i.e., total biosynthesis) but also for elucidating cryptic biosynthesis pathways.

Development of an auto-inducible expression system by nitrogen sources switching based on the nitrogen catabolite repression regulation

“…In recent years, a nitrate inducible expression system based on the bidirectional promoter P niiA /P niaD has been applied in A. nidulans [ 25 , 26 ]. A. nidulans utilizes NH 4 + as a primary nitrogen source and NO 3 − as a secondary nitrogen source via NCR.…”

Anthraquinone-fused enediynes: discovery, biosynthesis and development

“…With the rapid advances in genome mining strategies 111 and the better understanding of regulatory networks in natural product biosynthesis, 112 more AFEs will be discovered by cultivating new strains that contain the AFE BGCs, manipulating the regulatory genes for pathway activation, 113 or the reconstitution of new BGCs using the synthetic biology approach, and then expressing the reconstituted pathway in heterologous hosts. 114,115 It is reasonable to expect that these new compounds will provide more opportunities to elucidate the biosynthetic pathways for AFEs and develop AFE-based ADCs.…”

Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi

“…Unfortunately, individual replacement of all native promoters in a large BGC with strong promoters is an elaborate and timeconsuming task, further complicated by the limited availability of well characterized fungal promoters. Nonetheless, such extensive refactoring can still be attempted with a filamentous fungal host that shows a high HR rate that facilitates recombination of DNA fragments in vivo (Chiang et al, 2020;Pohl et al, 2020). Single promoter replacement is much more practical when it is employed to overexpress an in-cluster regulator, which in turn results in complete BGC activation with minimal engineering efforts (Bromann et al, 2012;Zabala et al, 2012).…”

“…In these strains, up to 6 or 7 genes of interest (GOIs) can be placed under the control of the native regulatory elements of the asperfuranone pathway, whose genes have been removed, while the inducible promoter PalcA controls the major TF regulating the BGC pathway. This elegant approach allows for the induction of entire heterologous BGCs upon the addition of methyl ethyl ketone, and its potential was demonstrated by the successful production of citreoviridin, mutilin, and pleuromutilin (Chiang et al, 2020).…”