Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

Abstract

Sesquiterpenoids are important secondary metabolites with various pharma-and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established. Here, we introduce Ustilago maydis as a novel production host. This model fungus is a close relative of higher basidiomycetes. It offers the advantage of metabolic compatibility and potential tolerance for substances toxic to other microorganisms. We successfully implemented a heterologous pathway to produce the carotenoid lycopene that served as a straightforward read-out for precursor pathway engineering. Overexpressing genes encoding enzymes of the mevalonate pathway resulted in increased lycopene levels. Verifying the subcellular localization of the relevant enzymes revealed that initial metabolic reactions might take place in peroxisomes: despite the absence of a canonical peroxisomal targeting sequence, acetyl-CoA C-acetyltransferase Aat1 localized to peroxisomes. By expressing the plant (+)-valencene synthase CnVS and the basidiomycete sesquiterpenoid synthase Cop6, we succeeded in producing (+)valencene and α-cuprenene, respectively. Importantly, the fungal compound yielded about tenfold higher titers in comparison to the plant substance. This proof of principle demonstrates that U. maydis can serve as promising novel chassis for the production of terpenoids.

Metabolic engineering of Synechocystis sp. PCC 6803 for the photoproduction of the sesquiterpene valencene

“…The highest product yield in yeast, ~540 mg/L valencene, was again achieved by a combination of genetic engineering and optimization of media composition and cultivation ( Chen et al, 2019 ). Recently, the corn smut fungus Ustilago maydis was explored as a microbial production host for sesquiterpenes, due to previous successes in biotechnological applications using this host ( Lee et al, 2020 ).…”

Identification of Feldin, an Antifungal Polyyne from the Beefsteak Fungus Fistulina hepatica

“…Moreover, if an understanding of polyyne biosynthesis were desirable, heterologous expression, including a derivatization approach, could be meaningful. Following identification of potential polyyne biosynthesis genes in the F. hepatica genome sequence of Floudas et al [16], overexpression of respective candidate genes should be attempted, similar to the proof-of-principle approach recently accomplished by Lee et al [66] on sesquiterpene production. Simultaneous expression of plant enzymes for a coupling approach might, eventually, allow the engineering of polyyne-derivatives, which are relatively stable and secreted into the culture medium.…”

Recent developments of tools for genome and metabolome studies in basidiomycete fungi and their application to natural product research

“…Among fungi of the phylum Basidiomycota, the corn smut U. maydis has recently been developed into a heterologous host for the production of terpenoids through the overexpression of endogenous mevalonate pathway genes (Lee et al, 2020). The chassis was validated through the heterologous expression of fungal and plant sesquiterpenoid synthases from C. cinerea and the Nootka cypress Callitropsis nootkatensis, respectively.…”

“…Ascomycete hosts are easier to transform and engineer than basidiomycete fungi, therefore they are the preferred chassis for the study of basidiomycete NP gene clusters. However, a first step toward the use of U. maydis has been achieved recently when its mevalonate pathway was overexpressed, and the resulting strain was used to express fungal and plant sesquiterpenoid synthases (Lee et al, 2020). Expression of entire biosynthetic gene clusters will likely follow in this host.…”