Facile assembly and fluorescence-based screening method for heterologous expression of biosynthetic pathways in fungi

Abstract

Heterologous expression of multi-gene biosynthetic pathways in eukaryotic hosts is limited by highly regulated individual monocistrons. Dissimilar to prokaryotes, each eukaryotic gene is strictly controlled by its own regulatory elements, such as promoter and terminator. Consequently, parallel transcription can occur only when a group of genes is synchronously activated. A strategy to circumvent this limitation is the concerted expression of multiple genes as a polycistron. By exploiting the "stop-carry on" mechanism of picornaviruses, we have designed a sophisticated, yet easy-to-assemble vector system to heterologously express multiple genes under the control of a single promoter. For facile selection of correctly transformed colonies by basic fluorescence microscopy, our vector includes a split gene for a fluorescent reporter protein. This method was successfully applied to produce the psychotropic mushroom alkaloid psilocybin in high yields by heterologous expression of the entire biosynthetic gene cluster in the mould Aspergillus nidulans.

Polycistronic gene expression in the model micro-organismUstilago maydis

“…One half of the fluorescence protein carried an NLS. Hence, expression of the corresponding polycistronic mRNA resulted in nuclear fluorescence (Hoefgen et al, 2018). In our set-up, we fused the NLS to the Gfp reporter (de Felipe and Ryan, 2004;Provost et al, 2007;Kim et al, 2011).…”

“…The strategy to use 2A peptides to co-express multiple genes of biosynthetic pathways has successfully been used before. (i) Carotenoids were produced in S. cerevisiae (Beekwilder et al, 2014), (ii) -lactam antibiotics or (iii) psychotropic mushroom alkaloids in A. nidulans (Unkles et al, 2014;Hoefgen et al, 2018}) as well as (iv) fungal toxins in A. niger (Schuetze and Meyer, 2017). This approach has now been expanded to the basidiomycete U. maydis underlining the broad applicability of polycistronic mRNAs in biotechnology.…”

Production Options for Psilocybin: Making of the Magic

“…Briefly, a tetracyclin‐inducible gene expression system (the so‐called Tet‐On cassette) was harnessed and combined with an advanced approach to produce the biosynthesis enzymes from a single transcript as a polycistron (i.e., expressing multiple genes as one mRNA molecule, which is still translated into discrete enzymes). In standard small‐volume Erlenmeyer shake flasks and without further optimization of the culture conditions and media, 1 accumulated in the biomass, and titers reached >100 mg L −1 …”

“…In standard small-volumeE rlenmeyer shake flasks and withoutf urther optimization of the culture conditions and media, 1 accumulated in the biomass,and titers reached > 100 mg L À1 . [16] Scheme2.a) In vitro enzymatic pathway to isonorbaeocystin ( 16). [12] b) Biotransformation of N,N-diethyltryptamine (17)i nto 4-phosphoryloxy-N,N-diethyltryptamine( 18)byP.…”

“…Detailed knowledge of the biosynthesis of 1 paved the way for the genetic engineering of a naïve microbial host for heterologous in vivo production. As a reliable and robust model organism, the Aspergillus nidulans mold was chosen . To reconstitute the biosynthesis, all four biosynthesis genes needed to be inserted into the host genome.…”