A universal gene expression system for fungi

Abstract

Biotechnological production of fuels, chemicals and proteins is dependent on efficient production systems, typically genetically engineered microorganisms. New genome editing methods are making it increasingly easy to introduce new genes and functionalities in a broad range of organisms. However, engineering of all these organisms is hampered by the lack of suitable gene expression tools. Here, we describe a synthetic expression system (SES) that is functional in a broad spectrum of fungal species without the need for host-dependent optimization. The SES consists of two expression cassettes, the first providing a weak, but constitutive level of a synthetic transcription factor (sTF), and the second enabling strong, at will tunable expression of the target gene via an sTF-dependent promoter. We validated the SES functionality in six yeast and two filamentous fungi species in which high (levels beyond organism-specific promoters) as well as adjustable expression levels of heterologous and native genes was demonstrated. The SES is an unprecedentedly broadly functional gene expression regulation method that enables significantly improved engineering of fungi. Importantly, the SES system makes it possible to take in use novel eukaryotic microbes for basic research and various biotechnological applications.

Profiling of chromatin accessibility across Aspergillus species and identification of transcription factor binding sites in the Aspergillus genome using filamentous fungi ATAC-seq

“…By constantly changing footprints and monitoring the transcription of downstream reporter genes, it was possible to identify whether the footprints containing the TFBs function in vivo . The 1 kb flanking sequence of the glucoamylase gene ( glaA ) acts as a homologous arm, and the aim was that the sequence successfully integrated into the glucoamylase gene locus, ensuring that all open reading frames were at the same position in the genome (Rantasalo et al 2018).…”

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

“…Synthetic promoter systems (SES) have been developed that are useful for several yeast and fungal species and that show yields comparable to P cbhI for cellulase production. SES consists of a promoter with a core sequence and of a heterologous transcription factor which binds to repeated activating sequences placed in the promoter (Rantasalo et al 2018). Other promoters like the Aspergillus tet-on/tet-off system are useful for research purposes rather than for industrial production (Wanka et al 2016;Kluge et al 2018).…”

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production

“…The native A. oryzae promoter of the oxidoreductase gene, kojA, which is involved in kojic acid biosynthesis successfully induced the expression of the polyketide synthase gene (wA) and production of the respective polyketide, YWA1 [36]. Whilst mainly native promoters are used for heterologous expression in filamentous fungi [37], in S. cerevisiae, universal expression systems for fungal genes comprising a set of synthetic promoters and transcription factors have been recently developed to synthesize a wide range of fungal natural products [38][39][40]. However, because A. oryzae possesses a variety of proteins and secretion systems for proteins and low-molecular-weight compounds that differ from those in S. cerevisiae [41][42][43], finding additional promoters that would be functional in A. oryzae is important for the use of this species as a heterologous expression host in addition to S. cerevisiae.…”