Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides

Abstract

Rhodotorula (Rhodosporidium) toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70% of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3, and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.

A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

“…To improve prospects for more advanced metabolic engineering in this fungal host, the development of robust genetic tools and resources is an immediate priority [7]. A major need on the tools front is for efficient and stable promoters to express genes of interest and selection markers.…”

“…A growing branch of synthetic biology involves the pursuit and improvement of standard parts that are reliable, orthogonal and robust for non-conventional hosts [8,9]. A small selection of promoters has already been characterized to modulate expression of heterologous genes in R. toruloides, the majority of them being metabolite-responsive promoters rather than constitutive [7,[10][11][12][13]. While promoters that are responsive to certain metabolites are valuable tools, a toolset of well characterized constitutive promoters remains necessary to explore the full potential of strain engineering.…”

Multiplexed CRISPR-Cas9 based genome editing ofRhodosporidium toruloides

“…The past four years have seen various researchers remedy this problem with the development of tools for transforming R. toruloides and efficiently expressing exogenous DNA (15, 56). While the toolkit has expanded to include useful promoters (53), drug markers (11), and targeted gene editing methods (18), CRISPR-Cas9 methods for advanced genome engineering have been lacking. This study outlines the strategies by which researchers can employ multiplexed CRISPR-Cas9 genome editing to manipulate R. toruloides.…”

Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides

“…For the CRISPR/Cas9 system to function, a Cas protein and RNA guide (either a single gRNA, or a crRNA and tracrRNA) must be coexpressed in the nucleus of the cell. While a number of constitutive and inducible RNA Polymerase (RNAP) II promoters, suitable for expression of proteins, have been reported (Johns, Love, & Aves, ; Wang et al, ), there are no reported RNAP III promoters in R. toruloides for expression of short RNA molecules with well‐defined ends, such as a gRNA. The commonly used RNAP III SNR52 promoter from Saccharomyces cerevisiae does not have a known ortholog in R. toruloides .…”

“…Expression cassettes were designed for RGR, tRNA Gly , and 5S‐based gRNA expression (Figure a and Table S1) and assembled into the integration region of an ATMT plasmid (pEGFP‐Rt‐YR‐HYG [Johns et al, ], replacing eGFP for the RGR cassette, inserting into pZPK‐PGPD_HYG‐Tnos [Wang et al, ] for the others) and integrated to R. toruloides strain NP11 with the hygromycin (Hyg) selection marker.…”