AUTHOR=Johns Alexander M. B. , Love John , Aves Stephen J. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.01666

DOI=10.3389/fmicb.2016.01666

ISSN=1664-302X

ABSTRACT=<p><italic>Rhodotorula</italic> (<italic>Rhodosporidium</italic>) <italic>toruloides</italic> 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 <italic>R. toruloides</italic> which include elements for <italic>Saccharomyces cerevisiae</italic> 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 <italic>NAR1, ICL1, CTR3</italic>, and <italic>MET16</italic>. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in <italic>R. toruloides</italic>, 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 <italic>NAR1</italic> promoter. The <italic>NAR1</italic> promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a <italic>leu2</italic> mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in <italic>R. toruloides</italic>.</p>