Microbial production of biofuels and bioproducts offers a sustainable
and economic alternative to petroleum-based fuels and chemicals. The basidiomycete
yeast Rhodosporidium toruloides is a promising platform organism for generating
bioproducts due to its ability to consume a broad spectrum of carbon sources
(including those derived from lignocellulosic biomass) and to naturally accumulate
high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged
to produce a wide range of bioproducts. While R. toruloides has great potential,
it has a more limited set of tools for genetic engineering relative to more advanced
yeast platform organisms such as Yarrowia lipolytica and Saccharomyces
cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’
engineering capacity. Here we expand this capacity by demonstrating the first
use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression
cassette harboring nourseothricin resistance and selecting transformants
on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted
disruption of the native URA3 gene. While editing efficiencies were initially low
(0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying
these optimized design conditions enabled disruption of another native gene
involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies
of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient
multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation.
Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides
that will facilitate rapid and high-throughput genome engineering in this industrially
relevant organism.
Revised: April 24, 2019 |
Published: March 20, 2019
Citation
Otoupal P.B., M. Ito, A.P. Arkin, J.K. Magnuson, J.M. Gladden, and J.M. Skerker. 2019.Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides.mSphere 4, no. 2:Article No. e00099-19.PNNL-SA-142513.doi:10.1128/mSphere.00099-19