A Genomewide Evolution-Based CRISPR/Cas9 with Donor-Free (GEbCD) for Developing Robust and Productive Industrial Yeast

Developing more robust and productive industrial yeast
is crucial
for high-efficiency biomanufacturing. However, the challenges posed
by the long time required and the low abundance of mutations generated
through genomewide evolutionary engineering hinder the development
and optimization of desired hosts for industrial applications. To
address these issues, we present a novel solution called the Genomewide
Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which
nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition
of highly abundant yeast mutants. Together with modified rad52 of
the DNA double-strand break repair in Saccharomyces
cerevisiae, a hypermutation host was obtained with
a 400-fold enhanced mutation ability. Under multiple environmental
stresses the system could rapidly generate millions of mutants in
a few rounds of iterative evolution. Using high-throughput screening,
an industrial S. cerevisiae SISc-Δrad52-G4-72
(G4-72) was obtained that is strongly robust and has higher productivity.
G4-72 grew stably and produced ethanol efficiently in multiple-stress
environments, e.g. high temperature and high osmosis. In a pilot-scale
fermentation with G4-72, the fermentation temperature was elevated
by 8 °C and ethanol production was increased by 6.9% under the
multiple stresses posed by the industrial fermentation substrate.
Overall, the GEbCD system presents a powerful tool to rapidly generate
abundant mutants and desired hosts, and offers a novel strategy for
optimizing microbial chassis with regard to demands posed in industrial
applications.