(102d) Harnessing Alternative Splicing for Gene Regulation in Saccharomyces Cerevisiae

Authors: 
Cui, X., Singapore-MIT Alliance for research and technology
Ma, X., Singapore-MIT Alliance for research and technology, DiSTAP
Zhou, K., Singapore-MIT Alliance for research and technology, DiSTAP
Stephanopoulos, G., Massachusetts Institute of Technology

In eukaryotic organisms,
introns are well-conserved elements that play important roles in regulating
gene expression. The presence of introns in eukaryotes increases the complexity
of gene regulation by alternative splicing, which diversifies mRNAs and proteins.
However, the mechanism behind alternative splicing in eukaryotic microorganisms
was still not fully understood and has not been widely used in bioengineering.

In this study, we aim to
harness introns as basic elements to regulate gene expression in Saccharomyces
cerevisiae
. We developed a synthetic promoter library of 72 intron-containing
promoters and
20 intron-containing
terminators
in S.
cerevisiae
,
which were designed and constructed in the structure of “[Promoter]-[Start
codon]-[Intron]-[The rest of a gene]” and “[Gene excluding stop codon]-[Intron]-[Stop
codon]-[Terminator]”. We observed that the inclusion of native introns in gene
expression cassette could
expand the dynamic range of protein expression level. It was notable that the
protein expression level of Yellow Fluorescence Protein (YFP) could be enhanced
over 7-fold when the weakest intron-containing promoter was replaced by the
strongest one. More importantly, a few intron-containing promoters and
terminators exhibited enhanced strength compared to corresponding native
promoters and terminators. The results offered us opportunities to enhance
protein expression in S. cerevisiae by utilizing these intron-promoters
and terminators, and further improve product titres in pathway engineering.
Those synthetic intron-containing promoters and terminators with low strength
were also useful in regulating protein expression for complex pathways. In the future,
we will use machine-learning to learn the rules behind the performance of the
synthetic intron-containing promoters and terminators. In addition, we will use
the intron-containing promoters to express multiple genes in metabolic
engineering applications.

margin-left:0cm;text-align:justify;text-justify:inter-ideograph">Furthermore, we also
discovered that a set of stem loop-containing artificial introns could be used
to auto-induce gene expression in S. cerevisiae. The synthetic introns
we designed contain a fixed 5’ Splicing Site (5’SS), a branch point (BP) as
well as a 3’ Splicing Site (3’SS). At 5’SS, a stem loop structure was built artificially.
By inserting the artificial introns into the middle of YFP gene, it was
observed that the YFP expression was automatically induced along the growth of
yeast cells. This means that the specific fluorescence increases substantially
along the cell growth. The results have demonstrated the utility of an
orthogonal gene regulation tool other than promoters for S. cerevisiae for
the first time. This tool could be utilized in producing the proteins
that may be toxic or inhibit cell growth, and thus be valuable to genetic
engineering research.

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