Cdk1-regulated diphosphodegron tags for controlled protein expression
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Tartu Ülikool
Abstract
Cyclin dependent kinases (CDKs) regulate the cell cycle by phosphorylating downstream
proteins in an ordered manner. In yeast S. cerevisiae, Cdk1 regulates the cell cycle
progression from G1 to M phase. Cdk1 phosphorylation can have various effects on the
proteins, for example, they can be activated or targeted for degradation. The latter was used in
this work to design a regulatory network for controlled protein expression that could be used
in cell factories. For this, Far1-based diphosphodegron tags that are degraded in response to
phosphorylation by Cdk1, were used to differentially regulate the levels of the transcriptional
repressor TetR. Furthermore, as cell factories require dynamic control of protein levels over
the cultivation process, the effect of cell culture density on cell cycle and the degron tags was
studied. The work shows that Far1-based degron tags can be used as modular tags to regulate
exogenous proteins by switching them on or off in an OD-dependent manner using the
endogenous Cdk1 machinery. Finally, it was shown that more complex patterns of regulation
can be achieved by inducing expression of cyclins at high OD.
Description
Keywords
cell cycle, phosphorylation, synthetic biology, protein degradation