Akademik Veri Yönetim Sistemi
Methods in Enzymolology - Recombinant Protein Expression: Eukaryotic Hosts, WILLIAM B. O’DELL,ZVI KELMAN, Editör, Academic Press , London, ss.105-128, 2019
Hybrid-architectured
promoter design to deregulate expression in yeast under modulating power of
carbon sources involves replacing native cis-acting DNA sequence(s) with
de novo synthetic tools in coordination with master regulator
transcription factor (TF) to alter crosstalk between signaling pathways,
and consequently, transcriptionally rewires the expression. Hybrid-promoter
architectures can be designed to mimic native promoter
architectures in yeast’s preferred carbon source utilization pathway. The
method aims to generate engineered promoter variants (EPVs) that combine the
advantages of being an exceptionally stronger EPV(s) than the naturally
occurring promoters and permit ′′green-and-clean′′ production on a non-toxic carbon source. To implement
the method, a predetermined essential part of the general transcription machinery is
targeted. This targeting involves cis-acting
DNA sequences to be replaced with synthetic cis-acting
DNA sites in coordination with the
targeted TF that must bind,
required for transcription machinery
activation. The method needs genomic
and functional information that can lead to the discovery
of the master TF(s) and synthetic cis-acting DNA elements, which enable
the engineering of binding of master regulator TF(s). By introducing our recent work on the engineering of Pichia pastoris (syn. Komagataella
phaffii) alcohol oxidase 1 (AOX1) hybrid-promoter architectures, we provide the method and protocol for the
hybrid-architectured EPV design to deregulate expression in yeast. The method
can be adapted to other promoters in different substrate utilization pathways
in P. pastoris, as well as in other yeasts.