Akademik Veri Yönetim Sistemi
Methods in Enzymology - Recombinant Protein Expression: Eucaryotic Hosts, William B O'Dell,Zvi Kelman, Editör, Academic Press , London, ss.81-104, 2021
Engineered promoters are key components that allow engineered
expression of genes in the cell-factory design. Promoters having exceptional
strength are attractive candidates for designing metabolic engineering
strategies for tailoring de novo
production strategies that require directed evolution methods by engineering
with de novo synthetic biology tools.
Engineered promoter variants (EPVs) of naturally occurring promoters (NOPs) can
be designed using metabolic engineering strategies and synthetic biology tools
if the genes encoding the activating transcription factors (TFs) exist in the
genome and are expressed and synthesized at non-limiting concentrations within the
cell. The hybrid-architectured EPV design
method targets an essential and predetermined part of the general transcription machinery. That is cis-acting
DNA site(s) in coordination with the trans-acting factor(s) that bound
on, required for the regulated 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, enabling the engineering of binding of master regulator TF(s). The
method aims to generate EPVs that combine the advantages of being an
exceptional stronger EPV(s) than the NOPs and green-and-clean production on a
non-toxic carbon source, such as ethanol or glucose. By introducing our recent
work on the engineering of ADH2 hybrid-promoter
architectures to
enhance
recombinant protein expression on ethanol, we provide the
method and protocol for the design of ADH2
hybrid-promoter architectures that can be adapted to other promoters in
different substrate utilization pathways in Pichia pastoris, as well as
in other yeasts.