Engineering promoter regulation.
Systems for easily controlled, conditional induction or repression of gene expression are indispensable tools in fundamental research and industrial-scale biotechnological applications. Both native and rationally designed inducible promoters have been widely used for this purpose. However, inherent regulation modalities or toxic, expensive or inconvenient inducers can impose limitations on their use. Tailored promoters with user-specified regulatory properties would permit sophisticated manipulations of gene expression. Here, we report a generally applicable strategy for the directed evolution of promoter regulation. Specifically, we applied random mutagenesis and a multi-stage flow cytometry screen to isolate mutants of the oxygen-responsive Saccharomyces cerevisiae DAN1 promoter. Two mutants were isolated which were induced under less-stringent anaerobiosis than the wild-type promoter enabling induction of gene expression in yeast fermentations simply by oxygen depletion during cell growth. Moreover, the engineered promoters showed a markedly higher maximal expression than the unmutated DAN1 promoter, under both fastidious anaerobiosis and microaerobisois.
Nevoigt
Elke
E
Department of Chemical Engineering, Massachusetts Institute of Technology, Room 56-469, Cambridge, Massachusetts 02139, USA.
Fischer
Curt
C
Mucha
Oliver
O
Matthäus
Falk
F
Stahl
Ulf
U
Stephanopoulos
Gregory
G
D000332:Aerobiosis / Q000502:physiology; D000693:Anaerobiosis / Q000502:physiology; D019020:Directed Molecular Evolution; D015966:Gene Expression Regulation, Fungal; D006023:Glycoproteins; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
C107098:DAN1 protein, S cerevisiae; D006023:Glycoproteins; D029701:Saccharomyces cerevisiae Proteins