Promoter library designed for fine-tuned gene expression in Pichia pastoris.
Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P(AOX1)) sequence. This first library initially spanned an activity range between approximately 6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a 'real case', i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in P(AOX1)-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new P(AOX1) synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.
Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
Franz S
FS
Hartner
Claudia
C
Ruth
David
D
Langenegger
Sabrina N
SN
Johnson
Petr
P
Hyka
Geoffrey P
GP
Lin-Cereghino
Joan
J
Lin-Cereghino
Karin
K
Kovar
James M
JM
Cregg
Anton
A
Glieder
D000429:Alcohol Oxidoreductases / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D019149:Bioreactors; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase