Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene.
Pseudomonas alcaligenes secretes a lipase with a high pH optimum, which has interesting properties for application in detergents. The expression of the lipase is strongly dependent on the presence of lipids in the growth medium such as soybean oil. The promoter of the gene was characterized and found to have resemblance to sigma54 controlled promoters, which are known to be tightly regulated. The transcription start was mapped precisely downstream of a sequence with close similarity to the -12/-24 consensus sequence of sigma54 controlled promoters. Interestingly, a hyperproducer mutant strain was isolated and found to have a C to T mutation in the -12/-24 promoter consensus region. In addition an Upstream Activating Sequence (UAS) with homology to sigma54 UAS consensus sequences was identified. It was demonstrated that an increase of the distance from the UAS to the transcription start or the deletion of the UAS results in significantly lower expression levels of lipase. A systematic mutational analysis of the UAS sequence has resulted in a variant with an increased lipase expression.
Cox
M
M
Protein Sciences Corporation, Meriden, CT 06450-7159, USA.
Gerritse
G
G
Dankmeyer
L
L
Quax
W J
WJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D004274:DNA, Recombinant; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008049:Lipase; C064053:CCTAGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases