Determinants for overproduction of the Pseudomonas oleovorans cytoplasmic membrane protein alkane hydroxylase in alk+ Escherichia coli W3110.
The Pseudomonas oleovorans alkB gene is expressed in alk+ Escherichia coli W3110 to 10 to 15% of the total cell protein, which is exceptional for a (foreign) cytoplasmic membrane protein. In other E. coli recombinants such as alk+ HB101, AlkB constitutes 2 to 3% of the total protein. In this study, we have investigated which factors determine the expression level of alkB in alk+ W3110. In particular, we have investigated the role of AlkB-induced stimulation of phospholipid synthesis. Blocking phospholipid synthesis in alk+ W3110 did not specifically alter the expression of alkB, and we conclude that stimulation of phospholipid synthesis is not a prerequisite for high-level expression of the membrane protein. W3110 is able to produce exceptionally high levels of alkane monooxygenase, because the rate of alkB mRNA synthesis in W3110 is an order of magnitude higher than that in HB101. This may be due in part to the higher copy number of pGEc47 in W3110 in comparison with HB101.
Nieboer
M
M
Institut of Biotechnology, ETH Hönggerberg (HPT), Zürich, Switzerland.
Gunnewijk
M
M
van Beilen
J B
JB
Witholt
B
B
D001426:Bacterial Proteins / Q000096:biosynthesis*; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000096:biosynthesis*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D008957:Models, Genetic; D010743:Phospholipids / Q000096:biosynthesis; D010957:Plasmids / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity
D001426:Bacterial Proteins; D008565:Membrane Proteins; D010743:Phospholipids; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A