Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter.
By using a beta-glucanase from Bacillus as a model protein, we investigated whether the secretion competence based on the action of the kil gene can be improved using stronger promoters for the expression of the kil gene. Since the production of extracellular target proteins also depends on the promoter strengths of the target gene, we constructed four expression vectors with all possible combinations of a weak and a strong stationary-phase promoter for the kil gene, and a weak and a strong constitutive promoter, respectively, for the beta-glucanase gene. The results of batch fermentations showed that the use of stronger promoters generally decreased the cell density. However, a drastic increase of productivity of the cells to produce and secrete beta-glucanase resulted in a significantly higher activity of extracellular beta-glucanase. The yield of extracellular beta-glucanase can be increased (to 168 %) by using a strong promoter for the beta-glucanase alone. However, the increase was much higher when the weak promoter of the kil gene was replaced by a strong stationary-phase promoter (to 221 %). An even higher yield of extracellular beta-glucanase was reached when beta-glucanase was expressed by a strong promoter in addition indicating a combinatorial effect. This shows that the extracellular production of a recombinant target gene can be optimized by tuning the promoter strengths of components, the kil gene and the target gene.
Beshay
Usama
U
Bioprocess Development Department, Genetic Engineering & Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research & Technology Applications, New Borg El-Arab, Alexandria, Egypt. ube@fermtech.techfak.uni-bielefeld.de
Miksch
Gerhard
G
Friehs
Karl
K
Flaschel
Erwin
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D044602:Cellulases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism*
D029968:Escherichia coli Proteins; D010455:Peptides; D011994:Recombinant Proteins; C069003:kil protein, E coli; D044602:Cellulases