Influence of induction and operation mode on recombinant rhamnulose 1-phosphate aldolase production by Escherichia coli using the T5 promoter.
Fed-batch culture techniques were employed to grow Escherichia coli at high-cell densities for the intracellular production of a recombinant rhamnulose 1-phosphate aldolase (RhuA) under the transcriptional control of the strong promoter T5, using a commercially available expression system (E. coli strain M15 and plasmid vector pQE40). A predetermined exponential feeding strategy at constant specific growth rate was selected to maintain carbon source limited growth using a defined medium. Growth rates below 0.36 h(-1) did not cause a severe formation of acetic acid while cell concentration increased exponentially up to 95 g(biomass)l(-1). The intrinsic biomass-substrate yield (gamma(X/S) = 0.48 gg(-1)) and the maintenance coefficient (mS = 0.10 g(substrate)g(biomass)(-1)h(-1)) were calculated from fed-batch growths at different specific growth rates. These values have been employed to determinate the addition profile during the fed-batch growth until IPTG induction, reaching a specific RhuA production levels of 565 AU g(biomass)(-1) which was lower than in batch (1250 AU g(biomass)(-1)). An inverse correlation between volumetric IPTG concentration and specific RhuA activity was found. A correlation between the ratio biomass/OD(600 nm) and the quantity of recombinant protein produced was found. Finally, the proposed process, after optimization of the IPTG concentration, led to significant increase in enzyme concentration and volumetric productivity compared to batch mode (2680 and 1338%, respectively).
Vidal
Luis
L
Departament d'Enginyeria Química, Escola Tècnica Superior d'Enginyeria, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.
Ferrer
Pau
P
Alvaro
Gregorio
G
Benaiges
M Dolors
MD
Caminal
Gloria
G
D000446:Aldehyde-Lyases / Q000096:biosynthesis* / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000502:physiology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017906:Siphoviridae / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology
D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D000446:Aldehyde-Lyases; C022526:rhamnulose-1-phosphate adolase