Suppressing posttranslational gluconoylation of heterologous proteins by metabolic engineering of Escherichia coli.
Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.
Aon
Juan C
JC
Microbial and Cell Culture Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA. juan.c.aon@gsk.com
Caimi
Richard J
RJ
Taylor
Alexander H
AH
Lu
Quinn
Q
Oluboyede
Femi
F
Dally
Jennifer
J
Kessler
Michelle D
MD
Kerrigan
John J
JJ
Lewis
Tia S
TS
Wysocki
Lisa A
LA
Patel
Pramatesh S
PS
D002265:Carboxylic Ester Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005470:Fluorometry; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005942:Gluconates / Q000378:metabolism*; D013058:Mass Spectrometry; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism*
C114004:6-phosphogluconolactone; D005942:Gluconates; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022363:6-phosphogluconolactonase