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Applied and Environmental Microbiology, February 2008, p. 950-958, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.01790-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli{triangledown} ,{dagger}

Juan C. Aon,1* Richard J. Caimi,2 Alexander H. Taylor,1 Quinn Lu,3 Femi Oluboyede,1 Jennifer Dally,2 Michelle D. Kessler,1 John J. Kerrigan,3 Tia S. Lewis,3 Lisa A. Wysocki,3 and Pramatesh S. Patel1

Microbial and Cell Culture Development,1 Biopharmaceutical Analytical Sciences,2 Gene Expression and Protein Biochemistry, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 194063

Received 1 August 2007/ Accepted 2 December 2007

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.

* Corresponding author. Mailing address: Microbial and Cell Culture Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406. Phone: (610) 270-5802. Fax: (610) 270-7449. E-mail: juan.c.aon{at}gsk.com

{triangledown} Published ahead of print on 14 December 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, February 2008, p. 950-958, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.01790-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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