Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

Abstract

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.

Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis

“…With the need for control of post‐translational gluconoylation in recombinant proteins becoming more significant in the production of proteins of pharmaceutical and medical applications 31, mP‐AGE analysis could provide an important tool for easy detection and separation of δ‐gluconolactone modified proteins and for the development of methods and substances that can prevent this modification.…”

Conserved Network of Proteins Essential for Bacterial Viability

“…Therefore, we speculate that the proteins may need to be modified in some way to mark them as targets for YgjD. Clearly, glycosylation may be one such way to modify the proteins, but other modifications also exist, some of which, such as acetylation (69) or nonenzymatic glycation (4,45), have been observed in eubacteria. Interestingly, glycated proteins arise when intermediates in sugar metabolism, such as methylglyoxal (67) or 6-phosphogluconolactone (4), accumulate, resulting in protein inactivation and toxic side effects such as protein precipitation (47), suggesting that a cellular mechanism should exist for the targeted degradation of such products.…”

Characterization of S‐thiolation on secreted proteins fromE. coliby mass spectrometry

“…The less accurate measurement at protein level was due to the decreased accuracy associated with determining larger masses. It has been known that modifications of +178 Da and +258 Da at the N‐terminus of E. coli expressed proteins, especially those expressed by the BL21 (DE3) strain, are due to gluconoylation and phosphogluconoylation 27, 28. The same modifications are assumed to occur on MP without further verification.…”