Proteomic Profiling of Escherichia coli Proteins under High Cell Density Fed-Batch Cultivation with Overexpression of Phosphogluconolactonase Yonghui Wang, † Shiaw-Lin Wu, † William S. Hancock,* ,† Robin Trala, ‡ Michelle Kessler, ‡ Alexander H. Taylor, ‡ Pramatesh S. Patel, and Juan C. Aon* ,‡ Barnett Institute, Northeastern University, Boston, Massachusetts 02115, and Microbial and Cell Culture Development, GlaxoSmithKline, UE0447C, 709 Swedeland Road, King of Prussia, Pennsylvania 19406 In this study, we used proteomics to better understand the growth on glucose of Escherichia coli in high cell density, fed-batch cultures and the response to overex- pression of plasmid-encoded 6-phosphogluconolactonase (PGL). Using liquid chroma- tography coupled to electrospray mass spectrometry, at least 300 proteins were identified in the cytosolic fraction of the six time points used to monitor the fermentation. The relative abundance changes of selected proteins were obtained by comparing the peak area of the corresponding peptides at a particular m/z (mass over charge ratio) value. During the time course of samples collected during the rapid growth achieved under batch and fed-batch conditions, both the control and recombinant E. coli strains showed up-regulation of proteins participating in the tricarboxylic acid (TCA) cycle, particularly acetyl-CoA synthetase (AcCoAS), malate dehydrogenase (MDH), and succinyl-CoA synthetase (SuccCoAS). In the recombinant strain culture, fumarase was up-regulated until 35 h after inoculation but was not in the control strain culture. In addition, the proteomic measurement detected up-regulation of three well-characterized binding transport proteins in both control and recombinant strains. The up-regulation of TCA cycle enzymes is consistent with the increase in growth rate observed in the cell culture. In addition, up-regulation of these proteins demonstrated the importance of both the pentose-phosphate shunt and TCA cycle to the increased biosynthetic activity required by a high level protein synthesis. This study shows the potential of proteomics using shotgun sequencing (LC/MS of tryptic digests) to measure global changes in protein abundance during a fermentation process and will facilitate the development of robust manufacturing systems. Introduction The measurement of quantitative proteomic changes can be used to characterize a disease state for biomarker discovery (1, 2) or the effect of drug treatment (3, 4), as well as the characterization of biological systems. In such a study the understanding of metabolic pathways is crucial, and in addition to feedback mechanisms, changes of protein concentration can have a significant influence on the activity of such pathways. In this study, we have explored the use of proteomics to study the different stages in cell cultivation with the goal of optimizing biomass yields and growth rate at high cell densities. High cell density cultivation (HCDC) has been used as a way to enhance the yield of desired products (5). However, the achievement of high growth rates and maximized product yield in microbial systems under high cell density is still a challenging topic for the biotech- nology industry (6-8). The synthesis of plasmid-encoded proteins, as well as plasmid-DNA replication, in cultured cells often generates a significant metabolic burden, which usually results in reduced growth rates (9). This metabolic burden may be related to the limited capacity of a cell to supply the extra demand for building blocks and energy. Also, industrial microorganisms are fre- quently challenged by substrate or oxygen limitation during HCDC. These unfavorable conditions have been known to induce stress responses in microbial cells, e.g., byproduct synthesis, and consequent changes in cellular protein composition, as well as other components in the biomass. Effects of different stressful culture conditions on cell growth (10) and approaches to recover the reduced growth rate (11-14) have been extensively studied. One particular approach to overcome the reduced growth rates observed in HCDC is to modulate the central carbon metabolism, which is supported by the study of Flores et al. (14). In this study, the authors engineered the pentose phosphate shunt (PP shunt) by overexpressing the zwf gene, which encodes for glucose-6-phosphate dehydrogenase, and observed a recovery in the growth rate of E. coli cultures. To better understand cell growth and intracellular responses, proteomic profiling can provide valuable knowl- edge that can be used for the development of metabolic and cellular engineering strategies, which can help optimize both yield and productivity in the bioreactor process (15, 16). We, therefore, carried out such analyses * To whom correspondence should be addressed. (W.D.H.) Ph: (617) 373-4881. E-mail: wi.hancock@neu.edu. (J.C.A.) Ph: (610) 270-5802. Fax: (610) 270-7449. E-mail: juan.c.aon@gsk.com. † Northeastern University. ‡ GlaxoSmithKline. 1401 Biotechnol. Prog. 2005, 21, 1401-1411 10.1021/bp050048m CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 08/20/2005