Enzyme and Microbial Technology 39 (2006) 391–398 Global transcriptional analysis of metabolic burden due to plasmid maintenance in Escherichia coli DH5 during batch fermentation Dave Siak-Wei Ow a,b,∗ , Peter Morin Nissom a , Robin Philp a , Steve Kah-Weng Oh a , Miranda Gek-Sim Yap a,b a Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore b NUS Graduate School for Integrative Sciences and Engineering, 10 Medical Drive, #01-10, National University of Singapore, Singapore 117597, Singapore Received 6 September 2005; received in revised form 26 October 2005; accepted 10 November 2005 Abstract DNA plasmids of Escherichia coli are common vectors for recombinant protein and metabolite production and have potential therapeutic applications as genetic vaccines and therapeutics. However, plasmid maintenance imposes a metabolic burden on the host cells, resulting in reduced growth rate and cell density. In 2 L batch fermentation, DH5 cells carrying a 7.3 kb NS3 plasmid had a lower specific growth rate than the non-plasmid-bearing host (0.64 h -1 versus 0.87 h -1 ). In this work, global transcriptional analysis was combined with proteomics studies to evaluate the effect of plasmid maintenance on gene expression. Global transcriptional expression analysis of plasmid-bearing cells over host showed a general trend of downregulated biosynthetic/energy metabolism genes, differentially expressed transport genes and upregulated heat shock proteins. In the central metabolic pathways, most glycolytic genes were downregulated, while less expression difference was found in the pentose phosphate pathway. Expression ratios of 19 proteins identified from proteomics studies were consistent with these observations. Our findings suggest that plasmid maintenance alone perturbs global gene regulation, and leads to significant changes in central metabolic pathways in the host. This work contributes to our understanding of plasmid metabolic load at the gene expression level and could potentially aid in future metabolic engineering efforts. © 2006 Elsevier Inc. All rights reserved. Keywords: Metabolic burden; DH5; Fermentation; Plasmid maintenance 1. Introduction In biotechnology, high copy DNA plasmids of Escherichia coli serve as important vectors for production of valuable metabolites and proteins, and also have potential therapeutic applications as genetic vaccines. However, the maintenance and synthesis of such plasmids impose an uncharacterized metabolic burden on the bacterial host, widely observed as reduced host growth and viability [1]. Because of the detrimental effect of this plasmid-imposed metabolic burden, cells that lose plasmid are able to grow faster during fermentation. Over time, these non-productive cells become a significant fraction of the popu- lation [2]. This contributes to problems such as plasmid loss and reduced productivity, which are of relevance to the industry [3]. ∗ Corresponding author. Tel.: +65 64788859; fax: +65 64789561. E-mail address: dave ow@nus.edu.sg (D.S.-W. Ow). The central metabolic pathways of E. coli provide precur- sors, cofactors and energy for biosynthesis and other metabolic processes. Therefore, they have been the targets of cell line engi- neering efforts for bioprocess improvements for both native and recombinant bio-products. Metabolic burden could arise due to the extra biosynthetic demands for plasmid synthesis [4], or the perturbation of the E. coli host regulatory system affecting central metabolic pathways [1,5]. In this work, global tran- scriptional analysis was conducted in parallel with proteomics studies to examine expression changes resulting from plasmid maintenance on central metabolism and global gene regulation. Although DNA microarray technology has been applied to eluci- date the metabolic response of E. coli to protein overexpression previously [6], this is the first global transcriptome-proteome study of metabolic changes attributed solely to the maintenance of plasmid during batch fermentation. We believe that a better understanding of plasmid-imposed metabolic burden could be useful for improving industrial E. coli strains in the future. 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.11.048