Transcriptome analysis of a shikimic acid producing strain of Escherichia coli W3110 grown under carbon- and phosphate-limited conditions
Introduction
Shikimic acid is a chiral compound found in the shikimate pathway (aromatic amino acid metabolism) of plants and microorganisms (Fig. 1). This compound is industrially interesting as it serves as starting material for the production of several chemical substances, e.g. the anti-influenza agent Tamiflu® (Bongaerts et al., 2001, Kim et al., 1997, Kramer et al., 2003, Rohloff et al., 1998). One of the major issues concerning the production of shikimic acid has been to avoid shikimate pathway by-product formation since this reduces shikimic acid yield and quality (Chandran et al., 2003, Draths et al., 1999, Knop et al., 2001, Kramer et al., 2003). It has been found that a carbon-limited growth condition aggravates by-product formation, whereas growth under carbon-rich conditions (e.g. phosphate-limitation) favors shikimate production over that of by-products (Chandran et al., 2003, Johansson et al., 2005, Knop et al., 2001). Different hypotheses have been presented to explain this phenomenon, such as the hydroaromatic equilibration hypothesis (Knop et al., 2001), and the intracellular equilibration hypothesis (Johansson et al., 2005). However, the reasons for the by-product formation are still not completely understood.
Global transcriptome analysis is a powerful tool that can be used to study regulation of cellular metabolism. In relation to the aromatic metabolism in Escherichia coli, transcriptome analysis has previously been used for investigating the transcriptional response to addition of phenylalanine and shikimic acid (Polen et al., 2005), and to investigate the tryptophan metabolism in E. coli (Khodursky et al., 2000). These two studies reported important effects, such as a strong upregulation of the trp-operon under tryptophan starved conditions. In the present paper (of which an abstract was presented at the ECB12-meeting, Johansson and Lidén, 2005), transcriptome analysis was used to elucidate by-product formation from the shikimate pathway. Gene expression of a shikimic acid producing strain (W3110.shik1) was compared to that of a control strain (W3110) under carbon as well as under phosphate-limited conditions. In addition, differences between expression levels at phosphate- and carbon-limitation for the two strains were analyzed, with focus on the central carbon metabolism and amino acid metabolism.
Section snippets
Strains
W3110 was received from American Type Culture Collection (ATCC 27325) and was also used as host for the shikimic acid producing strain (referred to as W3110.shik1), with the following genetic modifications: ΔaroL, tryptophan and phenylalanine feedback resistant aroGFBR, tryptophan feedback resistant trpEFBR and tnaA. In addition, W3110.shik1 was cloned with plasmid pSGs26 (derived from pBR322) containing tyrosine and phenylalanine feedback resistant aroFFBR and two antibiotic resistance markers
Statistical considerations
The results presented are based on steady state samples from a total of eight chemostat experiments all made at a dilution rate around 0.23 h−1 (two of each strain in both phosphate (P-) and carbon (C-) limitation). Technical duplicates were made for one of the W3110 samples from both the P- and the C-limited chemostats, i.e. a total of 10 chips were compared. The comparisons of expression levels were made in two dimensions and were presented in form of fold changes (Fig. 2).
Several methods are
Concluding remarks
Understanding by-product formation deriving from the shikimate pathway is a significant challenge in order to obtain efficient shikimic acid production in E. coli. C-limited condition has been shown to aggravate the problem of by-product formation when compared to C-rich conditions (P-limitation)—a phenomenon not yet fully understood. In the present study, transcriptome analyses of a shikimate producing strain and a control strain of E. coli grown in C- and P-limited conditions was used in
Acknowledgements
This work was financially supported by the Swedish Agency for Innovation Systems (Vinnova) through the research contract Enabling Technologies for Industrial Fermentations and by Sparbanken Färs och Frosta. The Swegene Microarray resource center in Lund is acknowledged for help with the microarray analyses.
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