Elsevier

Journal of Biotechnology

Volume 126, Issue 4, 1 December 2006, Pages 528-545
Journal of Biotechnology

Transcriptome analysis of a shikimic acid producing strain of Escherichia coli W3110 grown under carbon- and phosphate-limited conditions

https://doi.org/10.1016/j.jbiotec.2006.05.007Get rights and content

Abstract

Shikimic acid, which is produced in the aromatic amino acid pathway in plants and microorganisms, is an industrially interesting chiral starting material for the synthesis of many chemical substances, e.g. the influenza medicine Tamiflu®. When produced by genetically modified Escherichia coli it has previously been found that carbon-rich conditions (e.g. phosphate-limitation) favors production of shikimic acid over shikimate pathway by-products, whereas the situation is the opposite at carbon-(glucose-) limited conditions. In the present study, gene expression patterns of the shikimate producing strain W3110.shik1 (W3110 with aroL deletion and plasmid-overexpressed aroF) and the wild type strain W3110 grown under carbon- and phosphate-limited (carbon-rich) chemostat conditions (D = 0.23 h−1) were analyzed. The study suggests that the by-product formation under carbon-limitation is explained by a set of upregulated genes coupled to the shikimate pathway. The genes, ydiB, aroD and ydiN, were strongly induced only in carbon-limited W3110.shik1. Compared to W3110 the lg2-fold changes were: 6.25 (ydiB); 3.93 (aroD) and 8.18 (ydiN). In addition, the transcriptome analysis revealed a large change in the gene expression when comparing phosphate- to carbon-limitation, which to a large part could be explained by anabolic–catabolic uncoupling, which is present under phosphate-limitation but not under carbon-limitation. Interestingly, there was also a larger difference between the two strains under carbon-limitation than under phosphate-limitation. The reason for this difference is interpreted in terms of starvation for aromatic amino acids under carbon-limitation which is relieved under phosphate-limitation due to an upregulation of aroK and aroA.

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.

References (63)

  • T. Polen et al.

    The global gene expression response of Escherichia coli to l-phenylalanine

    J. Biotechnol.

    (2005)
  • M.J. Teixeira de Mattos et al.

    Bioenergetic consequences of microbial adaptation to low-nutrient environments

    J. Biotechnol.

    (1997)
  • M.J. Whipp et al.

    Cloning and analysis of the shiA gene, which encodes the shikimate transport system of Escherichia coli K-12

    Gene

    (1998)
  • P. Baldi et al.

    A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes

    Bioinformatics

    (2001)
  • B.R. Bochner et al.

    Positive selection for loss of tetracycline resistance

    J. Bacteriol.

    (1980)
  • G. Bogosian et al.

    Trp repressor protein is capable of intruding into other amino acid biosynthetic systems

    Mol. Gen. Genet.

    (1983)
  • M. Cashel et al.

    The stringent response

  • S.S. Chandran et al.

    Phosphoenolpyruvate availability and the biosynthesis of shikimic acid

    Biotechnol. Prog.

    (2003)
  • D.-E. Chang et al.

    Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival

    J. Bacteriol.

    (1999)
  • I.P. Crawford et al.

    Regulation of tryptophan biosynthesis

    Annu. Rev. Biochem.

    (1980)
  • Y. Cui et al.

    A consensus sequence for binding of Lrp to DNA

    J. Bacteriol.

    (1995)
  • M. Dauner et al.

    Bacillus subtilis metabolism and energetics in carbon-limited and excess-carbon chemostat culture

    J. Bacteriol.

    (2001)
  • J. Delgado et al.

    Inverse flux analysis for reduction of acetate excretion in Escherichia coli

    Biotechnol. Prog.

    (1997)
  • K.M. Draths et al.

    Shikimic acid and quinic acid: replacing isolation from plant sources with recombinant microbial biocatalysis

    J. Am. Chem. Soc.

    (1999)
  • B. Ely et al.

    Aromatic amino acid biosynthesis: Regulation of shikimate kinase in Escherichia coli K12

    J. Bacteriol.

    (1979)
  • B.R. Ernsting et al.

    Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli

    J. Bacteriol.

    (1992)
  • B.R. Ernsting et al.

    Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein?

    J. Bacteriol.

    (1993)
  • Q. Hua et al.

    Analysis of gene expression in Escherichia coli in response to changes of growth-limiting nutrient in chemostat cultures

    Appl. Environ. Microbiol.

    (2004)
  • L. Johansson et al.

    Shikimic acid production by a modified strain of Escherichia coli (W3110.shik1) under phosphate-limited and carbon-limited conditions

    Biotechnol. Bioeng.

    (2005)
  • L. Johansson et al.

    Transcriptome analysis of a shikimic acid producing strain of Escherichia coli W3110 at carbon- and phosphate-limited conditions

    J. Biotechnol.

    (2005)
  • D.I. Johnson et al.

    Evidence that repression mechanisms can exert control over the thr, leu, and ilv operons of Escherichia coli K-12

    J. Bacteriol.

    (1983)
  • Cited by (0)

    View full text