title: Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite.
abstract: BACKGROUND
  High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. However, metabolic engineering can result in unintended phenotypes that may go unnoticed in initial screening. The red pigment lycopene, a carotenoid with antioxidant properties, has been used as a reporter of isoprenoid pathway flux in metabolic engineering for over a decade. Lycopene production is known to vary between wild-type Escherichia coli hosts, but the reasons behind this variation have never been fully elucidated.
  
  
  RESULTS
  In an examination of six E. coli strains we observed that strains also differ in their capacity for increased lycopene production in response to metabolic engineering. A combination of genetic complementation, quantitative SWATH proteomics, and biochemical analysis in closely-related strains was used to examine the mechanistic reasons for variation in lycopene accumulation. This study revealed that rpoS, a gene previously identified in lycopene production association studies, exerts its effect on lycopene accumulation not through modulation of pathway flux, but through alteration of cellular oxidative status. Specifically, absence of rpoS results in increased accumulation of reactive oxygen species during late log and stationary phases. This change in cellular redox has no effect on isoprenoid pathway flux, despite the presence of oxygen-sensitive iron-sulphur cluster enzymes and the heavy redox requirements of the methylerythritol phosphate pathway. Instead, decreased cellular lycopene in the ΔrpoS strain is caused by degradation of lycopene in the presence of excess reactive oxygen species.
  
  
  CONCLUSIONS
  Our results demonstrate that lycopene is not a reliable indicator of isoprenoid pathway flux in the presence of oxidative stress, and suggest that caution should be exercised when using lycopene as a screening tool in genome-wide metabolic engineering studies. More extensive use of systems biology for strain analysis will help elucidate such unpredictable side-effects in metabolic engineering projects.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, 4072, Australia. m.bongers1@uq.edu.au.
authors.forename: Mareike
authors.initials: M
authors.lastname: Bongers
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, 4072, Australia. p.chrysanthopoulos@uq.edu.au.|Metabolomics Australia (Queensland Node), The University of Queensland, St. Lucia, QLD, 4072, Australia. p.chrysanthopoulos@uq.edu.au.
authors.forename: Panagiotis K
authors.initials: PK
authors.lastname: Chrysanthopoulos
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, 4072, Australia. james.behrendorff@gmail.com.
authors.forename: James B Y H
authors.initials: JB
authors.lastname: Behrendorff
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, 4072, Australia. m.hodson1@uq.edu.au.|Metabolomics Australia (Queensland Node), The University of Queensland, St. Lucia, QLD, 4072, Australia. m.hodson1@uq.edu.au.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Hodson
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, 4072, Australia. c.vickers@uq.edu.au.
authors.forename: Claudia E
authors.initials: CE
authors.lastname: Vickers
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, 4072, Australia. lars.nielsen@uq.edu.au.
authors.forename: Lars K
authors.initials: LK
authors.lastname: Nielsen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004896:Erythritol / Q000031:analogs & derivatives / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D000077276:Lycopene; D060847:Metabolic Engineering; D018384:Oxidative Stress; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism; D012808:Sigma Factor / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013729:Terpenes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D017382:Reactive Oxygen Species; D012808:Sigma Factor; D013729:Terpenes; C069567:sigma factor KatF protein, Bacteria; D002338:Carotenoids; D004896:Erythritol; D000077276:Lycopene