title: Quantitative analysis of aromatics for synthetic biology using liquid chromatography.
abstract: The replacement of petrochemical aromatics with bio-based molecules is a key area of current biotechnology research. To date, a small number of aromatics have been produced by recombinant bacteria in laboratory scale while industrial production still requires further strain development. While each study includes some distinct analytical methodology to quantify certain aromatics, a method that can reliably quantify a great number of aromatic products and relevant pathway intermediates is needed to accelerate strain development. In this study, we developed a robust reverse phase high performance liquid chromatography method to quantify a wide range of aromatic metabolites present in host microorganisms using the shikimate pathway, which is the major metabolic pathway for biosynthesis of aromatics. Twenty-three metabolites can be quantified precisely with the optimized method using standard HPLC equipment and UV detection, with the mobile phase used for chromatography also compatible with mass spectrometry (MS). The limit of quantification/detection is as low as 10-10to 10-13mol, respectively, which makes this method feasible for quantification of intracellular metabolites. This method covers most metabolic routes for aromatics biosynthesis, it is inexpensive, robust, simple, precise and sensitive, and has been demonstrated on cell extracts from S. cerevisiae genetically engineered to overproduce aromatics.
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.
authors: Bin
authors: B
authors: Lai
authors: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, Queensland, Australia.|Metabolomics Australia (Queensland Node), The University of Queensland, Brisbane, Queensland, Australia.
authors: Manuel R
authors: MR
authors: Plan
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.
authors: Nils J H
authors: NJ
authors: Averesch
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.
authors: Shiqin
authors: S
authors: Yu
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.
authors: Frauke
authors: F
authors: Kracke
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.
authors: Nicolas
authors: N
authors: Lekieffre
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.|Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, Queensland, Australia.
authors: Sarah
authors: S
authors: Bydder
authors: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, Queensland, Australia.|Metabolomics Australia (Queensland Node), The University of Queensland, Brisbane, Queensland, Australia.
authors: Mark P
authors: MP
authors: Hodson
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.
authors: Gal
authors: G
authors: Winter
authors: Centre for Microbial Electrochemical Systems (CEMES), The University of Queensland, Brisbane, Queensland, Australia.|Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane, Queensland, Australia.
authors: Jens O
authors: JO
authors: Krömer
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005818:Genetic Engineering; D006841:Hydrocarbons, Aromatic / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D057230:Limit of Detection; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012765:Shikimic Acid / Q000378:metabolism*; D058615:Synthetic Biology / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D006841:Hydrocarbons, Aromatic; D012765:Shikimic Acid
keywords: Aromatics; High performance liquid chromatography; Quantitative metabolomics; Shikimate pathway; Synthetic biology