Quantitative analysis of aromatics for synthetic biology using liquid chromatography

Lai, Bin, Plan, Manuel R. , Averesch, Nils J. H. , Yu, Shiqin, Kracke, Frauke, Lekieffre, Nicolas, Bydder, Sarah, Hodson, Mark P. , Winter, Gal and Kroemer, Jens O. (2017) Quantitative analysis of aromatics for synthetic biology using liquid chromatography. Biotechnology Journal, 12 1: . doi:10.1002/biot.201600269


Author Lai, Bin
Plan, Manuel R.
Averesch, Nils J. H.
Yu, Shiqin
Kracke, Frauke
Lekieffre, Nicolas
Bydder, Sarah
Hodson, Mark P.
Winter, Gal
Kroemer, Jens O.
Title Quantitative analysis of aromatics for synthetic biology using liquid chromatography
Journal name Biotechnology Journal   Check publisher's open access policy
ISSN 1860-7314
1860-6768
Publication date 2017-01-01
Year available 2016
Sub-type Article (original research)
DOI 10.1002/biot.201600269
Open Access Status Not yet assessed
Volume 12
Issue 1
Total pages 9
Place of publication Weinheim, Germany
Publisher Wiley - V C H Verlag GmbH & Co. KGaA
Language eng
Subject 2402 Applied Microbiology and Biotechnology
1313 Molecular Medicine
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 to 10 mol, 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.
Formatted 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 10−10 to 10−13 mol, 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. acccharomyces cerevisiae genetically engineered to overproduce aromatics.
Keyword Aromatics
High performance liquid chromatography
Quantitative metabolomics
Shikimate pathway
Synthetic biology
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID DE120101549
Institutional Status UQ

 
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