title: Targeted cofactor quantification in metabolically engineered E. coli using solid phase extraction and hydrophilic interaction liquid chromatography-mass spectrometry.
abstract: Quantification of energy and redox cofactors is of great value to synthetic biologists to infer the balance of energy metabolism in engineered microbial strains and assess each strain's potential for further improvement. Most currently used methods for intracellular cofactor measurement suffer from incomplete coverage, low reproducibility, suboptimal sensitivity or specificity. In this study, we described an SPE-HILIC/MS approach for simultaneous determination of six cofactor targets (ATP, ADP, NAD, NADH, NADP, NADPH) in Escherichia coli cells. Sufficient linearity, precision and metabolite recoveries of this new approach justified its reliability in targeted cofactor quantification. Our approach was then compared with conventional enzymatic assays to demonstrate its superior performance. We applied the SPE-HILIC/MS approach to profile shift of cofactor balances in several engineered E. coli strains with varying isobutanol production. Our cofactor analysis clearly revealed that optimal energy fitness was achieved in the highest-yield strain through combined modulation of a transhydrogenase and a NAD(+) kinase. Apart from the targeted cofactors, the SPE enrichment procedure also allowed for confident identification of 39 groups of polar metabolites mainly involved in central carbon metabolism in E. coli cells.
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Zhucui
authors.initials: Z
authors.lastname: Li
authors.affiliation: College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China.
authors.forename: Afang
authors.initials: A
authors.lastname: Yang
authors.affiliation: College of Life Sciences, Nankai University, Tianjin 300071, China.
authors.forename: Yujing
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Liu
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Zhidan
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: zhang_xl@tib.cas.cn.
authors.forename: Xueli
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: shui_wq@tib.cas.cn.
authors.forename: Wenqing
authors.initials: W
authors.lastname: Shui
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods; D060847:Metabolic Engineering; D009243:NAD / Q000032:analysis* / Q000378:metabolism; D009249:NADP / Q000032:analysis* / Q000378:metabolism; D052616:Solid Phase Extraction / Q000379:methods
chemical_list: D000440:Butanols; D009243:NAD; D009249:NADP; C040507:isobutyl alcohol; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate
keywords: Cofactor; Energy metabolism; HILIC–MS; Isobutanol production; SPE; Targeted metabolome