A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids
Abstract
Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs), key components of crucial nutrients, soaps, industrial chemicals, and fuels. Yet, our ability to control the specificity of microbially synthesized FFAs is still limited for medium-chain fatty acids (MCFAs), which is mainly due to lack of high-throughput approaches for FFA analysis. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. Specially, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and the m/z peak ratio at 730 and 758 was used as an index for improved MCFA production. This yeast colony-based method can be performed at a rate of ~2 second per sample, which is much faster than the gold standard gas chromatography (GC)-MS (typically > 30 min per sample). To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified 9 mutants that resulted in improved MCFA production relative to the wild-type strain. This colony-based MALDI-ToF MS screening method offers an alternative way for engineering microbial fatty acid compositions in a high-throughput manner.
- Authors:
-
- Univ. of Illinois at Urbana-Champaign, IL (United States)
- Univ. of Illinois at Urbana-Champaign, IL (United States); Chinese Academy of Sciences (CAS), Shenzhen (China)
- Publication Date:
- Research Org.:
- Univ. of Illinois at Urbana-Champaign, IL (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Contributing Org.:
- Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
- OSTI Identifier:
- 1608255
- Alternate Identifier(s):
- OSTI ID: 1608744
- Grant/Contract Number:
- SC0018420
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Biotechnology and Bioengineering
- Additional Journal Information:
- Journal Volume: 117; Journal Issue: 7; Journal ID: ISSN 0006-3592
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Free fatty acids; Mass spectrometry; High-throughput screening; Directed evolution; Metabolic engineering
Citation Formats
Xue, Pu, Si, Tong, Mishra, Shekhar, Zhang, Linzixuan, Choe, Kisurb, Sweedler, Jonathan V., and Zhao, Huimin. A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids. United States: N. p., 2020.
Web. doi:10.1002/bit.27343.
Xue, Pu, Si, Tong, Mishra, Shekhar, Zhang, Linzixuan, Choe, Kisurb, Sweedler, Jonathan V., & Zhao, Huimin. A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids. United States. https://doi.org/10.1002/bit.27343
Xue, Pu, Si, Tong, Mishra, Shekhar, Zhang, Linzixuan, Choe, Kisurb, Sweedler, Jonathan V., and Zhao, Huimin. Thu .
"A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids". United States. https://doi.org/10.1002/bit.27343. https://www.osti.gov/servlets/purl/1608255.
@article{osti_1608255,
title = {A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids},
author = {Xue, Pu and Si, Tong and Mishra, Shekhar and Zhang, Linzixuan and Choe, Kisurb and Sweedler, Jonathan V. and Zhao, Huimin},
abstractNote = {Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs), key components of crucial nutrients, soaps, industrial chemicals, and fuels. Yet, our ability to control the specificity of microbially synthesized FFAs is still limited for medium-chain fatty acids (MCFAs), which is mainly due to lack of high-throughput approaches for FFA analysis. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. Specially, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and the m/z peak ratio at 730 and 758 was used as an index for improved MCFA production. This yeast colony-based method can be performed at a rate of ~2 second per sample, which is much faster than the gold standard gas chromatography (GC)-MS (typically > 30 min per sample). To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified 9 mutants that resulted in improved MCFA production relative to the wild-type strain. This colony-based MALDI-ToF MS screening method offers an alternative way for engineering microbial fatty acid compositions in a high-throughput manner.},
doi = {10.1002/bit.27343},
journal = {Biotechnology and Bioengineering},
number = 7,
volume = 117,
place = {United States},
year = {Thu Mar 26 00:00:00 EDT 2020},
month = {Thu Mar 26 00:00:00 EDT 2020}
}
Web of Science
Works referenced in this record:
Exometabolomics and MSI: deconstructing how cells interact to transform their small molecule environment
journal, August 2015
- Silva, Leslie P.; Northen, Trent R.
- Current Opinion in Biotechnology, Vol. 34
Engineering Saccharomyces cerevisiae cells for production of fatty acid-derived biofuels and chemicals
journal, May 2019
- Hu, Yating; Zhu, Zhiwei; Nielsen, Jens
- Open Biology, Vol. 9, Issue 5
The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update
journal, May 2018
- Afgan, Enis; Baker, Dannon; Batut, Bérénice
- Nucleic Acids Research, Vol. 46, Issue W1
Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering
journal, September 2018
- Rigouin, Coraline; Croux, Christian; Borsenberger, Vinciane
- Microbial Cell Factories, Vol. 17, Issue 1
Engineering fungal de novo fatty acid synthesis for short chain fatty acid production
journal, March 2017
- Gajewski, Jan; Pavlovic, Renata; Fischer, Manuel
- Nature Communications, Vol. 8, Issue 1
DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways
journal, December 2008
- Shao, Zengyi; Zhao, Hua; Zhao, Huimin
- Nucleic Acids Research, Vol. 37, Issue 2
Directed evolution as a powerful synthetic biology tool
journal, March 2013
- Cobb, Ryan E.; Sun, Ning; Zhao, Huimin
- Methods, Vol. 60, Issue 1
A novel function of yeast fatty acid synthase: Subunit α is capable of self-pantetheinylation
journal, May 2000
- Fichtlscherer, Fabienne; Wellein, Christian; Mittag, Maria
- European Journal of Biochemistry, Vol. 267, Issue 9
Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase
journal, June 2017
- Rigouin, Coraline; Gueroult, Marc; Croux, Christian
- ACS Synthetic Biology, Vol. 6, Issue 10
Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
journal, April 2003
- Al-Saad, Khalid A.; Siems, William F.; Hill, H. H.
- Journal of the American Society for Mass Spectrometry, Vol. 14, Issue 4
High throughput screening of enzyme activity with mass spectrometry imaging
journal, February 2015
- de Rond, Tristan; Danielewicz, Megan; Northen, Trent
- Current Opinion in Biotechnology, Vol. 31
Fatty Acid Biosynthesis: Chain‐Length Regulation and Control
journal, June 2019
- Heil, Christina S.; Wehrheim, S. Sophia; Paithankar, Karthik S.
- ChemBioChem, Vol. 20, Issue 18
pymzML v2.0: introducing a highly compressed and seekable gzip format
journal, January 2018
- Kösters, M.; Leufken, J.; Schulze, S.
- Bioinformatics, Vol. 34, Issue 14
Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase
journal, December 2006
- Christensen, C. E.; Kragelund, B. B.; von Wettstein-Knowles, P.
- Protein Science, Vol. 16, Issue 2
A sample preparation method for recovering suppressed analyte ions in MALDI TOF MS: A method for recovering analyte ions in MALDI MS
journal, April 2015
- Lou, Xianwen; de Waal, Bas F. M.; Milroy, Lech-Gustav
- Journal of Mass Spectrometry, Vol. 50, Issue 5
A sensitive GC/MS detection method for analyzing microbial metabolites short chain fatty acids in fecal and serum samples
journal, May 2019
- Zhang, Shuming; Wang, Hongbin; Zhu, Mei-Jun
- Talanta, Vol. 196
Engineering fatty acid synthases for directed polyketide production
journal, February 2017
- Gajewski, Jan; Buelens, Floris; Serdjukow, Sascha
- Nature Chemical Biology, Vol. 13, Issue 4
Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis
journal, June 2012
- Kille, Sabrina; Acevedo-Rocha, Carlos G.; Parra, Loreto P.
- ACS Synthetic Biology, Vol. 2, Issue 2
Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
journal, August 2017
- Si, Tong; Li, Bin; Comi, Troy J.
- Journal of the American Chemical Society, Vol. 139, Issue 36
A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry
journal, January 2020
- Topolska, Magdalena; Martínez-Montañés, Fernando; Ejsing, Christer S.
- Biomolecules, Vol. 10, Issue 1
Self-cloning Yeast Strains Containing Novel FAS2 Mutations Produce a Higher Amount of Ethyl Caproate in Japanese Sake
journal, January 2004
- Aritomi, Kazuo; Hirosawa, Isao; Hoshida, Hisashi
- Bioscience, Biotechnology, and Biochemistry, Vol. 68, Issue 1
Engineering microbial fatty acid metabolism for biofuels and biochemicals
journal, April 2018
- Marella, Eko Roy; Holkenbrink, Carina; Siewers, Verena
- Current Opinion in Biotechnology, Vol. 50
Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals
journal, January 2014
- Runguphan, Weerawat; Keasling, Jay D.
- Metabolic Engineering, Vol. 21
Measuring the peptides in individual organelles with mass spectrometry
journal, February 2000
- Rubakhin, Stanislav S.; Garden, Rebecca W.; Fuller, Robert R.
- Nature Biotechnology, Vol. 18, Issue 2
Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009
- Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
- Nature Methods, Vol. 6, Issue 5, p. 343-345
Advances in analytical tools for high throughput strain engineering
journal, December 2018
- Marcellin, Esteban; Nielsen, Lars Keld
- Current Opinion in Biotechnology, Vol. 54
Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids
journal, May 2017
- Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.
- ACS Catalysis, Vol. 7, Issue 6
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns
journal, January 2002
- Al-Saad, Khalid A.; Zabrouskov, Vladimir; Siems, William F.
- Rapid Communications in Mass Spectrometry, Vol. 17, Issue 1
Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry
journal, March 2013
- Nemes, Peter; Rubakhin, Stanislav S.; Aerts, Jordan T.
- Nature Protocols, Vol. 8, Issue 4
Inhibition of the fungal fatty acid synthase type I multienzyme complex
journal, August 2008
- Johansson, P.; Wiltschi, B.; Kumari, P.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 35