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Title: 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:
ORCiD logo [1];  [2];  [1];  [1];  [1];  [1]; ORCiD logo [1]
  1. Univ. of Illinois at Urbana-Champaign, IL (United States)
  2. 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. doi: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. doi: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 = {2020},
month = {3}
}

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