A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids

Xue, Pu; Si, Tong; Mishra, Shekhar; Zhang, Linzixuan; Choe, Kisurb; Sweedler, Jonathan V.; Zhao, Huimin

doi:10.1002/bit.27343

Title: A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids

Journal Article · Thu Mar 26 00:00:00 EDT 2020 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.27343· OSTI ID:1608255

^[1]; Si, Tong ^[2]; Mishra, Shekhar ^[1]; Zhang, Linzixuan ^[1]; Choe, Kisurb ^[1]; Sweedler, Jonathan V. ^[1];

^[1]

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)

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.

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Research Organization:: Univ. of Illinois at Urbana-Champaign, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Contributing Organization:: Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)

Grant/Contract Number:: SC0018420

OSTI ID:: 1608255

Alternate ID(s):: OSTI ID: 1608744

Journal Information:: Biotechnology and Bioengineering, Vol. 117, Issue 7; ISSN 0006-3592

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 20 works

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59 BASIC BIOLOGICAL SCIENCES
Free fatty acids
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High-throughput screening
Directed evolution
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