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

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 ^[3]; Zhang, Linzixuan ^[3]; Choe, Kisurb ^[3]; Sweedler, Jonathan V. ^[3]; ^[3]

Univ. of Illinois at Urbana-Champaign, IL (United States); CABBI
Univ. of Illinois at Urbana-Champaign, IL (United States); Chinese Academy of Sciences (CAS), Shenzhen (China)
Univ. of Illinois at Urbana-Champaign, IL (United States)

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.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

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

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

References (30)

Fatty Acid Biosynthesis: Chain‐Length Regulation and Control Heil, Christina S.; Wehrheim, S. Sophia; Paithankar, Karthik S. ChemBioChem, Vol. 20, Issue 18 https://doi.org/10.1002/cbic.201800809	journal	June 2019
A sample preparation method for recovering suppressed analyte ions in MALDI TOF MS: A method for recovering analyte ions in MALDI MS Lou, Xianwen; de Waal, Bas F. M.; Milroy, Lech-Gustav Journal of Mass Spectrometry, Vol. 50, Issue 5 https://doi.org/10.1002/jms.3587	journal	April 2015
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns Al-Saad, Khalid A.; Zabrouskov, Vladimir; Siems, William F. Rapid Communications in Mass Spectrometry, Vol. 17, Issue 1 https://doi.org/10.1002/rcm.858	journal	January 2002
Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Al-Saad, Khalid A.; Siems, William F.; Hill, H. H. Journal of the American Society for Mass Spectrometry, Vol. 14, Issue 4 https://doi.org/10.1016/S1044-0305(03)00068-0	journal	April 2003
High throughput screening of enzyme activity with mass spectrometry imaging de Rond, Tristan; Danielewicz, Megan; Northen, Trent Current Opinion in Biotechnology, Vol. 31 https://doi.org/10.1016/j.copbio.2014.07.008	journal	February 2015
Exometabolomics and MSI: deconstructing how cells interact to transform their small molecule environment Silva, Leslie P.; Northen, Trent R. Current Opinion in Biotechnology, Vol. 34 https://doi.org/10.1016/j.copbio.2015.03.015	journal	August 2015
Engineering microbial fatty acid metabolism for biofuels and biochemicals Marella, Eko Roy; Holkenbrink, Carina; Siewers, Verena Current Opinion in Biotechnology, Vol. 50 https://doi.org/10.1016/j.copbio.2017.10.002	journal	April 2018
Advances in analytical tools for high throughput strain engineering Marcellin, Esteban; Nielsen, Lars Keld Current Opinion in Biotechnology, Vol. 54 https://doi.org/10.1016/j.copbio.2018.01.027	journal	December 2018
A sensitive GC/MS detection method for analyzing microbial metabolites short chain fatty acids in fecal and serum samples Zhang, Shuming; Wang, Hongbin; Zhu, Mei-Jun Talanta, Vol. 196 https://doi.org/10.1016/j.talanta.2018.12.049	journal	May 2019
Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals Runguphan, Weerawat; Keasling, Jay D. Metabolic Engineering, Vol. 21 https://doi.org/10.1016/j.ymben.2013.07.003	journal	January 2014
Directed evolution as a powerful synthetic biology tool Cobb, Ryan E.; Sun, Ning; Zhao, Huimin Methods, Vol. 60, Issue 1 https://doi.org/10.1016/j.ymeth.2012.03.009	journal	March 2013
Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B. ACS Catalysis, Vol. 7, Issue 6 https://doi.org/10.1021/acscatal.7b00408	journal	May 2017
Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase Rigouin, Coraline; Gueroult, Marc; Croux, Christian ACS Synthetic Biology, Vol. 6, Issue 10 https://doi.org/10.1021/acssynbio.7b00034	journal	June 2017
Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Si, Tong; Li, Bin; Comi, Troy J. Journal of the American Chemical Society, Vol. 139, Issue 36 https://doi.org/10.1021/jacs.7b04641	journal	August 2017
Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis Kille, Sabrina; Acevedo-Rocha, Carlos G.; Parra, Loreto P. ACS Synthetic Biology, Vol. 2, Issue 2 https://doi.org/10.1021/sb300037w	journal	June 2012
Measuring the peptides in individual organelles with mass spectrometry Rubakhin, Stanislav S.; Garden, Rebecca W.; Fuller, Robert R. Nature Biotechnology, Vol. 18, Issue 2 https://doi.org/10.1038/72622	journal	February 2000
Engineering fatty acid synthases for directed polyketide production Gajewski, Jan; Buelens, Floris; Serdjukow, Sascha Nature Chemical Biology, Vol. 13, Issue 4 https://doi.org/10.1038/nchembio.2314	journal	February 2017
Engineering fungal de novo fatty acid synthesis for short chain fatty acid production Gajewski, Jan; Pavlovic, Renata; Fischer, Manuel Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms14650	journal	March 2017
Enzymatic assembly of DNA molecules up to several hundred kilobases Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan Nature Methods, Vol. 6, Issue 5, p. 343-345 https://doi.org/10.1038/nmeth.1318	journal	April 2009
Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry Nemes, Peter; Rubakhin, Stanislav S.; Aerts, Jordan T. Nature Protocols, Vol. 8, Issue 4 https://doi.org/10.1038/nprot.2013.035	journal	March 2013
A novel function of yeast fatty acid synthase: Subunit α is capable of self-pantetheinylation Fichtlscherer, Fabienne; Wellein, Christian; Mittag, Maria European Journal of Biochemistry, Vol. 267, Issue 9 https://doi.org/10.1046/j.1432-1327.2000.01282.x	journal	May 2000
Inhibition of the fungal fatty acid synthase type I multienzyme complex Johansson, P.; Wiltschi, B.; Kumari, P. Proceedings of the National Academy of Sciences, Vol. 105, Issue 35 https://doi.org/10.1073/pnas.0805827105	journal	August 2008
pymzML v2.0: introducing a highly compressed and seekable gzip format Kösters, M.; Leufken, J.; Schulze, S. Bioinformatics, Vol. 34, Issue 14 https://doi.org/10.1093/bioinformatics/bty046	journal	January 2018
DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways Shao, Zengyi; Zhao, Hua; Zhao, Huimin Nucleic Acids Research, Vol. 37, Issue 2 https://doi.org/10.1093/nar/gkn991	journal	December 2008
The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update Afgan, Enis; Baker, Dannon; Batut, Bérénice Nucleic Acids Research, Vol. 46, Issue W1 https://doi.org/10.1093/nar/gky379	journal	May 2018
Engineering Saccharomyces cerevisiae cells for production of fatty acid-derived biofuels and chemicals Hu, Yating; Zhu, Zhiwei; Nielsen, Jens Open Biology, Vol. 9, Issue 5 https://doi.org/10.1098/rsob.190049	journal	May 2019
Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase Christensen, C. E.; Kragelund, B. B.; von Wettstein-Knowles, P. Protein Science, Vol. 16, Issue 2 https://doi.org/10.1110/ps.062473707	journal	December 2006
Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering Rigouin, Coraline; Croux, Christian; Borsenberger, Vinciane Microbial Cell Factories, Vol. 17, Issue 1 https://doi.org/10.1186/s12934-018-0989-5	journal	September 2018
Self-cloning Yeast Strains Containing Novel FAS2 Mutations Produce a Higher Amount of Ethyl Caproate in Japanese Sake Aritomi, Kazuo; Hirosawa, Isao; Hoshida, Hisashi Bioscience, Biotechnology, and Biochemistry, Vol. 68, Issue 1 https://doi.org/10.1271/bbb.68.206	journal	January 2004
A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry Topolska, Magdalena; Martínez-Montañés, Fernando; Ejsing, Christer S. Biomolecules, Vol. 10, Issue 1 https://doi.org/10.3390/biom10010118	journal	January 2020

Similar Records

Data from A Mass Spectrometry-Based High-Throughput Screening Method for Engineering Fatty Acid Synthases with Improved Production of Medium Chain Fatty Acids

Dataset · Wed Mar 25 20:00:00 EDT 2020 · OSTI ID:3013824

Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids

Journal Article · Tue Feb 28 19:00:00 EST 2023 · Metabolic Engineering · OSTI ID:1993841

Data for Evaluation of Strategies to Narrow the Product Chain-Length Distribution of Microbially Synthesized Free Fatty Acids

Dataset · Tue Feb 28 19:00:00 EST 2023 · OSTI ID:3014475

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Directed evolution
Free fatty acids
High-throughput screening
Mass spectrometry
Metabolic engineering

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

Citation Formats

References (30)

Similar Records

Related Subjects