Modification of substrate specificity of thioesterase, reductase, desaturase by MALDI-tof-MS analysis of microbial colonies

Choe, Kisurb

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https://hdl.handle.net/2142/117595 Copy

Description

Title

Modification of substrate specificity of thioesterase, reductase, desaturase by MALDI-tof-MS analysis of microbial colonies

Author(s)

Choe, Kisurb

Issue Date

2022-12-02

Director of Research (if dissertation) or Advisor (if thesis)

Sweedler, Jonathan

Doctoral Committee Chair(s)

Sweedler, Jonathan

Committee Member(s)

• Nair, Satish
• Procko , Erik
• Zhao, Huimin

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Enzymes
• mass spectrometry
• MALDI-MS

Abstract

Chemical heterogeneity in biological systems reveal important clues to the inner workings of the fundamental processes such as lipid metabolism and physiology. High throughput screening of microbes and mutant libraries resolve such complexities enabling multiple biochemical studies such as directed evolution, functional genomics, microbial identification, and genome mining. A promising screening approach is measurement of biochemical products directly from microbial colonies via optically guided matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Measuring the compounds from microbial colonies bypasses liquid culture while minimizing the automation processes, and the wide chemical coverage and the high sensitivity of MALDI-MS can enable diverse biochemical screening projects such as modifying enzymes and functional genomics survey of gene activation/inhibition libraries. The thesis first presents the informatics and the experimental methods that were created to develop the MALDI-MS based colony screen. The macroMS software was developed to perform informatics tasks such as image analysis finding samples, MALDI-MS automation targeting the samples, and data analysis / visualization to find an analyte of interest. Then an experimental method for colony sample preparation, constructing sprayer for applying MALDI matrix, and running MALDI-MS is described. Based on the colony screening method, random libraries of an acyl-ACP thioesterase, an acyl-ACP desaturase, an acyl-CoA reductase were scanned, revealing novel variants with increased specificity for specific products.

Graduation Semester

2022-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Kisurb Choe

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