Optically guided mass spectrometry to screen microbial colonies for directed enzyme evolution

Pu Xue, Tong Si, Huimin Zhao

Research output: Chapter in Book/Report/Conference proceedingChapter


Directed evolution is a well-established and powerful tool for enzyme engineering, which consists of iterative rounds of creating and screening a library of variants. In many cases, the ability to characterize these variants in high-throughput remains a bottleneck. In addition, profiling of desired candidates becomes even more challenging when engineering multiple enzymes in a biochemical pathway. In this chapter, we describe a label-free, high-throughput method for the engineering of multistep enzymatic reactions in bacterial colonies via optically guided matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). This method is able to detect products, reactants, and byproducts with high sensitivity and accuracy. We demonstrate its effectiveness in two applications related to natural product biosynthesis, including facile creation of analog of the peptidic antibiotic plantazolicin and rapid profiling of congeners of rhamnolipid. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. In both cases, improved MS acquisition efficiency and information-rich insights were obtained through this technique on large populations of colonies at a rate of 1–2.5 s per colony. This method should be generally applicable to high-throughput phenotyping of microbial libraries from a wide range of enzymatic reactions.

Original languageEnglish (US)
Title of host publicationEnzyme Engineering and Evolution
Subtitle of host publicationSpecific Enzyme Applications
EditorsDan S. Tawfik
PublisherAcademic Press Inc.
Number of pages19
ISBN (Print)9780128244319
StatePublished - 2020

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Directed enzyme evolution
  • High-throughput screening
  • Mass spectrometry

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry


Dive into the research topics of 'Optically guided mass spectrometry to screen microbial colonies for directed enzyme evolution'. Together they form a unique fingerprint.

Cite this