Enhancing 2-Pyrone Synthase Efficiency by High-Throughput Mass-Spectrometric Quantification and In Vitro/In Vivo Catalytic Performance Correlation

Yu Zhou, Shuaizhen Zhou, Scott Lyons, Haoran Sun, Jonathan V. Sweedler, Yi Lu

Research output: Contribution to journalArticlepeer-review

Abstract

Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.
Original languageEnglish (US)
Article numbere202300849
JournalChemBioChem
Volume25
Issue number5
Early online dateDec 20 2023
DOIs
StatePublished - Mar 1 2024

Keywords

  • biocatalysis
  • Directed evolution
  • High-throughput method
  • Polyketides
  • Synthetic Biology
  • Biocatalysis

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Biochemistry
  • Organic Chemistry

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