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

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

doi:10.1002/cbic.202300849

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

Journal Article · Mon Feb 05 00:00:00 EST 2024 · ChemBioChem: a European journal of chemical biology

DOI:https://doi.org/10.1002/cbic.202300849· OSTI ID:2290380

^[1]; ^[2]; Lyons, Scott ^[3]; Sun, Haoran ^[3]; ^[4]; ^[1]

Department of Chemistry The University of Texas at Austin 105 E 24th St Austin TX 78712 USA, DOE Center for Advanced Bioenergy and Bioproducts Innovation University of Illinois at Urbana-Champaign 1206 W Gregory Dr Urbana IL 61801 USA
DOE Center for Advanced Bioenergy and Bioproducts Innovation University of Illinois at Urbana-Champaign 1206 W Gregory Dr Urbana IL 61801 USA, Carl R. Woese Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 W Gregory Dr Urbana IL 61801 USA
Department of Molecular Bioscience The University of Texas at Austin 100 E 24th St Austin TX 78712 USA
DOE Center for Advanced Bioenergy and Bioproducts Innovation University of Illinois at Urbana-Champaign 1206 W Gregory Dr Urbana IL 61801 USA, Carl R. Woese Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 W Gregory Dr Urbana IL 61801 USA, Department of Chemistry University of Illinois at Urbana-Champaign 505 S Mathews Avenue Urbana IL 61801 USA

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 k _cat (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.

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

Sponsoring Organization:: Robert A. Welch Foundation; USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0018420

OSTI ID:: 2290380

Alternate ID(s):: OSTI ID: 2377398
OSTI ID: 2290384

Journal Information:: ChemBioChem: a European journal of chemical biology, Journal Name: ChemBioChem: a European journal of chemical biology Journal Issue: 5 Vol. 25; ISSN 1439-4227

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: France

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
Biocatalysis
Directed evolution
High-throughput method
Polyketides
Synthetic Biology

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

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