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

Dataset · Wed Dec 20 00:00:00 EST 2023

DOI:https://doi.org/10.13012/B2IDB-1645677_V1· OSTI ID:3014704

Lu, Yi ^[1]; Sweedler, Jonathan ^[2]; Zhou, Shuaizhen ^[3]; Zhou, Yu ^[1]; Lyons, Scott ^[4]; Sun, Haoran ^[4]

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1. Department of Chemistry, The University of Texas at Austin, 105 E 24th St, Austin, TX 78712 (USA); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
2. 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); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
3. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W Gregory Dr, Urbana, IL 61801 (USA); Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
4. Department of Molecular Bioscience, The University of Texas at Austin, 100 E 24th St, Austin, TX 78712 (USA)

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.

• Research Organization: Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States); University of Illinois Urbana-Champaign
• Sponsoring Organization: U.S. Department of Energy (DOE)
• DOE Contract Number: SC0018420
• OSTI ID: 3014704
• Country of Publication: United States
• Language: English

Related Subjects

catalysis
mass spectrometry
metabolic engineering