Integrated Bioprocessing for the pH-Dependent Production of 4- Levulinate in Pseudomonas putida KT2440
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Prather-Integrated Bioprocessing.pdf
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Author(s)
Martin, Collin H.
Wu, Danyi
Prather, Kristala L. Jones
Date Issued
January 2010
Journal
Applied and Environmental Microbiology
Publisher
American Society for Microbiology
Citation
Martin, Collin H., Danyi Wu and Kristala L. Jones Prather."Integrated Bioprocessing for the pH-Dependent Production of 4-Valerolactone from Levulinate in Pseudomonas putida KT2440." Applied and Environmental Microbiology, January 2010,76.2, p. 417-424.
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Author's final manuscript
Abstract
Enzymes are powerful biocatalysts capable of performing specific chemical transformations under mild conditions, yet as catalysts they remain subject to the laws of thermodynamics, namely, that they cannot catalyze chemical reactions beyond equilibrium. Here we report the phenomenon and application of using extracytosolic enzymes and medium conditions, such as pH, to catalyze metabolic pathways beyond their intracellular catalytic limitations. This methodology, termed “integrated bioprocessing” because it integrates intracellular and extracytosolic catalysis, was applied to a lactonization reaction in Pseudomonas putida for the economical and high-titer biosynthesis of 4-valerolactone from the inexpensive and renewable source levulinic acid. Mutant paraoxonase I (PON1) was expressed in P. putida, shown to export from the cytosol in Escherichia coli and P. putida using an N-terminal sequence, and demonstrated to catalyze the extracytosolic and pH-dependent lactonization of 4-hydroxyvalerate to 4-valerolactone. With this production system, the titer of 4-valerolactone was enhanced substantially in acidic medium using extracytosolically expressed lactonase versus an intracellular lactonase: from <0.2 g liter−1 to 2.1 ± 0.4 g liter−1 at the shake flask scale. Based on these results, the production of 4-hydroxyvalerate and 4-valerolactone was examined in a 2-liter bioreactor, and titers of 27.1 g liter−1 and 8.2 g liter−1 for the two respective compounds were achieved. These results illustrate the utility of integrated bioprocessing as a strategy for enabling production from novel metabolic pathways and enhancing product titers.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
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DOI of Published Version
https://doi.org/10.1128/AEM.01769-09
