Integrated Bioprocessing for the pH-Dependent Production of 4-Valerolactone from Levulinate in Pseudomonas putida KT2440

• Published in: Applied and Environmental Microbiology Vol. 76; no. 2; pp. 417 - 424
• Main Authors: Martin, Collin H, Wu, Danyi, Prather, Kristala L. Jones
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.01.2010; American Society for Microbiology (ASM)
• Subjects: Alkaline Phosphatase - analysis; Amino Acid Sequence; Aryldialkylphosphatase; Aryldialkylphosphatase - analysis; Bacteria; Bacteriology; biochemical pathways; Biological and medical sciences; bioprocessing; Bioreactors; Biosynthesis; Biotechnology; Catalysis; catalysts; catalytic activity; Chemical reactions; cytosol; E coli; Enzymes; Escherichia coli; Escherichia coli Proteins - analysis; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Lactones - metabolism; levulinic acid; Levulinic Acids - metabolism; Microbiology; Molecular Sequence Data; production technology; Pseudomonas putida; Pseudomonas putida - metabolism; Reaction kinetics; thermodynamics; Valerates - metabolism; Pseudomonadales; pH; Bacteria; Pseudomonadaceae; Pseudomonas putida; Production
• ISSN: 0099-2240; 1098-5336; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.01769-09

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.