(346a) Integrated Bioprocessing for the Production of Hydroxyvalerates and 4-Valerolactone in Pseudomonas Putida

Enzymes are powerful biocatalysts capable of carrying out 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 combines 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 carbon source levulinic acid, which is produced from the treatment of cellulosic materials with dilute acid. 4-valerolactone sees extensive use as a flavor and fragrance molecule and has been reported to be suitable for use as a fuel additive and dispersant.

In this work, enzymes indigenous to P. putida were used to reduce levulinic acid to 3- and 4-hydroxyvalerate, and multi-gram-per-liter titers of both hydroxyvalerates were achieved at the shake flask scale. Mutant paraoxonase I was expressed in P. putida and shown to catalyze the extracytosolic and pH-dependent lactonization of cytosolically produced 4-hydroxyvalerate to 4-valerolactone. With this production system, 4-valerolactone could be synthesized biochemically at the multi-gram-per-liter scale in shake flasks without the need for harsh conditions or solvents, whereas with an intracellular lactonase only milligram-per-liter quantities of lactone could be synthesized. These results illustrate the utility of integrated bioprocessing as a strategy for enabling recombinant pathways and enhancing product titers, and to our knowledge this is the first report on the biosynthesis of hydroxyvalerates and 4-valerolactone from a renewable carbon source.