Improvement of 4-Hydroxyvalerate Production in Pseudomonas putida Using Adaptive Laboratory Evolution

Abstract

4-Hydroxyvalerate (4HV) is a precursor for synthesis of gammavalerolactone, which can be used as a potential future energy source and variety of industrial raw materials. Recently, levulinic acid (LA) catabolic pathway is discovered and 4HV-CoA was revealed to be one of the intermediates in the pathway, that can readily converted to 4HV by a single reaction. However, except the recent discovery on the LA catabolic pathway, metabolic regulations and transporter genes involved with it in P. putida are not well studied. Therefore, we have performed adaptive laboratory evolution (ALE) to find new genes involved in LA uptake and regulation in P. putida. For the ALE, we cultured wild type P. putida in a minimal media with high concentrations of LA and 4HV. After several months, we selected fast growing mutants which are expected to show increased LA utilization, and analyzed their genome to figure out mutations associated with LA uptake and metabolism. In addition to this, we constructed a biosensor to detect the 4HV concentration and a random transposon mutagenesis to find additional genetic change that is negatively involved in 4HV production.