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

Abstract

4-Hydroxyvalerate (4HV) is a precursor of gamma-valerolactone that can be used as a potential future energy source and a variety of industrial raw materials. Recent studies have shown that 4HV is produced as an intermediate in the metabolism of levulinic acid (LA) in Pseudomonas putida. However, its metabolic pathway and transporter genes of P. putida are not well studied. Therefore, we have performed adaptive laboratory evolution (ALE) to find new genes involved in LA metabolism in P. putida. For this purpose, we are adapting wild type P. putida for a long time on minimal media with various concentrations of LA to get fast growing mutants and 4HV. We will analyze their genome to figure out mutations associated with increasing cell growth resulted from increased LA metabolism. Finally, we will engineer P. putida that produce high amount of 4HV by reverse engineering. Together with this, a random transposon (Tn library) mutagenesis is used to find addition genes negatively involved in 4HV production. To do that, we already constructed a biosensor that detects elevated levels of 4HV.