Biosynthesis of 4-hydroxyvalerate from glycerol in engineered Pseudomonas putida KT2440 and its application for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxy valerate) (P(3HB-co-3HV-co-4HV)) terpolymer

The 4-hydroxyvalerate (4HV) is a key intermediate that serves as a precursor for γ-valerolactone (GVL) and terpolymers, contributing to the synthesis of liquid fuels and biodegradable plastics with improved properties. While 4HV is typically derived from levulinic acid (LA) via chemical catalysis, these methods are limited by high costs and operational complexity. In our previous study, we designed an engineered Pseudomonas putida KT2440 with double knockout of pcaIJ and lvaR and introduction of aroG , asbF , and adc , enabling high production of LA from glycerol. Herein, we introduced acyl-CoA synthetase gene ( lvaE ) and 4-hydroxyvaleryl-CoA dehydratase gene ( lvaD ) into LA producing strain to construct a 4HV production pathway from glycerol. The strain harboring both lvaD and lvaE consumed the highest amount of LA, and, subsequently, showed the highest 4HV production. In fed batch fermentation , P. putida KT2440 HP403 produced 28.77 mM of 4HV from glycerol. To expand its potential applications as a PHA precursor, biologically synthesized 4HV was supplied to the high-performing PHA producer Cupriavidus necator PHB -4 / phaC BP-M-CPF4 for polymer synthesis. This produced a terpolymer with the composition P(54.49 mol% 3HB- co -6.98 mol% 3HV- co -38.53 mol% 4HV) with a PHA content of 60.21%. The terpolymer exhibited improved elongation at break and lower glass transition and crystallization temperatures due to increased amorphicity. This study suggests a 4HV bioconversion strategy derived from glycerol by engineered P. putida KT2440 HP403 and demonstrates the production of a high-content P(3HB- co -3HV- co -4HV) terpolymer in C. necator containing high-mole fraction of 4HV, thereby providing a new sustainable approach for PHA production.