Enhanced production of microbial levulinic acid through deletion of the levulinic acid transcriptional regulator (lvaR) in engineered Pseudomonas putida KT2440.

Levulinic acid (LA) is a platform compound regarded as a promising organic intermediate for the synthesis of various chemicals such as fuel additives, plasticizers, solvents, and pharmaceuticals. Traditionally, LA is produced via acid-catalyzed dehydration and hydrolysis of lignocellulosic biomass, but this process involves challenges such as high temperatures and pressures, the use of strong acids, byproducts formation, and limitations in recovery and purification. To provide an alternative for chemical synthesis, we previously designed an integrated process to produce LA from glucose using genetically engineered Pseudomonas putida KT2440. However, as the consumption of the produced LA could not be completely prevented, its overall yield was limited. Therefore, in this study we constructed P. putida strains with additional knock-out of the lva operon genes (lvaAB, lvaE, and lvaR) in a pcaIJ knock-out strain, and introduced the aroG, asbF, and adc genes to design an LA production pathway. The pcaIJ, lvaR double knock-out strain P. putida HP205 produced 20.42 mM of LA from glycerol, and culture condition including temperature, glucose concentration, and nitrogen source were optimized. Under optimal conditions, P. putida HP205 produced 73.9 mM (8.58 g/L) LA in fed-batch fermentation. When crude glycerol was used as the substrate, both LA production and cell growth were enhanced. This study presents the impact of the LA transcriptional regulator and demonstrates a strategy for enhanced LA production in P. putida.