Engineering the lva operon and Optimization of Culture Conditions for Enhanced Production of 4-Hydroxyvalerate from Levulinic Acid in Pseudomonas putida KT2440

Abstract

Monomeric 4-hydroxyvalerate is a versatile chemical used to produce various commodities and fine chemicals. In the present study, the lvaAB gene was deleted from the lva operon in Pseudomonas putida KT2440 and tesB, obtained from Escherichia coli, was overexpressed under the control of the lva operon system, which is induced by the substrate levulinic acid and the product 4-hydroxyvalerate to produce 4-hydroxyvalerate from levulinic acid. The lvaAB-deleted strain showed almost complete conversion of levulinic acid to 4-hydroxyvalerate, compared with 24% conversion in the wild-type strain. In addition, under optimized culture conditions, the final engineered strain produced a maximum of 50 g/L 4-hydroxyvalerate with 97% conversion from levulinic acid. The system presented here could be applied to produce high titers of 4-hydroxyvalerate in a cost-effective manner at a large scale from renewable cellulosic biomass.

Inducible and tunable gene expression systems for Pseudomonas putida KT2440

“…Because it is inexpensive, LA can be used as a starting material to produce a wide range of chemicals, including polymers, plasticizers, fuels, resins, pharmaceuticals, anti-freeze agents, and solvents 27 – 30 . The lvaAB- deleted P. putida KT2440 strain produced a maximum of 50 g/L of 4HV with 97% conversion from LA using the LA-inducible LvaR/P lvaA system without the addition of any chemical inducers 21 . LA can be used as a carbon source for strain KT2440 21 , 25 .…”

“…The lvaAB- deleted P. putida KT2440 strain produced a maximum of 50 g/L of 4HV with 97% conversion from LA using the LA-inducible LvaR/P lvaA system without the addition of any chemical inducers 21 . LA can be used as a carbon source for strain KT2440 21 , 25 . As the lva operon-disturbed strain did not grow on LA, the added LA could not be intrinsically catabolized by the other endogenous system 21 , 25 .…”

“…LA can be used as a carbon source for strain KT2440 21 , 25 . As the lva operon-disturbed strain did not grow on LA, the added LA could not be intrinsically catabolized by the other endogenous system 21 , 25 . It is noteworthy that in strain KT2440, LA is catabolized to the central metabolites, acetyl-CoA and propionyl-CoA.…”

“…These low-cost substrates are already used as potent starting materials for both cell growth and the production of a wide range of biochemicals. Although the microbial production of industrially relevant biochemicals using Glu- and LA-inducible expression systems has been previously reported 20 , 21 , the promoters have not been characterized in detail.…”

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

“…As LA contains both ketone and carboxylic acid functional groups, a wide range of valuable chemicals and fuels can be produced from it ( Pileidis and Titirici, 2016 ). Moreover, low-cost LA is considered to be a promising organic intermediate for the synthesis of several chemicals such as polymers, plasticizers, fuels, resins, fragrances, pharmaceuticals, anti-freeze agents, and solvents ( Bozell et al, 2000 ; Rackemann and Doherty, 2011 ; Pileidis and Titirici, 2016 ; Sathesh-Prabu and Lee, 2019 ; Cha et al, 2020 ; Hayes and Becer, 2020 ).…”