Identifying Bottlenecks in High-Titer Microbial Production of 4-Hydroxyvaleric Acid from Levulinic Acid in Escherichia coli

Abstract

The microbial conversion of renewable feedstocks into bio-based bulk chemicals offers a sustainable alternative to traditional petrochemical processes. 4-Hydroxyvaleric acid (4-HV) is a promising platform chemical, serving as a monomer for biodegradable polyesters and a precursor for liquid fuels. In this study, we engineered an Escherichia coli strain to produce high levels of 4-HV from levulinic acid using an antibiotic-free, substrate-inducible expression system. A bicistronic ribosome binding site design was employed to fine-tune enzyme expression, resulting in enhanced productivity during fed-batch cultivation. Despite achieving high 4-HV titers and volumetric productivity, a production plateau was observed, prompting a systematic investigation of potential constraints. Key limiting factors—including enzyme activity, medium composition, ionic strength, and product-associated toxicity—were identified and assessed. Furthermore, we evaluated the reuse of recombinant biomass from earlier fermentations to improve process sustainability and reduce production costs. This work highlights critical challenges in scaling 4-HV biosynthesis and provides valuable insights for optimizing microbial production of valueadded chemicals from biomass-derived substrates.