Enhancing translation efficiency and exploring constraints in high-level 4-hydroxyvaleric acid production from levulinic acid in Escherichia coli

4-Hydroxyvaleric acid (4-HV) holds promise as a sustainable monomer for biodegradable polyesters and liquid transportation fuels. This study achieved high-level 4-HV production from levulinic acid using an antibiotic-free, substrate-inducible system in Escherichia coli. Enzymes involved in the conversion of levulinic acid to 4-HV were expressed with a bicistronic design of ribosome binding sites. The engineered strain demonstrated a 28% higher productivity compared to its counterpart, reaching a significant concentration of 107 g/L 4-HV with a production rate of 4.5 g/L/h and a molar conversion of 95% from levulinic acid in fed-batch cultivation. Recombinant cells from the initial cultivation were reused for a second round of biotransformation, demonstrating 73% efficiency of fresh cells. The study identified specific factors contributing to decreased system efficiency, including medium conditions, increased ionic strength, and high product concentration. Overall, the reported system and our findings hold significant potential for cost-effective microbial production of 4-HV at scale from levulinic acid.