Substrate-inducible and antibiotic-free high-level 4-hydroxyvaleric acid production in engineered .

In this study, we developed a levulinic acid (LA)-inducible and antibiotic-free plasmid system mediated by HpdR/P and -complementation to produce 4-hydroxyvaleric acid (4-HV) from LA in an engineered strain. The system was efficiently induced by the addition of the LA substrate and resulted in tight dose-dependent control and fine-tuning of gene expression. By engineering the 5' untranslated region (UTR) of mRNA, the gene expression of green fluorescent protein (GFP) increased by at least two-fold under the promoter. Furthermore, by evaluating the robustness and plasmid stability of the proposed system, the engineered strain, IRV, expressing the engineered 3-hydroxybutyrate dehydrogenase (3HBDH) and formate dehydrogenase (FDH), produced 82 g/L of 4-HV from LA, with a productivity of 3.4 g/L/h and molar conversion of 92% in the fed-batch cultivation (5 L fermenter) without the addition of antibiotics or external inducers. Overall, the reported system was highly beneficial for the large-scale and cost-effective microbial production of value-added products and bulk chemicals from the renewable substrate, LA.