AUTHOR=Sathesh-Prabu Chandran , Tiwari Rameshwar , Lee Sung Kuk 

TITLE=Substrate-inducible and antibiotic-free high-level 4-hydroxyvaleric acid production in engineered Escherichia coli

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.960907

DOI=10.3389/fbioe.2022.960907

ISSN=2296-4185

ABSTRACT=<p>In this study, we developed a levulinic acid (LA)-inducible and antibiotic-free plasmid system mediated by HpdR/P<sub><italic>hpdH</italic></sub> and <italic>infA</italic>-complementation to produce 4-hydroxyvaleric acid (4-HV) from LA in an engineered <italic>Escherichia coli</italic> 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 <italic>hpdR</italic> mRNA, the gene expression of green fluorescent protein (GFP) increased by at least two-fold under the <italic>hpdH</italic> promoter. Furthermore, by evaluating the robustness and plasmid stability of the proposed system, the engineered strain, IRV<sup>750f</sup>, expressing the engineered 3-hydroxybutyrate dehydrogenase (3HBDH<sup>∗</sup>) and formate dehydrogenase (<italic>Cb</italic>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.</p>