Dynamic upregulation of the rate-limiting enzyme for valerolactam biosynthesis in Corynebacterium glutamicum

• Published in: Metabolic engineering 2023-05, Vol.77, p.89-99
• Main Authors: Zhao, Xixi, Wu, Yanling, Feng, Tingye, Shen, Junfeng, Lu, Huan, Zhang, Yunfeng, Chou, Howard H., Luo, Xiaozhou, Keasling, Jay D.
• Format: Article
• Language: English
• Subjects: Biosensor engineering; Caprolactam - metabolism; Corynebacterium glutamicum; Corynebacterium glutamicum - metabolism; Dynamic regulation; Fermentation; Lead - metabolism; Lysine; Metabolic Engineering; Up-Regulation; Valerolactam
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2023.02.005

Valerolactam is a monomer used to manufacture high-value nylon-5 and nylon-6,5. However, the biological production of valerolactam has been limited by the inadequate efficiency of enzymes to cyclize 5-aminovaleric acid to produce valerolactam. In this study, we engineered Corynebacterium glutamicum with a valerolactam biosynthetic pathway consisting of DavAB from Pseudomonas putida to convert L-lysine to 5-aminovaleric acid and β-alanine CoA transferase (Act) from Clostridium propionicum to produce valerolactam from 5-aminovaleric acid. Most of the L-lysine was converted into 5-aminovaleric acid, but promoter optimization and increasing the copy number of Act were insufficient to significantly improve the titer of valerolactam. To eliminate the bottleneck at Act, we designed a dynamic upregulation system (a positive feedback loop based on the valerolactam biosensor ChnR/Pb). We used laboratory evolution to engineer ChnR/Pb to have higher sensitivity and a higher dynamic output range, and the engineered ChnR-B1/Pb-E1 system was used to overexpress the rate-limiting enzymes (Act/ORF26/CaiC) that cyclize 5-aminovaleric acid into valerolactam. In glucose fed-batch culture, we obtained 12.33 g/L valerolactam from the dynamic upregulation of Act, 11.88 g/L using ORF26, and 12.15 g/L using CaiC. Our engineered biosensor (ChnR-B1/Pb-E1 system) was also sensitive to 0.01–100 mM caprolactam, which suggests that this dynamic upregulation system can be used to enhance caprolactam biosynthesis in the future. •Design of a dynamic upregulation system for the rate-limiting enzymes in the valerolactam biosynthetic pathway.•Laboratory evolution to engineer ChnR/Pb to have higher sensitivity and a higher dynamic output range.•The highest titer of Valerolactam reached 12.33 g/L in a 1.2 L fermenter.