Center for Synthetic Biochemistry, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China; CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.
Center for Synthetic Biochemistry, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China; CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Metab Eng. 2023 May;77:89-99. doi: 10.1016/j.ymben.2023.02.005. Epub 2023 Mar 16.
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.
正戊内酰胺是一种用于制造高价值尼龙-5 和尼龙-6,5 的单体。然而，由于酶将 5-氨基戊酸环化生成正戊内酰胺的效率不足，因此生物法生产正戊内酰胺受到限制。在这项研究中，我们通过工程改造谷氨酸棒杆菌，构建了一个正戊内酰胺生物合成途径，该途径由恶臭假单胞菌的 DavAB 酶将 L-赖氨酸转化为 5-氨基戊酸，以及丙酸梭菌的 β-丙氨酸 CoA 转移酶（Act）将 5-氨基戊酸转化为正戊内酰胺。大多数 L-赖氨酸都转化为 5-氨基戊酸，但启动子优化和 Act 拷贝数的增加不足以显著提高正戊内酰胺的产量。为了消除 Act 的瓶颈，我们设计了一个动态调控系统（基于正戊内酰胺生物传感器 ChnR/Pb 的正反馈回路）。我们利用实验室进化工程改造 ChnR/Pb，使其具有更高的灵敏度和更大的动态输出范围，并使用工程化的 ChnR-B1/Pb-E1 系统过表达限速酶（Act/ORF26/CaiC），将 5-氨基戊酸环化生成正戊内酰胺。在葡萄糖补料分批培养中，通过 Act 的动态调控，我们获得了 12.33 g/L 的正戊内酰胺，使用 ORF26 获得了 11.88 g/L，使用 CaiC 获得了 12.15 g/L。我们工程化的生物传感器（ChnR-B1/Pb-E1 系统）对 0.01-100 mM 己内酰胺也很敏感，这表明该动态调控系统将来可用于增强己内酰胺的生物合成。
