l-Alanyl-l-glutamine (AQ) is a functional dipeptide with high water solubility, good thermal stability and high bioavailability. It is widely used in clinical treatment, post-operative rehabilitation, sports health care and other fields. AQ is mainly produced via chemical synthesis which is complicated, time-consuming, labor-intensive, and have a low yield accompanied with the generation of by-products. It is therefore highly desirable to develop an efficient biotechnological process for the industrial production of AQ. A metabolically engineered E. coli strain for AQ production was developed by over-expressing l-amino acid α-ligase (BacD) from Bacillus subtilis, and inactivating the peptidases PepA, PepB, PepD, and PepN, as well as the dipeptide transport system Dpp. In order to use the more readily available substrate glutamic acid, a module for glutamine synthesis from glutamic acid was constructed by introducing glutamine synthetase (GlnA). Additionally, we knocked out glsA–glsB to block the first step in glutamine metabolism, and glnE–glnB involved in the ATP-dependent addition of AMP/UMP to a subunit of glutamine synthetase, which resulted in increased glutamine supply. Then the glutamine synthesis module was combined with the AQ synthesis module to develop the engineered strain that uses glutamic acid and alanine for AQ production. The expression of BacD and GlnA was further balanced to improve AQ production. Using the final engineered strain p15/AQ10 as a whole-cell biocatalyst, 71.7 mM AQ was produced with a productivity of 3.98 mM/h and conversion rate of 71.7%. A metabolically engineered strain for AQ production was successfully developed via inactivation of peptidases, screening of BacD, introduction of glutamine synthesis module, and balancing the glutamine and AQ synthesis modules to improve the yield of AQ. This work provides a microbial cell factory for efficient production of AQ with industrial potential.

中文翻译：

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用于高效生产 L-丙氨酰-L-谷氨酰胺的大肠杆菌代谢工程。


L-丙氨酰-L-谷氨酰胺（AQ）是一种功能性二肽，具有高水溶性、良好的热稳定性和高生物利用度。广泛应用于临床治疗、术后康复、运动保健等领域。 AQ主要通过化学合成法生产，工艺复杂、费时、劳动强度大、收率低，并有副产物产生。因此，非常需要开发一种用于工业生产 AQ 的有效生物技术方法。通过过表达来自枯草芽孢杆菌的 l-氨基酸 α-连接酶 (BacD)，并灭活肽酶 PepA、PepB、PepD 和 PepN 以及二肽转运系统，开发了用于生产 AQ 的代谢工程大肠杆菌菌株副总裁。为了使用更容易获得的底物谷氨酸，通过引入谷氨酰胺合成酶（GlnA）构建了由谷氨酸合成谷氨酰胺的模块。此外，我们敲除 glsA–glsB 以阻断谷氨酰胺代谢的第一步，而 glnE–glnB 参与 AMP/UMP 向谷氨酰胺合成酶亚基的 ATP 依赖性添加，从而导致谷氨酰胺供应增加。然后将谷氨酰胺合成模块与AQ合成模块相结合，开发出利用谷氨酸和丙氨酸生产AQ的工程菌株。 BacD 和 GlnA 的表达进一步平衡，以提高 AQ 产量。使用最终的工程菌株 p15/AQ10 作为全细胞生物催化剂，产生了 71.7 mM AQ，生产率为 3.98 mM/h，转化率为 71.7%。 通过肽酶灭活、BacD筛选、引入谷氨酰胺合成模块、平衡谷氨酰胺和AQ合成模块以提高AQ产量，成功开发了用于AQ生产的代谢工程菌株。这项工作为高效生产 AQ 提供了一个具有工业潜力的微生物细胞工厂。