Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document.When citing, please reference the published version. Take down policyWhile the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive.

Background: 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.Results: 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 %.Conclusion: 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.

Background 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 medicine, post-operative rehabilitation, sports health care and other fields. AQ is mainly produced by chemical synthesis which is complicated, time-consuming, labor-consuming, low yield and accompany with by-products. It is highly desirable to develop an efficient biotechnological process for AQ production. Results A metabolic engineered E. coli strain for AQ production was developed by over-expressing L-amino acid-ligase (BacD) from Bacillus subtilis , peptidases including PepA, PepB, PepD, PepN and dipeptide transport system Dpp were inactivated. In order to use the more readily available substrate, glutamic acid, a glutamine synthetic module from glutamic acid to glutamine was constructed by introducing glutamine synthetase (GlnA), glsA-glsB catalyze the first step in glutamine metabolism and glnE-glnB involved in the ATP-dependent addition of AMP/UMP to a subunit of glutamine synthetase were blocked which resulted in increased glutamine supply. This glutamine synthetic module combined with AQ synthetic module to develope the engineered strain that using glutamic acid and alanine for AQ production. The expression of BacD and GlnA was further balanced to improve the AQ production. The engineered strain p15/AQ10 was used in the whole-cell biocatalysis and 65.6 mM AQ was produced with productivity of 7.29 mM/h and conversion rate of 65.6%. Conclusion Metabolic engineered strains were developed for AQ production. Strategies including inactivation of peptidases, screening of BacD, introducing glutamine synthetic module, and balancing the glutamine and AQ synthetic modules were applied to improve the yield of AQ. This work provides the biosynthetically industrial potential for efficient production of AQ by microbial cell factory.