Metabolic engineering of Escherichia coli for efficient production of l-alanyl-l-glutamine

Jiangming Zhu,Yong Tao,Xiaotong Zhong,Bohua Wang,Quanxiu Gao,Baixue Lin,Jiezheng Liu,Wei Yang,Qun Liu,Jianzhong Huang

doi:10.1186/s12934-020-01369-2

Abstract

Backgroundl-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.ResultsA 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%.ConclusionA 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.

Highlights

Glutamine (l-Gln) plays an important role in maintaining intestinal function [1,2,3,4], promoting immune function [5, 6], maintaining homeostasis of the internal environment [7] and improving the adaptability of organism to stress [8]
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 native peptidases
Construction of the AQ synthesis module The E. coli strain p01/BW25113, overexpressing BacD from Bacillus subtilis (BsBacD) which catalyzes the formation of AQ from alanine and glutamine, was

Summary

Introduction

Glutamine (l-Gln) plays an important role in maintaining intestinal function [1,2,3,4], promoting immune function [5, 6], maintaining homeostasis of the internal environment [7] and improving the adaptability of organism to stress [8]. The supply of exogenous l-glutamine or glutamyl dipeptide is an important nutritional solution to reduce glutamine deficiency in vivo, and can be applied in clinical treatment. Some characteristics of glutamine such as low solubility in water, easy decomposition and poor thermal stability, as well as production of toxic pyroglutamate during heat sterilization restricted. L-Alanyl-l-glutamine (abbreviated as AQ) is a dipeptide of glutamine and alanine, which is more stable and water-soluble than glutamine. AQ is hydrolyzed to release glutamine and alanine in vivo. While it acts as a source of glutamine, AQ has higher bioavailability and a short half-life [11], and does not cause cumulative damage to the body, so it is used as a substitute for glutamine in clinical practice

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