Metabolic Engineering of Escherichia Coli For
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Zhu et al. Microb Cell Fact (2020) 19:129 https://doi.org/10.1186/s12934-020-01369-2 Microbial Cell Factories RESEARCH Open Access Metabolic engineering of Escherichia coli for efcient production of L-alanyl-L-glutamine Jiangming Zhu1 , Wei Yang1, Bohua Wang1, Qun Liu1,2, Xiaotong Zhong1,2, Quanxiu Gao3, Jiezheng Liu1,2, Jianzhong Huang3*, Baixue Lin1,2* and Yong Tao1,2 Abstract 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 felds. 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 efcient 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 frst 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 fnal 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 efcient production of AQ with industrial potential. Keywords: AQ, Metabolic engineering, Whole-cell biocatalysis, L-amino acid α-ligase, Glutamine synthase Background [7] and improving the adaptability of organism to stress Glutamine (l-Gln) plays an important role in maintaining [8]. During disease or stress conditions, demand out- intestinal function [1–4], promoting immune function [5, paces supply, and glutamine becomes conditionally 6], maintaining homeostasis of the internal environment essential [9]. Te supply of exogenous l-glutamine or glutamyl dipeptide is an important nutritional solution to *Correspondence: [email protected]; [email protected] reduce glutamine defciency in vivo, and can be applied 1 Chinese Academy of Sciences Key Laboratory of Microbial Physiological in clinical treatment. However, some characteristics of and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China glutamine such as low solubility in water, easy decompo- 3 National Engineering Research Center of Industrial Microbiology sition and poor thermal stability, as well as production of and Fermentation Technology, College of Life Sciences, Fujian Normal toxic pyroglutamate during heat sterilization restricted University, Fuzhou 350117, Fujian, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhu et al. Microb Cell Fact (2020) 19:129 Page 2 of 10 its application in medicine. Otherwise, as a result of its produced more than 100 mM AQ in 47 h of fermenta- unstable nature, the low yield of glutamine during the tion [20]. Whole-cell biocatalysis can be used to perform purifcation process is a great challenge [10]. enzyme cascade reaction, improve catalytic efciency, l-Alanyl-l-glutamine (abbreviated as AQ) is a dipeptide of and simplify the preparation process. glutamine and alanine, which is more stable and water-sol- A metabolically engineered E. coli strain for AQ pro- uble than glutamine. AQ is hydrolyzed to release glutamine duction was developed by over-expressing l-amino acid and alanine in vivo. While it acts as a source of glutamine, α-ligase (BacD) from Bacillus subtilis and inactivating AQ has higher bioavailability and a short half-life [11], and native peptidases. In order to use a more readily avail- does not cause cumulative damage to the body, so it is used able substrate, the glutamine synthesis module based as a substitute for glutamine in clinical practice. on introducing glutamine synthetase (GlnA) was con- Te commercial demand for AQ is increasing with the structed and optimized. Ten glutamine synthesis mod- expansion of new applications and the development of ule was combined with the AQ synthesis module to use new products based on AQ. Chemical and biotechno- glutamic acid and alanine for AQ production. Te pro- logical methods for AQ production have been developed tein expression of BacD and GlnA was further balanced [12–16]. AQ can be synthesized by chemical condensa- to improve the AQ production. Finally, we used the engi- tion of benzyloxycarbonyl-l-alanine and methyl glu- neered strain to develop a whole-cell biocatalytic process tamate via activated ester [17] or triphenyl phosphine/ for AQ production (Fig. 1). Tis work provides an envi- hexachloroethane condensation, which used to require ronmentally friendly, highly efcient and cost-efective complex steps such as amino acid activation, subunit process for industrial biosynthesis of AQ. protection, and removing the protective groups. Enzy- matic processes for AQ production have been developed Results using an α-amino acid ester acyltransferase [18, 19]. Construction of the AQ synthesis module Tabata and Hashimoto engineered Escherichia coli by Te E. coli strain p01/BW25113, overexpressing BacD expressing l-amino acid α-ligase (BacD), which catalyzes from Bacillus subtilis (BsBacD) which catalyzes the the formation of AQ in an ATP-dependent manner, and formation of AQ from alanine and glutamine, was Fig. 1 Schematic presentation of the AQ metabolic pathway in engineered E. coli. The GlnA and BacD were co-expressed. Discontinued arrows represent the enzymatic activities that have been eliminated. GlsA glutaminase 1, GlsB glutaminase 2, GlnE fused glutamine synthetase deadenylase/adenylyltransferase, GlnB nitrogen regulatory protein PII-1, PepA aminopeptidase A/I, PepB aminopeptidase B, PepD peptidase D, PepN aminopeptidase N, Dpp dipeptide ABC transporter DppABCDF Zhu et al. Microb Cell Fact (2020) 19:129 Page 3 of 10 Fig. 3 AQ production by engineered strains over-expressing BacD homologs from diferent species. Ba, BacD from Bacillus altitudinis; Fig. 2 Extracellular titers of AQ produced by engineered strains Bs, BacD from Bacillus subtilis; Bv, BacD from Beta vulgaris; Vc, BacD overexpressing BsBacD. WT wild type; BW, AQ06, AQ09, AQ10 were from Vibrio campbellii; Pm, BacD from Perkinsus marinus; Sr, BacD transformed with plasmid p01. The engineered strains were induced from Streptomyces rubrolavendulae; Bsa, BacD from Bacillus safensis; and suspended in a reaction mixture containing 50 mM glutamine, Blo, BacD from Bifdobacterium longum subsp. Infantis; Pf, BacD from 50 mM alanine, and 10 mM magnesium chloride. The bioconversion Pseudomonas fuorescens reactions were performed at 30 °C and 200 rpm for 18 h. Glucose was supplemented at a concentration of 10 mM every 3 h previously reported BacD homologs and those used in constructed for the production of AQ. Extracellular AQ this study (Additional fle 1: Fig. S1). Te genes encod- concentration was measured by HPLC, and 2.0 mM AQ ing BacD homologs from diferent species were codon- was obtained (Fig. 2). optimized by Nanjing Generay (China) and cloned into In E. coli, peptidases encoded by pepA, pepB, pepD and strain AQ10 (BW25113, ∆glnEB, ∆glsAB, ∆lpxM, ∆pep- pepN, have been reported to degrade a broad spectrum of ABDN, ∆dpp). Te performance of diferent BacD pro- dipeptides [20, 21], and inactivating them might reduce teins was investigated in vivo, using the respective strains AQ degradation. It was reported that deletion of dpp, as a whole-cell biocatalysts. Te result showed that the encoding a dipeptide ABC transporter increased AQ accu- strain overexpressing BaBacD (from Bacillus altitudinis) mulation [20, 22, 23]. By knocking out the genes pepN, produced higher amount of AQ (19.2 mM) than strains pepA, pepB, pepD and dpp, the degradation of AQ was with other BacD homologs. By comparison, 7.9 mM AQ alleviated. In the starting host BW25113, 20 mM AQ was was obtained using the strain overexpressing BsBacD completely degraded after 3 h, compared to only 1.3 mM (from Bacillus subtilis) (Fig. 3). Although there was solu- in the chassis AQ09 (BW25113ΔpepN, ΔpepA, ΔpepB, ble expression of BvBacD (from Beta vulgaris), VcBacD ΔpepD, Δdpp) after 6 h.