As a compatible solute, ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylicacid) was commonly found in halophilic and halotolerant microorganisms. It was first discovered in Ectothiorhodospira halochloris by Galinski et al.  and its structure was identified to be a cyclic amino acid derivative. In addition to its primary function of maintaining cell osmotic balance and resisting the impact of high osmotic pressure, an increasing attention was focused on its remarkable ability in protecting biological compounds [2, 3]. Therefore the potential applications of ectoine lies in food biotechnology, agriculture, skin caring, and medical fields [4–8].
As shown in Figure. 1, ectoine is synthesized from the precursor L-aspartate-β-semialdehyde (ASA), a central hub in microbial amino acid production, by three successive enzymatic steps that are catalyzed by the L-2,4-diaminobutyrate transaminase (EctB), the 2,4-diaminobutyrate acetyltransferase (EctA) and the ectoine synthase (EctC) [9–12]. The ectoine biosynthetic genes are normally organized in an operon (ectABC) that might also comprise the ectD gene . Similar gene clusters involved in ectoine biosynthesis were disclosed in strain Marinococcus halophilus , Halobacillus dabanensis D-8T , Methylomicrobium alcaliphilum 20Z , Nesterenkonia halobia DSM20541  and so on.
Commercially, ectoine production is realized through fermentation of halophiles in a special and complex process called “bacterial milking” [18, 19]. Although this method can be used to obtain ectoine in large scale, the use of large amounts of salt will corrode the equipment, which requires higher corrosion resistance of the fermentation equipment. The ability of bacteria to resist osmotic pressure shock needs to be higher in the process. To address the shortcomings of this process, great efforts have been made, such as optimizing process conditions, improving ectoine production performance by breeding of halophilic bacteria. However, using transgenic nonhalophilic bacteria for ectoine production is more efficient in recent years.
Therefore, Becker et al. successfully integrated the ectABCD gene operon of Pseudomonas stutzeri A1501 into Corynebacterium glutamicum based on systematic metabolic engineering, and mutated aspartate kinase to ensure sufficient supply of ASA, and the final overall spacetime yield achieved 6.7 g/L ectoine per day . In 2015, ectABC gene cluster of Halomonas elongata DSM 2581 was over expressed in E. coli K-12/BW25113, and the titer of ectoine reached 25.1 g/L, with a productive yield of 4048 mg/g DCW by a whole cell biocatalytic process using aspartate and glycerol as substrates . Subsequently, an engineered strain E. coli ECT05 was constructed through a series of metabolic engineering strategies, besides the final titer reached 25.1 g/L, and overall ectoine yield was 0.11 g/g of glucose . Recently, production of ectoine was up to 65 g/L within 56 h by transcriptional balancing of the ectoine pathway in Corynebacterium. glutamicum .
Although the ectoine production can be realized by fermentation of engineered strains, there are still problems of low glucose conversion rate and low ectoine production efficiency. The recent reports mainly focus on metabolic modification of engineered strains. There were few studies on nutritional requirements optimization and fermentation regulation of engineered strains. Particularly, nitrogen is a constituent of cellular components such as proteins, nucleic acids and several cofactors. It also regulates primary and secondary metabolism in different bacteria. Xu et al. increased the yield of ε-poly-L-lysine through nitrogen source regulation and optimization . It was also reported that nitrogen source regulation has great effect on ethanol and antibiotic production [25, 26]. Ectoine is synthesized from aspartate, which acts as the direct precursor. The supply of amounts of nitrogen is essential for ectoine synthesis.
In this study, we constructed a metabolically engineered strain E. coli ET08 capable of producing ectoine efficiently. Further studies focus on the effects of complex nitrogen sources and amino donors on ectoine production. Then the transcription levels of the key genes in ectoine and ammonium metabolic pathways were analyzed for description the function of the amino donor. Finally, ectoine production of engineered E. coli ET08 was evaluated by two-stage feeding fermentation with supplementing amino donor. This work provides a novel strategy for the synthesis of ectoine by engineered strain in industry.