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Ectoine is an important natural secondary metabolite in halophilic microorganisms. It protects cells against environmental stressors, such as salinity, freezing, drying, and high temperatures. Ectoine is widely used in medical, cosmetic, and other industries. Due to the commercial market demand of ectoine, halophilic microorganisms are the primary method for producing ectoine, which is produced using the industrial fermentation process “bacterial milking.” The method has some limitations, such as the high salt concentration fermentation, which is highly corrosive to the equipment, and this also increases the difficulty of downstream purification and causes high production costs. The ectoine synthesis gene cluster has been successfully heterologously expressed in industrial microorganisms, and the yield of ectoine was significantly increased and the cost was reduced. This review aims to summarize and update microbial production of ectoine using different microorganisms, environments, and metabolic engineering and fermentation strategies and provides important reference for the development and application of ectoine.

Ectoine is a compatible solute naturally produced in some halophilic bacteria as a protective agent for survival in salty environments. It has gained special interest as a therapeutic agent in the pharmaceutical and healthcare sectors for the treatment of different diseases. Ectoine mainly produced by bacterial milking, chemical, and fed-batch fermentation methods under a high-salt medium. Unfortunately, the ectoine yield through these methods is still too low to meet high industrial demand, causing salinity issues. The biobrick method was potentially utilized for efficient ectoine biosynthesis under a low-salt medium with different conditions in E. coli BL21(DE3) harboring the pET-22bNS-EctA-EctB-EctC plasmid. Firstly, three genes, L-2,4-diamino-butyric acid acetyltransferase (ectA), L-2,4-diaminobutyric acid transaminase (ectB), and ectoine synthase (ectC) from Bacillus pseudofirmus OF4, were precisely assembled and expressed into E. coli BL21(DE3). After optimizing the reaction conditions in a whole-cell catalytic reaction [50 mM of the sodium phosphate buffer (pH~7.5) containing 300 mM L-aspartic acid, 100 mM glycerol, 1/20 g/mL cell pellets], the amount of ectoine in the plasmid pET-22bNS-ALacBTacCTac reached the maximum level of 167.2 mg/mL/d (6.97 mg/mL/h). Moreover, Western blot analysis revealed that high expression levels of EctA and EctC had a significant effect on ectoine biosynthesis, indicating that both proteins might be the key enzymes in ectoine production. We conclude that a high amount of ectoine achieved through the biobrick method and efficiently used for different industrial applications.

The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ⁷⁰-dependent promoter and a δ³⁸-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.

The ectABC genes encoding the biosynthesis of ectoine were identified from Nesterenkonia halobia DSM 20541. The intergenic regions of the ectABC genes from N. halobia DSM 20541 were more loosely spaced than those that had been reported before. The amino acid sequence deduced from ectABC of the strain was highly homologous to the EctABC of Brevibacterium linens BL2 (EctA 50%, EctB 70%, and EctC 68% identities). The osmoprotection of ectABC was studied in the Escherichia coli KNabc and E. coli XL1-Blue. The results revealed that ectABC could shorten the lag phase and enhance the final OD600 of E. coli XL1-Blue in MM63 medium containing 0.68 M NaCl, and could initiate KNabc growth in 0.2 M NaCl. Ectoine was proven to be accumulated in E. coli KNabc/pGEM-Nect using HPLC-UV, and validated by LC-MSD-Trap-VL.

The moderately halophilic bacterium Virgibacillus halodenitrificans PDB-F2 copes with salinity by synthesizing or taking up compatible solutes. The main compatible solutes in this strain were ectoine and hydroxyectoine, as determined by 1 H nuclear magnetic resonance spectroscopy ( 1 H-NMR). A high-performance liquid chromatography (HPLC) analysis showed that ectoine was the major solute that was synthesized in response to elevated salinity, while hydroxyectoine was a minor solute. However, the hydroxyectoine/ectoine ratio increased from 0.04 at 3 % NaCl to 0.45 at 15 % NaCl in the late exponential growth phase. A cluster of ectoine biosynthesis genes was identified, including three genes in the order of ectA , ectB , and ectC . The hydroxyectoine biosynthesis gene ectD was not part of the ectABC gene cluster. Reverse transcription–quantitative polymerase chain reactions (RT-qPCR) showed that the expression of the ect genes was salinity dependent. The expression of ectABC reached a maximum at 12 % NaCl, while ectD expression increased up to 15 % NaCl. Ectoine and hydroxyectoine production was growth phase dependent. The hydroxyectoine/ectoine ratio increased from 0.018 in the early exponential phase to 0.11 in the stationary phase at 5 % NaCl. Hydroxyectoine biosynthesis started much later than ectoine biosynthesis after osmotic shock, and the temporal expression of the ect genes differed under these conditions, with the ectABC genes being expressed first, followed by ectD gene. Increased culture salinity triggered ectoine or hydroxyectoine uptake when they were added to the medium. Hydroxyectoine was accumulated preferentially when both ectoine and hydroxyectoine were provided exogenously.