Hydroxyectoine, a vital compatible solute, is widely utilized in cosmetics, food, pharmaceutical industries, and biologics. However, the current microbial fermentation methods for hydroxyectoine production face challenges including insufficient precursor supply and low yields. Therefore, developing engineering microbial strains capable of efficiently synthesizing hydroxyectoine is of great significance. In this study, we first constructed a high-yield ectoine-producing strain ECT04 by multi-copy integration of the ectoine synthesis genes ectABC into the pseudogene loci of Escherichia coli MG1655(DE3), achieving an ectoine titer of 6.03 g/L. Subsequently, we employed plasmids with varying copy numbers to express ectD from Chromohalobacter salexigens to enable the conversion for hydroxyectoine production. We further investigated the effects of promoter, co-substrate ɑ-ketoglutarate, Fe²⁺ concentration, and dissolved oxygen on hydroxyectoine synthesis. Through fed-batch fermentation in a 7-L bioreactor, we significantly enhanced the hydroxyectoine production efficiency, attaining a final titer of 8.58 g/L and a productivity of 0.24 g/(L·h). This work successfully achieved the de novo synthesis of hydroxyectoine in E. coli, laying a foundation for the efficient bioproduction of this compound.
Escherichia coli/genetics* ; Fermentation ; Amino Acids, Diamino/biosynthesis* ; Bioreactors/microbiology* ; Metabolic Engineering/methods* ; Chromohalobacter/genetics* ; Plasmids/genetics*

Bacillus alcalophilus DTY1, one moderate halophytic bacterium isolated from saline soil in Loess Plateau of China, was characterized with efficient production of ectoine. In this study, the gene cluster ectABC taking in charge of biosynthesizing ectoine was cloned from the genomic library of strain DTY1. Nucleotide sequencing indicated that ectA, ectB and ectC were predicted to encode peptides of 169, 428 and 132 amino acids, respectively. The deduced amino acid sequences of EctA, EctB and EctC share 59%, 81% and 81% identity to 2,4-diaminobutyric acid acetyltransferase, 2,4-diaminobutyric acid transaminase and ectoine synthase of B. halodurans C-125, respectively. A fragment containing ectABC genes was introduced into B. cereus Z, which made the transgenic Z cells increased tolerance to salt, remarkably. HPLC analysis of ectoine in the transgenic Z cells revealed that 70.1 mg/g ectoine was detected in 1.0% NaCl medium and 118.6 mg/g ectoine in 5.0% NaCl medium. Furthermore, as the concentration of salt increased, transgenic Z cells accumulated more ectoine. These results suggest that ectoine is an important facet in B. alcalophilus DTY1 to high-osmolarity surroundings, and the expression of ectABC is induced by salt strength.
Amino Acid Sequence ; Amino Acids, Diamino ; biosynthesis ; genetics ; physiology ; Bacillus ; classification ; genetics ; Bacillus cereus ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; Cloning, Molecular ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; genetics ; Molecular Sequence Data ; Osmotic Pressure ; Sodium Chloride ; metabolism ; pharmacology