New insights into hydroxyectoine synthesis and its transcriptional regulation in the broad‐salt growing halophilic bacterium Chromohalobacter salexigens

• Published in: Microbial biotechnology Vol. 14; no. 4; pp. 1472 - 1493
• Main Authors: Argandoña, Montserrat, Piubeli, Francine, Reina‐Bueno, Mercedes, Nieto, Joaquín J., Vargas, Carmen
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01-07-2021; John Wiley and Sons Inc; Wiley
• Subjects: Accumulation; Amino Acids, Diamino; Bacteria; Chromohalobacter - genetics; Chromohalobacter salexigens; Design optimization; Divergence; Ectoine; Enzymes; Gene regulation; Genes; Genetic engineering; Genomes; Heat stress; Heat tolerance; Hydroxylase; Inactivation; Metabolic engineering; Microorganisms; Proteins; Salinity; Sodium Chloride; Stationary phase; Synthesis; Transcription
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/1751-7915.13799

Summary
Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS‐dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC‐GlxA‐like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post‐transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyectoine production.
Besides the main ectoine hydroxylase EctD, the halophilic bacterium Chromohalobacter salexigens possess a secondary ectoine hydroxylases, EctE, which contributes to C. salexigens thermoprotection. Expression of ectD and ectE is dependent on the growth phase and the general stress factor RpoS. In addition, the EctZ transcriptional regulator plays a dual function under exponential growth as both an activator of osmorregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD.