EctD-mediated biotransformation of the chemical chaperone ectoine into hydroxyectoine and its mechanosensitive channel-independent excretion

Abstract

BackgroundEctoine and its derivative 5-hydroxyectoine are cytoprotectants widely synthesized by microorganisms as a defense against the detrimental effects of high osmolarity on cellular physiology and growth. Both ectoines possess the ability to preserve the functionality of proteins, macromolecular complexes, and even entire cells, attributes that led to their description as chemical chaperones. As a consequence, there is growing interest in using ectoines for biotechnological purposes, in skin care, and in medical applications. 5-Hydroxyectoine is synthesized from ectoine through a region- and stereo-specific hydroxylation reaction mediated by the EctD enzyme, a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenases. This chemical modification endows the newly formed 5-hydroxyectoine with either superior or different stress- protecting and stabilizing properties. Microorganisms producing 5-hydroxyectoine typically contain a mixture of both ectoines. We aimed to establish a recombinant microbial cell factory where 5-hydroxyectoine is (i) produced in highly purified form, and (ii) secreted into the growth medium.ResultsWe used an Escherichia coli strain (FF4169) defective in the synthesis of the osmostress protectant trehalose as the chassis for our recombinant cell factory. We expressed in this strain a plasmid-encoded ectD gene from Pseudomonas stutzeri A1501 under the control of the anhydrotetracycline-inducible tet promoter. We chose the ectoine hydroxylase from P. stutzeri A1501 for our cell factory after a careful comparison of the in vivo performance of seven different EctD proteins. In the final set-up of the cell factory, ectoine was provided to salt-stressed cultures of strain FF4169 (pMP41; ectD+). Ectoine was imported into the cells via the osmotically inducible ProP and ProU transport systems, intracellularly converted to 5-hydroxyectoine, which was then almost quantitatively secreted into the growth medium. Experiments with an E. coli mutant lacking all currently known mechanosensitive channels (MscL, MscS, MscK, MscM) revealed that the release of 5-hydroxyectoine under osmotic steady-state conditions occurred independently of these microbial safety valves. In shake-flask experiments, 2.13 g l−1 ectoine (15 mM) was completely converted into 5-hydroxyectoine within 24 h.ConclusionsWe describe here a recombinant E. coli cell factory for the production and secretion of the chemical chaperone 5-hydroxyectoine free from contaminating ectoine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0525-4) contains supplementary material, which is available to authorized users.

Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis

“…It is outside the scope of this overview to address in depth the biotechnological production of ectoines in natural and synthetic microbial cell factories, or to describe in detail the varied practical applications for these compounds. Insightful reviews covering these topics have been published [ 86 , 91 , 94 , 145 ], and recent reports have summarized the current status of efforts to improve the productivity of natural and synthetic microbial cell factories for ectoines [ 116 , 123 , 151 , 152 , 153 , 154 , 155 , 156 , 157 ].…”

Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory

“…It partially releases the newly produced trehalose also into the periplasmic space, where this disaccharide is hydrolyzed by an osmotically inducible trehalase (TreA), and the glucose monomers are then recovered again by the E. coli cells through a phosphotransferase transport system (PTS) for their use as carbon sources (84). To avoid the contamination of newly produced ectoine/ hydroxyectoine by trehalose and to provide an incentive to the osmotically stressed cells to enhance the production of ectoines in response to increased salinity to provide physiologically adequate osmostress protection for E. coli (2), we used for the setup of the cell factory a strain that is defective in trehalose synthesis (FF4169; otsA1::Tn10) (83,85). The use of such a host strain proved to be beneficial for the expression of the ect-lacZ reporter fusions carrying either the wild-type ect promoter or its Mut 12 and Mut 15 derivatives in comparison with its otsBA ϩ parent strain, MC4100 (Fig.…”

“…The extracted samples and the cell-free supernatant derived from the corresponding cell cultures were diluted 10-fold with distilled water and acetonitrile (the final concentration of acetonitrile was 50%) and analyzed for their ectoine/5-hydroxyectoine content by isocratic high-performance liquid chromatography (HPLC) (24). For these measurements, we employed an Agilent 1260 Infinity LC system (Agilent, Waldbronn, Germany) and a Grom-Sil Amino 1PR column (Grom, Rottenburg-Hailfingen, Germany) essentially as described previously (24) with the exception that a 1260 Infinity diode array detector (DAD) (Agilent) was used instead of the previously used UV/visible detector system (24,85). The ectoine and 5-hydroxyectoine contents of HPLC-analyzed samples were quantified using the OpenLAB software suite (Agilent).…”

Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata

“…A channel-independent export of compatible solutes was also described for B. subtilis and E. coli. In B. subtilis none of the known MS channels of the MscL-and MscStype participated in export of the endogenous compatible solute proline (Hoffmann et al 2012), and in osmotically adapted E. coli cells hydroxyectoine export is independent of MS channels as well (Czech et al 2016). We are not aware that the export systems have been identified in any of these organisms.…”

“…In contrast to C. glutamicum, none of the known MscL-and MscStype of MS channels in B. subtilis participate in export of the endogenous compatible solute proline (Hoffmann et al 2012). Moreover, in osmotically adapted E. coli cells export of the compatible solutes ectoine and hydroxyectoine is also independent of MS channels (Czech et al 2016).…”