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

Abstract

Ectoine and hydroxyectoine are widely synthesized by members of the and a few as potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-roots-associated bacterium by transferring it into, an enterobacterium that does not produce ectoines naturally. Using reporter fusions, we found that the heterologous promoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the build-up of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the -10, -35, and spacer regions of the Sigma-70 type promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in the promoter sequence that increase its resemblance to house-keeping Sigma-70-type promoters of afforded substantially enhanced expression, both in absence and in the presence of osmotic stress. Building on this set of promoter mutants, we engineered an chassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions.Ectoines are compatible solutes, organics osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited the promoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact that does not synthesize ectoines naturally, the promoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation of transcription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116 bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of the promoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds, into the growth medium.

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

“…Ectoine and 5-hydroxyectoine are produced by microorganisms in response to true osmotic stress, and not just in response to increases in the external salinity [ 116 ]. In cases where the build-up of ectoine/5-hydroxyectoine pools has been studied in more detail, there is often a linear relationship between the cellular content of these solutes and the external salinity/osmolarity [ 116 , 117 , 118 ].…”

Compatible Solute Synthesis and Import by the Moderate Halophile Spiribacter salinus: Physiology and Genomics

“…Either because of the superior physico-chemical properties of this chemical chaperon ( Zaccai et al, 2016 ; Stadmiller et al, 2017 ) or to save precursors and biosynthetic resources used for ectoine production ( Pastor et al, 2010 ; Oren, 2011 ; Widderich et al, 2014 ). It is known from other studies, that the down-regulation of the ectoine pool in response to the import of glycine betaine or other compatible solutes is primarily the consequence of the down-regulation of the transcription of the ectoine biosynthetic genes ( Bestvater et al, 2008 ; Czech et al, 2018 ).…”

With a pinch of extra salt—Did predatory protists steal genes from their food?

“…All 5-hydroxyectoine producers are aerobic, or at least O 2 tolerant, microorganisms because the ectoine hydroxylase (EctD; EC 1.14.11.55) is an oxygen-dependent enzyme [ 34 , 36 ]. The ectoine biosynthetic genes ( ectABC ) are typically organized as an osmotically inducible operon [ 37 ] that may also contain the gene ( ectD ) for the ectoine hydroxylase and a specialized aspartokinase (Ask_Ect) involved in synthesizing the ectoine precursor L-aspartate-beta-semialdehyde [ 34 , 36 ]. Effective osmostress protection can also be accomplished through the import of ectoines, and several types of osmotically induced ectoine/5-hydroxyectoine import systems have been identified in microorganisms (for an example see [ 38 ] and additional references therein).…”