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

Ectoine and hydroxyectoine are widely synthesized by members of theBacteria and a few members of theArchaea 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-root-associated bacteriumPseudomonas stutzeri by transferring it intoEscherichia coli , an enterobacterium that does not produce ectoines naturally. Usingect-lacZ reporter fusions, we found that the heterologousect 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 buildup 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-typeect promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in theect promoter sequence that increase its resemblance to housekeeping sigma-70-type promoters ofE. coli afforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set ofect promoter mutants, we engineered anE. coli 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.IMPORTANCE Ectoines are compatible solutes, organic 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 theect promoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact thatE. coli does not synthesize ectoines naturally, theect promoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation ofect 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 theect promoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.