High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli

Abstract

BackgroundRecently, the compatible solute 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) has attracted considerable interest due to its great potential as a protecting agent. To overcome the drawbacks of high salinity in the traditional bioprocess of ectoine using halophilic bacteria, various attempts have been made to engineer ectoine biosynthesis in nonhalophilic bacteria. Unfortunately, the yields of ectoine in these producers are still low and hardly meet the demands of large scale production. In this paper, the whole-cell biocatalytic process using aspartate and glycerol as substrates was tried for high production of ectoine in nonhalophilic bacteria.ResultsThe ectoine genes ectABC from the halophilic bacterium Halomonas elongata were successfully introduced into Escherichia coli K-12 strain BW25113 under the arabinose-inducible promoter. To our delight, a large amount of ectoine was synthesized and excreted into the medium during the course of whole-cell biocatalysis, when using aspartate and glycerol as the direct substrates. At the low cell density of 5 OD/mL in flask, under the optimal conditions (100 mM sodium phosphate buffer (pH 7.0), 100 mM sodium aspartate, 100 mM KCl and 100 mM glycerol), the concentration of extracellular ectoine was increased to 2.67 mg/mL. At the high cell density of 20 OD/mL in fermentor, a maximum titre of 25.1 g/L ectoine was achieved in 24 h. Meanwhile, the biomass productivity of ectoine is as high as 4048 mg per gram dry cell weight (g DCW)−1, which is the highest value ever reported. Furthermore, it was demonstrated that the same batch of cells could be used for at least three rounds. Finally, a total yield of 63.4 g ectoine was obtained using one litre cells.ConclusionUsing aspartate and glycerol as the direct substrates, high production of ectoine was achieved by the whole-cell biocatalysis in recombinant E. coli. Multiple rounds of whole-cell biocatalysis were established to further improve the production of ectoine. Our study herein provided a feasible biosynthesis process of ectoine with potential applications in large-scale industrial production.

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

“…Aspartate is the biosynthetic precursor of ectoine (20,86) (Fig. 1A), and its addition to the growth medium has already been proven beneficial for the recombinant production of ectoines (87). In exploratory experiments, we observed that the addition of 25 mM aspartate to our Ots Ϫ E. coli cell factory harboring plasmid pLC68 substantially enhanced hydroxyectoine production (see Fig.…”

“…9B and E). It shares this feature with other synthetic cell factories that rely on microbial chassis strains that do not produce ectoines naturally (61)(62)(63)(64)(65)87). Investigations into the release of hydroxyectoine from a recombinant E. coli strain expressing the ectD gene from P. stutzeri A1501 under the control of the tet promoter (85) demonstrated that this process was not mediated, in the absence of an osmotic downshock, by any of the mechanosensitive channels (MscL, MscS, and MscM) operating in E. coli (100).…”

The Potential Use of Ectoine Produced by a Moderately Halophilic Bacteria Chromohalobacter salexigens KT989776 for Enhancing Germination and Primary Seedling of Flax “Linum usitatissimum L.” under Salinity Conditions

“…The retention time: the spectra signal of LC-MS analysis (pseudo-molecular ion at m/z 143) (Fig1a) and LC-MS/MS analysis (product ions at m/z 143.3, 97.0, 68.2, 55.9, and 44.0) (Fig 1b) were all in agreement with the data of the authentic ectoine. Therefore, the compound in the cells alcoholic extract was identified as ectoine [38]. Seed germination is the most critical stage during the life span of most plants, a lot of plants can survive and tolerate abiotic stresses when safely pass this stage.…”

Optimized whole cell biocatalyst from acetoin to 2,3‐butanediol through coexpression of acetoin reductase with NADH regeneration systems in engineered Bacillus subtilis

“…In addition, it can be used in whole cell biocatalysis for production of metabolites . It was also used in ectoine production from aspartate‐semialdehyde and high ectoine concentration was achieved . In the current study using the same strategy, the cells were collected by centrifugation and subjected to whole cell biocatalysis.…”