High-efficiency production of 5-hydroxyectoine using metabolically engineered Corynebacterium glutamicum

Abstract

Background Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing. Results Here, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectDPST achieved a titre of 74 g L−1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABCopt, was successfully achieved without intermediate purification. Conclusions C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production

“…All bioreactor experiments involved two biological replicates. The latter was decided considering the high reproducibility of duplicate bioreactor fermentations previously demonstrated by us ,,− and others − using the same experimental setup. In addition, four operating reactors appear to be the maximum to ensure fast sampling and high-quality sample processing (including quenching, extraction, and storage), given the various analyses run in parallel.…”

Complete genome sequence of a haloalkaliphilic heterotrophic bacterium Halomonas salifodinae IM328

“…The sample was diluted and spread on HM-3.6 solid medium plates containing the following (g/L): sodium chloride 36, potassium chloride 2, magnesium sulfate heptahydrate 1, calcium chloride 0.27, sodium bromide 0.23, sodium bicarbonate 0.06, peptone 5, yeast extract 10, ferric chloride 0.001, pH 8.5 (adjusted with NaOH), and agar 1.5% (wt/vol) for solid medium. As a halophilic bacterium that could reach high optical density at 600 nm (above 2.0 with flask) and tolerate relatively high salinity, IM328 has the potential for a high yield of compatible solutes such as ectoine ( 2 ) and betaine ( 3 ). Since extremophiles have become a popular chassis for industrial bioproduction of high-value chemicals such as hydroxyectoine ( 4 ), which is costly to produce through chemical catalysis, complete genome sequencing is required for proper genetic modification in H. salifodinae IM328.…”

Rational engineering of Halomonas salifodinae to enhance hydroxyectoine production under lower-salt conditions

“…Hybrid biological-inorganic systems were also employed for the production of hydroxyectoine from CO 2 in halotolerant hydrogen-oxidizing Achromobacter xylosoxidans and Mycolicibacterium mageritense , which produced 208.77 µg/L and 9.49 µg/L hydroxyectoine at a salinity equivalent to seawater, respectively (Feng et al 2023 ). In addition, by heterologously expressing of codon-optimized ectD from P. stutzeri A1501, the recombinant Corynebacterium glutamicum produced 74 g/L hydroxyectoine from ectoine, representing the highest hydroxyectoine titer reported so far (Jungmann et al 2022 ). However, these processes still require high temperature sterilization, as these chassis cells are unable to survive in extreme conditions that can effectively prevent the growth of unwanted microorganisms.…”