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 ectD^PST 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 ectABC^opt, 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.

Keywords: 5-Hydroxyectoine; Biotransformation; Corynebacterium glutamicum; Ectoine; Ectoine hydroxylase; Extremolyte; High-value product; Intracellular metabolite; Proline; Trehalose.

Fig. 1

Genetic and metabolic engineering strategies to produce ectoine and hydroxyectoine in C. glutamicum. As part of the l-lysine biosynthetic pathway, l-aspartate is converted into l-aspartate-semialdehyde, involving aspartokinase (Ask; EC: 2.7.2.4) and l-aspartyl phosphate dehydrogenase (Asd; EC: 1.2.1.11). Synthesis of ectoines branches off from the l-aspartate-semialdehyde pool. First, l-2,4-diaminobutyrate transaminase (EctB; EC: 2.6.1.76) catalyzes the transamination to l-2,4-diaminobutyrate. Then, l-2,4-diaminobutyrate acetyltransferase (EctA; EC: 2.3.1.178) acetylates the intermediate to N-acetyl-2,4-diaminobutyrate. Finally, ectoine is cyclized by ectoine synthase (EctC; EC: 4.2.1.108). A fourth enzyme, i. e. ectoine hydroxylase (EctD; EC: 1.14.11), further converts ectoine into 5-hydroxyectoine via O₂-dependent hydroxylation, simultaneously decarboxylating the co-substrate α-ketoglutarate. Previously, over-production of a mixture of ectoine and 5-hydroxyectoine in engineered C. glutamicum ECT-2 [14] was achieved by genomic integration of the (codon-optimized) ectABCD cluster from Pseudomonas stutzeri under control of the promoter P_tuf (a). Pure ectoine production in C. glutamicum ectABC^opt [15] was based on episomal monocistronic expression of the codon-optimized ect^ABC genes from P. stutzeri, each under control of a distinct promoter (P), a bicistronic element (B) and a terminator sequence (T), respectively (b). Metabolic engineering strategy of this work to convert ectoine to hydroxyectoine in a biotransformation set-up using recombinant C. glutamicum that expresses ectD under control of P_tuf (c). Experimental proof of hydroxyectoine production using the biotransformation set-up. The C. glutamicum type strain ATCC 13032 episomally expressed the codon optimized ectD gene from Pseudomonas stutzeri A1501 (PST) in the pClik 5a (pClik) and pCES-PLPV (pCES) vector. Cultures were grown at 30 °C on minimal glucose medium in a microbioreactor with different initial levels of supplied ectoine (1, 5, 10, 15 mM) and analyzed for growth (on-line measurement of OD₆₂₀) and the conversion of ectoine into 5-hydroxyectoine (final titers after depletion of glucose) (d). n = 2

Fig. 2

Screening for optimal 5-hydroxyectoine production in recombinant C. glutamicum. The C. glutamicum type strain ATCC 13032 episomally expressed the codon optimized ectD genes from Pseudomonas stutzeri A1501 (PST), Mycobacterium smegmatis ATCC 19420 (MSM), Streptomyces coelicolor A3(2) (SCO), Halomonas elongata ATCC 33173 (HEL) and Virgibacillus salexigens ATCC 700290 with amino acid exchanges A163C and S244C (VSA), in the pCES-PLPV vector. Cultures were grown at 30 °C on minimal glucose medium in a microbioreactor and analyzed for growth (on-line measurement of OD₆₂₀) and the conversion of ectoine into hydroxyectoine (final titers after depletion of glucose). Screening at different initial ectoine concentrations (a), and different temperatures (b, c). n = 2. Screening of additional EctD enzyme variants in C. glutamicum at 37 °C and 5 mM ectoine (d). For the latter, C. glutamicum episomally expressed the codon optimized ectD genes from Gracilibacillus sp. SCU50 (GSP), Acidiphilium cryptum JF5 (ACR), Alkalihalobacillus clausii 7520–2 (ACL), Hydrocarboniclastica marina KCTC 62334 (HMA), Methylomicrobium alcaliphilum DSM 19304 (MAL), Neptunomonas concharum LHW37 (NCO), Leptospirillum ferriphilum ML-04 (LFE), Candidatus Nitrosopumilus sp. AR2 (CNS), Sphingopyxis alaskensis DSM 13593 (SAL), Paenibacillus lautus E7593-69 (PLA) and Chromohalobacter salexigens DSM 3043 (CSA), in the pCES-PLPV vector. The cultures were grown at 37 °C on minimal glucose medium in a microbioreactor and analyzed for growth (on-line measurement of OD₆₂₀) and the conversion of 5 mM initial ectoine into hydroxyectoine (final titers after depletion of glucose). n = 3

Fig. 3

Kinetics and stoichiometry of 5-hydroxyectoine production in metabolically engineered strains of C. glutamicum. The C. glutamicum type strain ATCC 13032 episomally expressed the codon optimized ectD genes from Pseudomonas stutzeri A1501 (a, c), Mycobacterium smegmatis ATCC 19420 (b, e) and Virgibacillus salexigens ATCC 700290 with amino acid exchanges A163C and S244C (c, f) in the pCES-PLPV vector. All strains were cultivated in shake flasks on glucose minimal medium with 5 mM of ectoine at 37 °C. Dark grey circles show conversion based on extracellularly detectable ectoines. Light grey circles indicate conversion additionally considering intracellular levels of ectoines. n = 3

Fig. 4

Impact of ectoine and 5-hydroxyectoine on intracellular metabolite levels in C. glutamicum. The C. glutamicum type strain ATCC 13032, episomally expressed the codon-optimized ectD genes from Pseudomonas stutzeri A1501, Mycobacterium smegmatis ATCC 19420 and Virgibacillus salexigens ATCC 700290 with amino acid exchanges A163C and S244C in the pCES-PLPV vector. All strains were cultivated in shake flasks on glucose minimal medium supplemented with 5 mM ectoine at 37 °C. Cells were harvested in mid-exponential phase (10 h) and disrupted for the analysis of intracellular metabolite levels. As a control, C. glutamicum harboring the empty vector pCES-PLPV was treated equally with (control + ectoine) and without (control) the addition of ectoine to the medium. n = 3

Fig. 5

Production of 5-hydroxyectoine in stirred tank bioreactors using metabolically engineered C. glutamicum. The C. glutamicum type strain ATCC 13032 episomally expressed the codon optimized ectD genes from Mycobacterium smegmatis ATCC 19420 (a) and Pseudomonas stutzeri A1501 (b–d). Fed-batch-mode process at 37 °C with initial sucrose and ectoine concentrations of 100 g L⁻¹ and 60 g L⁻¹, and the addition of feed (600 g L⁻¹ sucrose, 15 g L⁻¹ yeast extract) at constant rate, respectively (a, b). Batch-mode process at 37 °C with initial sucrose and ectoine concentrations of 100 g L⁻¹ and 50 g L⁻¹ (c) and 150 g L⁻¹ and 75 g L.⁻¹, respectively (d). n = 2

Fig. 6

De-novo synthesis of hydroxyectoine using a two-step bioprocess. Step one: cultivation of the ectoine producer C. glutamicum ectABC^opt [15] on 10 g L⁻¹ glucose minimal medium at 30 °C (a). Step two: cultivation of C. glutamicum episomally expressing the codon optimized ectD gene from Mycobacterium smegmatis ATCC 19420 at 37 °C. The medium displayed the final broth from step 1, clarified from the ectoine-producing cells, neutralized to pH 7.4, and replenished with glucose (20 g L.⁻¹) (b). Final ectoine and hydroxyectoine titers of phase 2 before and after cell disruption (c). n = 3

Fig. 7

Benchmarking the newly developed 5-hydroxyectoine production process based on metabolically engineered C. glutamicum. Comparison of fed-batch processes using C. glutamicum to produce l-aspartate derived chemicals. Product titers, achieved after the initial batch-phase, are plotted against final titers, reached after the feed-phase. Blue circles: de novo production of l-lysine and products derived thereof [–26, 30, 48, 86]; light orange circles: de novo synthesis of ectoine [14, 16]; dark orange circles: bioconversion from ectoine (this work) (a). Comparison of 5-hydroxyectoine titers of various microbial producers, namely C. glutamicum P_tuf ectD^PST in a 1.5-fold (74.4 g L⁻¹) and a onefold batch (47.8 g L⁻¹) (this work), C. glutamicum ECT-2 (0.3 g L⁻¹) [14], E. coli HECT31 (14.9 g L⁻¹) [22], E. coli FF5169 pMP41 (1.9 g L⁻¹) [23], E. coli DH5α pASK ectABCDask (1.6 g L⁻¹) [87], H. salina BCRC17875 (2.9 g L⁻¹) [21] and H. polymorpha ALU3/EctABCD (2.8 g L⁻¹) [88]. Bioconversions are marked with stars