Systems metabolic engineering upgrades Corynebacterium glutamicum for selective high-level production of the chiral drug precursor and cell-protective extremolyte L-pipecolic acid.

The nonproteinogenic cyclic metabolite l-pipecolic acid is a chiral precursor for the synthesis of various commercial drugs and functions as a cell-protective extremolyte and mediator of defense in plants, enabling high-value applications in the pharmaceutical, medical, cosmetic, and agrochemical markets. To date, the production of the compound is unfavorably fossil-based. Here, we upgraded the strain Corynebacterium glutamicum for l-pipecolic acid production using systems metabolic engineering. Heterologous expression of the l-lysine 6-dehydrogenase pathway, apparently the best route to be used in the microbe, yielded a family of strains that enabled successful de novo synthesis from glucose but approached a limit of performance at a yield of 180 mmol mol-1. Detailed analysis of the producers at the transcriptome, proteome, and metabolome levels revealed that the requirements of the introduced route were largely incompatible with the cellular environment, which could not be overcome after several further rounds of metabolic engineering. Based on the gained knowledge, we based the strain design on l-lysine 6-aminotransferase instead, which enabled a substantially higher in vivo flux toward l-pipecolic acid. The tailormade producer C. glutamicum PIA-7 formed l-pipecolic acid up to a yield of 562 mmol mol-1, representing 75% of the theoretical maximum. Ultimately, the advanced mutant PIA-10B achieved a titer of 93 g L-1 in a fed-batch process on glucose, outperforming all previous efforts to synthesize this valuable molecule de novo and even approaching the level of biotransformation from l-lysine. Notably, the use of C. glutamicum allows the safe production of GRAS-designated l-pipecolic acid, providing extra benefit toward addressing the high-value pharmaceutical, medical, and cosmetic markets. In summary, our development sets a milestone toward the commercialization of biobased l-pipecolic acid.

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

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.

Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus.

The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Šresolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Šresolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications.

Topical Ectoine Application in Children and Adults to Treat Inflammatory Diseases Associated with an Impaired Skin Barrier: A Systematic Review.

INTRODUCTION: Inflammatory skin diseases are a significant burden on affected patients. Inflammation is caused by various stress factors to the epidermis resulting in, e.g., dryness, redness, and pruritus. Emollients are used in basic therapy to restore the natural skin barrier and relieve symptoms. A systematic review was performed to evaluate the efficacy and safety of ectoine-containing topical formulations in inflammatory skin diseases characterized by an impaired skin barrier. METHODS: A systematic review was carried out in PubMed, the Cochrane Library, and Microsoft Academic up to October 2021. Inclusion criteria were ectoine-containing topical formulations (creams, emollients) used for (adjuvant) therapy of inflammatory skin diseases. Clinical studies of any design published in any language were included. RESULTS: A total of 230 references were screened for eligibility, of which six were selected for inclusion in the review (five studies on atopic dermatitis and one study on prevention and management of retinoid dermatitis). The application of topical formulations containing 5.5-7.0% ectoine positively influenced skin dryness and, consequently, pruritus and dermatitis-specific scores in patients with atopic dermatitis. Especially in infants and children, who belong to the most frequently affected group, the formulations were well-tolerated when applied for up to 4 weeks. In studies where ectoine was used as an adjuvant therapy, application was associated with a decreased need for pharmacological therapy and also improved the effectiveness of, e.g., topical corticosteroids. In patients undergoing isotretinoin therapy, ectoine was as effective as dexpanthenol in reducing retinoid dermatitis or improving symptoms. CONCLUSION: Ectoine is an effective natural substance with an excellent tolerability and safety profile, representing a beneficial alternative as basic therapy or to increase the efficacy of the pharmacological treatment regimen for patients with inflammatory skin diseases, including infants and children.

Metabolic Engineering of Corynebacterium glutamicum for High-Level Ectoine Production: Design, Combinatorial Assembly, and Implementation of a Transcriptionally Balanced Heterologous Ectoine Pathway.

Ectoine is formed in various bacteria as cell protectant against all kinds of stress. Its preservative and protective effects have enabled various applications in medicine, cosmetics, and biotechnology, and ectoine therefore has high commercial value. Industrially, ectoine is produced in a complex high-salt process, which imposes constraints on the costs, design, and durability of the fermentation system. Here, Corynebacterium glutamicum is upgraded for the heterologous production of ectoine from sugar and molasses. To overcome previous limitations, the ectoine pathway taken from Pseudomonas stutzeri is engineered using transcriptional balancing. An expression library with 185,193 variants is created, randomly combining 19 synthetic promoters and three linker elements. Strain screening discovers several high-titer mutants with an improvement of almost fivefold over the initial strain. High production thereby particularly relies on a specifically balanced ectoine pathway. In an optimized fermentation process, the new top producer C. glutamicum ectABCopt achieves an ectoine titer of 65 g L-1 and a specific productivity of 120 mg g-1 h-1 . This process is the first reported example of a simple fermentation process under low-salt conditions using well-established feedstocks to produce ectoine with industrial efficiency. There is a compelling case for more intensive implementation of transcriptional balancing in future metabolic engineering of C. glutamicum.