Systems metabolic engineering of Corynebacterium glutamicum eliminates all by-products for selective and high-yield production of the platform chemical 5-aminovalerate

Rohles, Christina Maria; Pauli, Sarah; Gießelmann, Gideon; Kohlstedt, Michael; Becker, Jörg; Wittmann, Christoph

doi:10.1016/j.ymben.2022.07.005

Cited by 16 publications

(10 citation statements)

References 67 publications

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“…Given the fact, that the immediate induction of the pedACD Cg cluster at process start apparently had no negative effects, we created the new strain CR099 pClik 5α P tuf pedACD Cg , an alternative producer that expressed the genes constitutively. For expression, we selected the promotor P tuf , the promotor of the gene of elongation factor TU, known to efficiently support gene expression in the microbe [ 59 – 61 ]. When grown under the same conditions, the constitutive producer performed well (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…5 ). Notably, C. glutamicum has been shown to be robust for the production of a range of demanding chemicals at high concentration, sometimes using quite toxic feedstocks [ 32 , 59 , 61 , 79 , 80 ]. The microbe performed well under suboptimal growth conditions such as oxygen limitation, required to synthetize lactate [ 81 ] and succinate [ 82 ], and acidic pH, promoting the production of diaminopentane [ 50 ].…”

Section: Discussionmentioning

confidence: 99%

“…Two novel pediocin PA-1 producers were constructed. C. glutamicum pClik P tuf pedACD Cg was derived by replacing the inducible promotor P tac by the constitutive P tuf promotor [ 61 ]. C. glutamicum pXMJ19 P tac pedA M31L CD Cg expressed a mutated pedA M31L gene that exhibited a substitution of l -methionine by l -leucine at position 31 [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

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High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

et al. 2023

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Background Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. Results Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L−1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. Conclusions The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

et al. 2023

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“…As shown here, the expression of a codon-optimized copy of ectD, encoding ectoine hydroxylase, in the wild type of C. glutamicum, supported by bioprocess development, enabled high-level production of hydroxyectoine. C. glutamicum is a well-known, safe, and industrially proven host [27,[43][44][45][46][47][48]. The microbe produces no endotoxins, does not undergo phage lysis, and is generally recognized as safe (GRAS), allowing the synthesis of a range of commercial products granted GRAS status by the United States Food and Drug Administration for the food and pharmaceutical industries [44], which appears as a great benefit towards the safe, industrialscale production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical application.…”

Section: Glutamicum P Tuf Ectd Pst Sets a Milestone Towards Industria...mentioning

confidence: 99%

“…Previously, our group has metabolically engineered the soil microbe C. glutamicum to overproduce different l -lysine-based chemicals, including l -lysine itself [ 24 ], diaminopentane [ 25 ], glutarate [ 26 ], 5-aminovalerate [ 27 ], and, last but not least, mixtures of ectoine and hydroxyectoine [ 14 ] as well as pure ectoine [ 15 ]. The latter two studies provided valuable proof-of-concept that C. glutamicum expresses heterologous proteins that convert l -aspartate semialdehyde, an intermediate of l -lysine biosynthesis [ 28 ], into the two ectoines (Fig.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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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.

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Recent advances in microbial production of diamines, aminocarboxylic acids, and diacids as potential platform chemicals and bio-based polyamides monomers

Son

Sohn

Baritugo

et al. 2023

Biotechnology Advances

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Systems metabolic engineering of Corynebacterium glutamicum eliminates all by-products for selective and high-yield production of the platform chemical 5-aminovalerate

Cited by 16 publications

References 67 publications

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

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

Recent advances in microbial production of diamines, aminocarboxylic acids, and diacids as potential platform chemicals and bio-based polyamides monomers

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