A bio-based route to the carbon-5 chemical glutaric acid and to bionylon-6,5 using metabolically engineered<i>Corynebacterium glutamicum</i>

Rohles, Christina Maria; Gläser, Lars; Kohlstedt, Michael; Gießelmann, Gideon; Pearson, Samuel; Campo, Aránzazu del; Becker, Jörg; Wittmann, Christoph

doi:10.1039/c8gc01901k

Cited by 85 publications

(67 citation statements)

References 82 publications

(100 reference statements)

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“…The concentrated feed was added pulsewise afterwards. This type of feeding is typically used during the industrial fermentation of C. glutamicum and has proven most valuable to achieve high production of, e.g., l ‐lysine, aminovalerate, and glutarate . During the feed phase, the ectoine level continuously increased from 20 g L −1 at the end of the batch phase to a final titer of more than 65 g L −1 after 56 h. The cells produced ectoine almost exclusively.…”

Section: Resultsmentioning

confidence: 99%

“…As shown, transcriptional balancing of the ectoine pathway in C. glutamicum provides the high‐value product at industrial efficiency, using the engineered cell factory in a simple, low‐salt fermentation process on well‐established feedstocks. In this regard, the created ectoine producer follows C. glutamicum cell factories previously described for the production of amino acids, amino acid‐related compounds, organic acids, and alcohols and continues the success story of the microbe as an industrial production host. Thus, transcriptional balancing boosted the flux through the ectoine pathway.…”

Section: Discussionmentioning

confidence: 99%

“…Shake flask cultivations were conducted as previously described . For screening purpose, the Biolector (m2p‐labs; Baesweiler, Germany) was used .…”

Section: Methodsmentioning

confidence: 99%

“…The fed‐batch production of ectoine was conducted in stirred bioreactors (DASGIP AG, Jülich, Germany) . Details are given in the Supporting Information.…”

Section: Methodsmentioning

confidence: 99%

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

Gießelmann

Dietrich

Jungmann

et al. 2019

Biotechnology Journal

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Shake flask cultivations were conducted as previously described . For screening purpose, the Biolector (m2p‐labs; Baesweiler, Germany) was used .…”

Section: Methodsmentioning

confidence: 99%

“…The fed‐batch production of ectoine was conducted in stirred bioreactors (DASGIP AG, Jülich, Germany) . Details are given in the Supporting Information.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Gießelmann

Dietrich

Jungmann

et al. 2019

Biotechnology Journal

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“…Microbial cell factories are a key to the bio-based industry [1]. Upgrading and streamlining their biocatalytic activity through systems metabolic engineering requires detailed understanding of the underlying metabolism [2][3][4][5]. Among other techniques, the assessment of intracellular metabolite levels and pathway fluxes, has proven valuable to understand metabolic network function and its regulation and derive novel targets for strain engineering [1,6].…”

Section: Introductionmentioning

confidence: 99%

A common approach for absolute quantification of short chain CoA thioesters in industrially relevant gram-positive and gram-negative prokaryotic and eukaryotic microbes

Gläser

Kuhl

Jovanović

et al. 2020

Preprint

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Background: Thioesters of coenzyme A participate in 5% of all enzymatic reactions and at least one third of all cellular carbon is typically metabolized through a CoA thioester. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5 – 10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance these metabolites explains the high interest to trace and quantify them in microbial cells.Results: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach detected CoA thioesters down to the level of 40 attomole and exhibited high precision and reproducibility for all microbes as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA-spectra. A succinyl-CoA synthase defective mutant of C. glutamicum, exhibited an unaffected level of succinyl-CoA, which indicated a complete compensation of the l-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the microbe. S. albus revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. Conclusions: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use on single protocol promises to facilitate automatized efforts, which rely on standardized workflows.

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Metabolic Engineering of Corynebacterium glutamicum

Becker

Wittmann

2021

Metabolic Engineering

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A bio-based route to the carbon-5 chemical glutaric acid and to bionylon-6,5 using metabolically engineeredCorynebacterium glutamicum

Abstract: Here, we present a green route to glutaric acid, a chemical of recognized industrial value, using a tailor-made cell factory.

Cited by 85 publications

References 82 publications

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

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

A common approach for absolute quantification of short chain CoA thioesters in industrially relevant gram-positive and gram-negative prokaryotic and eukaryotic microbes

Metabolic Engineering of Corynebacterium glutamicum

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