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

5-aminovalerate (AVA) is a platform chemical of substantial commercial value to derive nylon-5 and five-carbon derivatives like δ-valerolactam, 1,5-pentanediol, glutarate, and 5-hydroxyvalerate. Denovo bio-production synthesis of AVA using metabolically engineered cell factories is regarded as exemplary route to provide this chemical in a sustainable way. So far, this route is limited by low titers, rates and yields and suffers from high levels of by-products. To overcome these limitations, we developed a novel family of AVA producing C. glutamicum cell factories. Stepwise optimization included (i) improved AVA biosynthesis by expression balancing of the heterologous davBA genes from P. putida, (ii) reduced formation of the by-product glutarate by disruption of the catabolic y-aminobutyrate pathway (iii), increased AVA export, and (iv) reduced AVA re-import via native and heterologous transporters to account for the accumulation of intracellular AVA up to 300 mM. Strain C. glutamicum AVA-5A, obtained after several optimization rounds, produced 48.3 g L-1 AVA in a fed-batch process and achieved a high yield of 0.21 g g-1. Surprisingly in later stages, the mutant suddenly accumulated glutarate to an extent equivalent to 30% of the amount of AVA formed, tenfold more than in the early process, displaying a severe drawback toward industrial production. Further exploration led to the discovery that ArgD, naturally aminating N-acetyl-l-ornithine during l-arginine biosynthesis, exhibits deaminating side activity on AVA towards glutarate formation. This promiscuity became relevant because of the high intracellular AVA level and the fact that ArgD became unoccupied with the gradually stronger switch-off of anabolism during production. Glutarate formation was favorably abolished in the advanced strains AVA-6A, AVA-6B, and AVA-7, all lacking argD. In a fed-batch process, C. glutamicum AVA-7 produced 46.5 g L-1 AVA at a yield of 0.34 g g-1 and a maximum productivity of 1.52 g L-1 h-1, outperforming all previously reported efforts and stetting a milestone toward industrial manufacturing of AVA. Notably, the novel cell factories are fully genome-based, offering high genetic stability and requiring no selection markers

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

In the present work, we established the bio-based production of glutarate, a carbon-5 dicarboxylic acid with recognized value for commercial plastics and other applications, using metabolically engineered Corynebacterium glutamicum. The mutant C. glutamicum AVA-2 served as a starting point for strain development, because it secreted small amounts of glutarate as a consequence of its engineered 5-aminovalerate pathway. Starting from AVA-2, we overexpressed 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase (gabD) under the control of the constitutive tuf promoter to convert 5-aminovalerate further to glutarate. The created strain GTA-1 formed glutarate as a major product, but still secreted 5-aminovalerate as well. This bottleneck was tackled at the level of 5-aminovalerate re-import. The advanced strain GTA-4 overexpressed the newly discovered 5-aminovalerate importer NCgl0464 and formed glutarate from glucose in a yield of 0.27 mol mol−1. In a fed-batch process, GTA-4 produced more than 90 g L−1 glutarate from glucose and molasses based sugars in a yield of up to 0.70 mol mol−1 and a maximum productivity of 1.8 g L−1 h−1, while 5-aminovalerate was no longer secreted. The bio-based glutaric acid was purified to >99.9% purity. Interfacial polymerization and melt polymerization with hexamethylenediamine yielded bionylon-6,5, a polyamide with a unique structure

Systems metabolic engineering of Corynebacterium glutamicum for the production of L-lysine and ectoine

The soil bacterium Corynebacterium glutamicum has gained tremendous industrial interest, for the biotechnological production of L-lysine and L-lysine derived products. This work assessed the cellular function of the L-lysine producer C. glutamicum LYS-12 at high temperature. The interpretation of transcriptome and proteome- data together with previously generated fluxome data, provided a detailed picture of the regulatory adaption of the cells to 38°C and suggested potential targets for metabolic engineering. In line, the duplication of the lysGE gene cluster increased the L-lysine yield by 7% to 460 mmol mol-1. Additionally, the synthesis of the valuable compatible solute ectoine was optimized in C. glutamicum via transcriptional balancing of the heterologous ectoine cluster. The shuffling of synthetic promoter and spacer elements yielded a library of the terminal ectoine pathway. Strongly increased production was enabled by regulatory elements of medium strength, where the increased expression of the gene ectB, as compared to ectA and ectC, appeared crucial. The most advanced ectoine producer C. glutamicum ectABCopt achieved 65 g L-1 ectoine in a fed-batch process. In addition, transcriptome profiling of ectoine producing C. glutamicum revealed a yet unknown export mechanism for ectoine, an interesting target for further metabolic engineering.Das Gram-positive Bodenbakterium Corynebacterium glutamicum ist von großem industriellem Interesse und insbesondere für die biotechnologische Herstellung von L-Lysin und L-Lysin– verwandten Produkten von Bedeutung. Diese Arbeit befasst sich mit der Untersuchung des L-Lysin Produzenten C. glutamicum LYS-12, kultiviert bei erhöhten Temperaturen. Die Integration von Transkriptom-, Proteom- und bereits im Vorfeld generierten Fluxom-Daten, ergab neue Einsichten in die Adaptierung der Zellen an 38°C und brachte neue Strategien für die Stammoptimierung hervor. Die Verdopplung des Gen-Clusters lysGE führte zu einer Steigerung der L-Lysin Ausbeute um 7% auf 460 mmol mol-1. Des Weiteren wurde der terminale heterologe Ectoin-Synthese-Weg in C. glutamicum auf der Ebene des Transkriptoms optimiert. Durch das zufällige Abwechseln von Promotoren und regulatorischen Elementen konnte eine Ectoin-Plasmid Bibliothek erstellt werden. Eine hohe Produktion wurde durch regulatorische Elemente mittlerer Stärke erreicht, wobei die erhöhte Expression des Gens ectB, verglichen mit ectA und ectC, von besonderer Bedeutung zu sein schien. Der beste Produzent C. glutamicum ectABCopt erreichte einen Ectoin-Titer von 65 g L-1 in einem Fed-Batch Prozess. Anschließende Untersuchungen des Transkriptoms eines Ectoin produzierenden C. glutamicum Stammes, führten zu der Identifikation eines bislang unbekannten Export-Mechanismus für Ectoin, was ein interessantes Ziel für zukünftige Stammoptimierungen darstellt

Anodic electro-fermentation: anaerobic production of L-Lysine by recombinant Corynebacterium glutamicum

Microbial electrochemical technologies (MET) are promising to drive metabolic processes for the production of chemicals of interest. They provide microorganisms with an electrode as an electron sink or an electron source to stabilize their redox and/or energy state. Here, we applied an anode as additional electron sink to enhance the anoxic metabolism of the industrial bacterium Corynebacterium glutamicum through an anodic electro-fermentation. In using ferricyanide as extracellular electron carrier, anaerobic growth was enabled and the feedback-deregulated mutant Corynebacterium glutamicum lysC further accumulated L-lysine. Under such oxidising conditions we achieved L-lysine titers of 2.9 mM at rates of 0.2 mmol/L/h. That titer is comparable to recently reported L-lysine concentrations achieved by anaerobic production under reductive conditions (cathodic electro-fermentation). However unlike other studies, our oxidative conditions allowed anaerobic cell growth, indicating an improved cellular energy supply during anodic electrofermentation. In that light, we propose anodic electro-fermentation as the right choice to support C. glutamicum stabilizing its redox and energy state and empower a stable anaerobic production of L-lysine. This article is protected by copyright. All rights reserved