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 five-carbon platform chemicals

The increasing demand for plastics along with environmental concerns, drive the development of sustainable alternatives to petrochemically derived polymers: production of chemicals in biorefineries with stream lined microbial cell factories. In this regard, the microbe Corynebacterium glutamicum has evolved into an excellent production host throughout the years, due to its broad substrate and product spectrum. This work focused on systems metabolic engineering of C. glutamicum towards de novo production of 5 aminovalerate and glutarate, two promising building blocks for bioplastics. In a first step, conversion of 5 aminovalerate and the elimination of by-product formation were addressed. Additionally, the 5-aminovalerate export was enhanced via integration of the 4 aminobutyrate export protein (PP2911) from Pseudomonas putida. The final strain C. glutamicum AVA 6 produced 47 g L-1 5 aminovalerate in a fed batch fermentation process, without any side-product accumulation. High-level production of glutarate was subsequently achieved via extension of the endogenous 4 aminobutyrate pathway. The re assimilation of secreted 5 aminovalerate led to the establishment of the novel GTA 3 strain, exhibiting a titer of 90 g L 1 glutarate in a fed batch process. Finally, the complete value chain was proven by purification of the bio based glutarate and subsequent polymerization into a novel bionylon 6,5. The investigation of the polymer characteristics revealed unique material properties.Der stetig wachsende Plastikverbrauch und das steigende Umweltbewusstsein führen zur Entwicklung nachhaltiger Alternativen zu petrochemischen Polymeren wie z.B. der Produktion von Chemikalien in Bioraffinerien mittels mikrobieller Zellfabriken. In diesem Zusammenhang hat sich Corynebacterium glutamicum durch sein breites Substrat- und Produktspektrum zu einem exzellenten Produktionsorganismus entwickelt. Diese Arbeit befasst sich mit der de novo Herstellung von 5-Aminovalerat und Glutarat durch C. glutamicum, zwei vielversprechende Bausteine für die Bioplastikproduktion. Zunächst wurde die Degradierung von 5 Aminovalerat und die Produktion von Nebenprodukten eliminiert. Der Produktexport wurde durch Integration des 4-Aminobutyrat Transporters (PP2911) aus Pseudomonas putida verbessert. Der finale Stamm C. glutamicum AVA-6 produzierte fermentativ 47 g L 1 5 Aminovalerat. Anschließend wurde die Glutaratproduktion durch Erweiterung des endogenen 4 Aminobutyrat Stoffwechselweges optimiert. Die gesteigerte Wiederaufnahme von sekretiertem 5 Aminovalerat des neuen Stammes C. glutamicum GTA-3 ermöglichte die Produktion von 90 g L 1 Glutarat in einem Fermentationsprozess. Die komplette Wertschöpfungskette wurde durch Aufreinigung des bio-basierten Glutarats und anschließender Polymerisierung zu Bio-Nylon 6,5 vervollständigt. Die Materialeigenschaften des neuen Polymers erwiesen sich als bisher einzigartig