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

Abstract

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.

Keywords: Arginine; Bioeconomy; Corynebacterium glutamicum; Export; Fed-batch; GABA permease; Glutarate; Import; Lysine; Pseudomonas putida KT2440; Systems metabolic engineering.