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. De novo 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

5-氨基戊酸 (AVA) 是一种具有巨大商业价值的平台化学品，可用于衍生尼龙 5 和五碳衍生物，如 δ-戊内酰胺、1,5-戊二醇、戊二酸和 5-羟基戊酸。使用代谢工程细胞工厂从头生物生产 合成AVA 被认为是以可持续方式提供这种化学物质的示例性途径。到目前为止，这条路线受到低滴度、速率和产量的限制，并且副产物含量高。为了克服这些限制，我们开发了一个新的 AVA 家族，生产谷氨酸棒杆菌细胞工厂。逐步优化包括 (i) 通过平衡恶臭假单胞菌的异源davBA基因的表达来改善 AVA 生物合成，（ii）通过破坏分解代谢的γ-氨基丁酸途径减少副产物戊二酸的形成（iii），增加AVA输出，和（iv）通过天然和异源转运蛋白减少AVA再输入，以解释细胞内的积累AVA 高达 300 毫米。经过几轮优化获得的菌株谷氨酸棒杆菌AVA-5A在补料分批过程中产生了48.3 g L ^{-1 AVA，并实现了0.21 g g -1}的高产率。令人惊讶的是，突变体在后期突然积累了相当于形成的AVA量的30%的戊二酸，是早期过程的十倍，对工业化生产显示出严重的缺陷。进一步的探索导致发现 ArgD，自然胺化 N-乙酰-ll-精氨酸生物合成过程中的-鸟氨酸在 AVA 上表现出对戊二酸形成的脱氨基副活性。由于细胞内 AVA 水平高，并且 ArgD 不再关注生产过程中合成代谢的逐渐增强关闭，这种混杂变得相关。在缺乏argD的高级菌株 AVA-6A、AVA-6B 和 AVA-7 中，戊二酸的形成被有利地消除了。在分批补料过程中，谷氨酸棒杆菌AVA-7以 0.34 g g ^-1的产量和 1.52 g L ^-1 h ^-1的最大产率产生 46.5 g L ^{-1 AVA}，超越了之前报道的所有努力，并为 AVA 的工业制造迈出了里程碑。值得注意的是，新型细胞工厂完全基于基因组，提供高遗传稳定性且不需要选择标记