Metabolic engineering of Corynebacterium glutamicum for high-yield de novo biosynthesis of 5-aminovaleramide, a promising bio-based monomer

Abstract

Background: Lysine-derived C5 compounds are important intermediates in cellular metabolism and promising building blocks for sustainable polymer chemistry. Among these, 5-aminovalerate (5-AVA) has been extensively studied as a platform chemical produced via a two-step microbial pathway. However, its direct precursor, 5-aminovaleramide (5-AVD), generated from lysine by lysine 2-monooxygenase, remains largely unexplored. Notably, 5-AVD is an attractive product in its own right, as it provides a versatile intermediate for the synthesis of polyamides and other nitrogen-containing chemicals. Here, we establish the first de novo microbial production of 5-AVD by systematically engineering Corynebacterium glutamicum for optimized precursor flux, product export, and redox balance. Results: Trace secretion of 5-AVD was discovered in 5-AVA-producing strains, and tolerance studies showed that C. glutamicum can withstand high 5-AVD concentrations. To exploit this trait, the lysine-producing strain LYS-12 was engineered to express the davB gene from Pseudomonas putida under the constitutive tuf promoter, resulting in increased 5-AVD secretion. Pathway analysis revealed that the native exporter LysE is essential for efficient 5-AVD export, while heterologous GABA permeases provided no benefit. Mechanistic analysis further showed that LysE preferentially exports lysine over 5-AVD, establishing it as a flux gatekeeper that critically shapes product selectivity. Overexpression of heterologous NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) enhanced NADPH supply and improved redox balance, increasing the 5-AVD yield to 0.32 mol mol−1 in strain AVD-11. In fed-batch fermentation, AVD-11 reached a maximum productivity of 1.2 g L−1 h−1 and a final titer exceeding 36 g L−1 with > 97% selectivity, while chromosomally integrated davB remained genetically stable throughout the process. Conclusions: This study establishes C. glutamicum as a robust and industrially relevant platform for the sustainable production of 5-AVD. By combining rational pathway design, transporter control, and cofactor engineering, we deliver the first high-yield microbial route to this valuable amide and provide a blueprint for expanding the portfolio of lysine-derived monomers accessible through microbial cell factories.