Metabolic engineering of Corynebacterium glutamicum for the high-level production of valerolactam, a nylon-5 monomer.

ABSTRACT

Valerolactam (VL) is an important precursor chemical for nylon-5 and nylon 6,5. It has been produced by petroleum-based route involving harsh reaction conditions and generating toxic wastes. Here, we report the complete biosynthesis of VL by metabolically engineered Corynebacterium glutamicum overproducing L-lysine. The pathway comprising L-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) from Pseudomonas putida, and ß-alanine CoA transferase (act) from Clostridium propionicum was introduced into the C. glutamicum GA16 strain. To increase the VL flux, competitive pathways predicted from sRNA knockdown target screening were deleted. This engineered C. glutamicum strain produced VL as a major product, but still secreted significant amount of its precursor, 5-aminovaleric acid (5AVA). To circumvent this problem, putative 5AVA transporter genes were screened and engineered in the genome, thereby reuptaking 5AVA excreted. Also, multiple copies of the act gene were integrated into the genome to strengthen the conversion of 5AVA to VL. The final VL10 (pVL1) strain was constructed by enhancing glucose uptake system, which produced 9.68 g/L of VL in flask culture. Fed-batch fermentation of the VL10 (pVL1) strain produced 76.1 g/L of VL from glucose with the yield and productivity of 0.28 g/g and 0.99 g/L/h, respectively, showcasing a high potential for bio-based production of VL from renewable resources.