Substrate-Cofactor Synergistic Reorientation and Substrate Tunnel Engineering of SspLCD for Efficient l-Pipecolic Acid Biosynthesis

Homology screening and structural selection identified a novel lysine cyclodeaminase (SspLCD) with high substrate tolerance for l-pipecolic acid (l-PA) synthesis, rapamycin's key intermediate. Mutant M4 (I61V/I94V/I233V/A235S), developed via substrate-cofactor reorientation and tunnel engineering, exhibited 87.3-fold higher catalytic efficiency than the wild-type. At 1,000 mM substrate loading, M4 achieved >99.9% conversion in 3 h, yielding 1,033 g·L-1·day-1─the highest reported l-PA space-time yield to date. This study establishes an industrially viable pathway for efficient l-PA bioproduction.