Engineering of lysine cyclodeaminase conformational dynamics for relieving substrate and product inhibitions in the biosynthesis of l-pipecolic acid

Substrate and product inhibitions in enzyme-catalyzed reactions are major limitations in the preparative biosynthesis of valuable chemicals. In the present study, we described a modulation of the conformational dynamics of lysine cyclodeaminase from Streptomyces pristinaespiralis (SpLCD) for synchronously reducing substrate and product inhibitions. LCD is the key enzyme in the biosynthesis of piperidine derivatives, but incurs both severe substrate and product inhibitions due to steric hindrance by the narrow delivery tunnels of the substrate and product. Conformational dynamics studies via molecular simulations, which revealed the detailed atomic structures of both substrate and product delivery processes of SpLCD, indicated two separate intrinsic motions affected by the position of NAD ⁺ . Two key residues, Ile61 and Ile94, were observed to play a key role in regulating the shape of the substrate and product delivery processes. Via saturation mutagenesis studies, the Val61-Val94-SpLCD variant, which improved the K _M /k _cat , K _i -lys and K _i -LPA by 3.6, 19.4 and 9.2 times, respectively, was obtained. The structure analysis showed that the superior catalytic performance of the Val61-Val94-SpLCD variant is mainly due to the expanded substrate and product delivery tunnels when compared with the wild-type enzyme. By using recombinant Escherichia coli containing Val61-Val94-SpLCD as the whole-cell biocatalyst, a 2.5-fold higher substrate loading concentration with a total space-time yield of 0.83 g L ^-1 h ^-1 was achieved. Moreover, the l-pipecolic acid titer increased to 73.4 g L ^-1 without a decrease in yield, which was 4.2-fold higher than that achieved with the original recombinant whole-cell biocatalyst. The results of this study provide insights into the application of conformational dynamics of protein substrate and product delivery processes for simultaneously reducing both substrate and product inhibitions.