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Directed evolution of ornithine cyclodeaminase using an EvolvR-based growth-coupling strategy for efficient biosynthesis of L-proline.

L-proline takes a significant role in pharmaceutical and chemical industry as well as graziery. Typical biosynthesis of L-proline is from L-glutamate, involving three enzymes reaction as well as a spontaneous cyclisation. Alternatively, L-proline can be also synthesized in L-ornithine and/or L-arginine producing strains by an ornithine aminotransferase (OCD). In this study, a strategy of directed evolution combining rare codon selection and pEvolvR was developed to screen OCD with high catalytic efficiency, improving L-proline production from L-arginine chassis cells. The mutations were generated by CRISPR-assisted DNA polymerases and were screened by growth-coupled rare codon selection system. OCDK205G/M86K/T162A from Pseudomonas putida was identified with 2.85-fold increase in catalytic efficiency for the synthesis of L-proline. Furthermore, we designed and optimized RBS for the BaargI and Ppocd coupling cascade using RedLibs, as well as sRNA inhibition of argF to moderate L-proline biosynthesis in L-arginine overproducing Corynebacterium crenatum. The strain PS6 with best performance reached 15.3 g/L L-proline in the shake flask and showed a titer of 38.4 g/L in a 5-L fermenter with relatively low concentration of residual L-ornithine and/or L-arginine.