High-throughput screening of a nicotinate dehydrogenase producing Pseudomonas putida mutant for efficient biosynthesis of 6-hydroxynicotinic acid

Abstract A nicotinate dehydrogenase producing strain was isolated and identified to be Pseudomonas putida, which could bio-transform nicotinic acid into 6-hydroxynicotinic acid. The activity of nicotinate dehydrogenase towards nicotinic acid could reach up to 1.11 U/mL and the yield of 6-hydroxynicotinic acid accumulated to 176 g/L within 30 h with a space time yield of 1.36 g/(g•h). In order to further improve its catalytic potential, a mutant library was constructed through atmospheric and room temperature plasma (ARTP) and rapidly screened by a high-throughput screening strategy, which obtained a mutant strain mut-5 with activity of 2.68 U/mL. Via developing a nitrilase-nicotinate dehydrogenase enzymatic cascade system, 54.5 g/L 6-hydroxynicotinic acid could be obtained within 585 min with 3-cyanopyridine as the substrate, which would further reduce the production costs. The results indicated that the nicotinate dehydrogenase (separately or combining with nitrilase) could serve as a superior catalyst for 6-hydroxynicotinic acid production. To our knowledge, the nicotinate dehydrogenase activity reported in this study was the highest level in literature to date.