Breeding Of Nicotinate Dehydrogenase-producing Strain And Its Application In 6-hydroxynicotinic Acid Biosynthesis

6-Hydroxynicotinic acid is a high-value pharmaceutical intermediate,widely used in biomedical,dyeing,medical beauty and material synthesis industries.6-Hydroxynicotinic acid could be achieved directly from nicotinic acid in one step by biotransformation of nicotinate dehydrogenase,which was efficient and environmentally-friendly.However,low enzyme activity and low 6-hydroxynicotinic acid yields limit the large-scale industrial development of nicotinate dehydrogenase.In this study,nicotinate dehydrogenase producing strains could be rapidly screened out by the method of ultraviolet development and enzyme activity determination.The selected strain was applied to the biosynthesis of 6-hydroxynicotinic acid,with the product purified and characterized.In order to further improve the activity of nicotinate dehydrogenase,atmospheric and room temperature plasma（ARTP）mutation breeding system and high-throughput microbial clone screening system were employed.Fermentation conditions of the mutant was optimized to further improve the activity of nicotinate dehydrogenase and the potential of producing6-hydroxynicotinic acid.The nitrilase-nicotinate dehydrogenase cascade system was established to produce 6-hydroxynicotinic acid with 3-cyanopyridine as the substrate,which would reduce the production costs.Finally,composite immobilization of the mutant was employed to improve its ability to continuously transform 3-cyanopyridine into6-hydroxynicotinic acid.The main researches were as follows:（1）Screening of nicotinate dehydrogenase-producing strain and verifying its conversion potential.In this study,strains were isolated from the soil samples that were collected from different locations of Jiangsu Province.A nicotinate dehydrogenase producing strain Pseudomonas putida S14 was isolated and identified,based on the 6-hydroxynicotinic acid standards development figure and a method of 96-well plates nicotinate dehydrogenase activity determination.The activity of nicotinate dehydrogenase towards nicotinic acid could reach up to 1.11 U·m L^-1 and the yield of 6-hydroxynicotinic acid accumulated to 176 g·L^-1within 30 h with a space time yield of 1.36 g·（g·h）^-1.（2）Mutagenesis and high-throughput screening for high-yielding strains and fermentation process optimization.The random mutagenesis libraries of P.putida S14 were constructed by ARTP system to improve the activity of nicotinate dehydrogenase.When the mutation treatment time was 10 s,the lethality rate reached to 92.16%.The mutant P.putida S14 mut-5 was obtained by the high-throughput screening strategy.Its activity was 2.68U·m L^-1 and 2.4-fold that of the wild type.Using mut-5 free cells as the catalyst,244.9 g·L^-16-hydroxynicotinic acid could be accumulated within 38 hours,which was 139%of the wild-type transformation yield.After optimization of fermentation conditions,the results were as follows:the best carbon source was 20 g·L^-1 glycerin,the best nitrogen source was 10 g·L^-1corn steep liquor,and the best inducer concentration was 30 g·L^-1 nicotinic acid.The optimal initial p H was 7.0,the optimal fermentation temperature was 30℃ and the optimal inoculum amount was 30 g·L^-1.In addition,4.0 g·L^-1 magnesium sulfate could promote enzyme activity.After the optimization,the enzyme activity was further increased by 2.2 times.（3）Construction of a nitrilase-nicotinate dehydrogenase cascade system with Pseudomonas putida mut-D3 and P.putida S14 mut-5.In order to further reduce production costs,a nitrilase-nicotinate dehydrogenase cascade system was established,using cheaper3-cyanopyridine as the substrate.Via this cascade system,54.5 g·L^-1 6-hydroxynicotinic acid could be achieved within 585 min from 3-cyanopyridine.Sodium alginate（SA）and polyvinyl alcohol（PVA）were mixed for the immobilization of P.putida S14 mut-5.The concentrations of materials were optimized:1.0%SA,8.0%PVA and 0.6%Ca Cl₂.In addition,in order to further improve the transformation potential of immobilized cells,inorganic materials were added for composite immobilization,inclouding Si O₂,Ca CO₃,iron powder and bentonite.The results showed that 1.0%Si O₂ was the best additives.84.1 g·L^-16-hydroxynicotinic acid was accumulated within 820 min by the composite immobilized cells of mut-D3 and mut-5.The yield was increased 54%compared with free cells.