透明颤菌vgb基因在脱氮假单胞菌中的表达及对维生素B12合成的碳中心代谢流分析

在为维生素B₁₂生产菌株脱氮假单胞菌确立合适的接合转移操作条件的基础上，通过单交换的方式，将vgb基因整合到脱氮假单胞菌染色体上，获得了vgb重组菌株Pvgb-16，并通过¹³C同位素标记实验，探索VHb蛋白对脱氮假单胞菌碳中心代谢流变化和维生素B₁₂合成的影响。研究结果表明，在相同的供氧条件下，vgb重组菌株Pvgb-16拥有更高的比生长速率和比产物合成速率，与出发菌株相比分别提升了22%和52%。碳代谢通量分布分析表明，vgb重组菌株Pvgb-16的PP途径改善，提升了NADPH合成通量；甘氨酸由甜菜碱合成的通量上升，促进了前体物质氨基乙酰丙酸的合成，进一步加速维生素B₁₂的合成。总体来看，含vgb基因的重组菌株与出发菌株相比在促进菌体的生长、维生素B₁₂的合成速率及得率上都有显著效果，对进一步的发酵生产应用研究具有重要意义。

Expression of Vitreosicilla Hemoglobin Gene(vgb) In Pseudomonas denitrificans and the Central Carbon Metabolic Flux Analysis on Vitamin B12 Production

Based on the establishment of the conditions of conjugational transfer of Pseudomonas denitrificans, vgb gene was successfully integrated into the chromosome of P. denitrificans via single crossover to obtain vgb recombinant strain Pvgb-16, and its effects on the carbon center metabolic flux and vitamin B₁₂ biosynthesis were illuminated through the ¹³C isotope labeling experiment. The results demonstrated that the recombinant strain with vgb gene had the higher specific growth rate and specific vitamin B₁₂ production rate, which was increased by 22% and 52% respectively, compared to the original strain under the same oxygen supply condition. In addition, cultivations were carried out with 20% or 20% glucose as substrates, the labeling patterns of the proteinogenic amino acids were used to accurately estimate the fluxes in the central carbon metabolism of P. denitrificans. The results showed that the glucose consumption rate of recombinant strain was enhanced for 14.2%, and the further carbon metabolic flux distribution analysis revealed that the improvement of the PP pathway for higher NADPH generation and the biosynthesis rate of glycine from betaine for higher synthesis of precursor aminolevulinic acid greatly enhanced vitamin B₁₂ biosynthesis rate and productivity in the vgb recombinant strain. It can be concluded that the expression of vgb gene in P. denitrificans can effectively promote the cell growth rate, the accumulation rate and yield of vitamin B₁₂ production, which would be of great significance for the further research and application in industrial fermentation.