The Construction Of Genetic Recombinants Of P. Denitrificans And The Optimization Of Industrial Vitamin B₁₂ Fermentation Process

As the cofactors of methyltransferase, vitamin B₁₂ can enhance the metabolism of nutrition as well as improve the growth and maturity of erythrocyte, which is important to the hematopoietic system. It is proved to have the function of preventing pernicious anemia and maintaining the health of the nervous system. Therefore, with the development of human's life, the need of vitamin B₁₂ will increase gradually and how to improve the productivity and quality of vitamin B₁₂ is the question that should be solved urgently. The present research focused on the aerobic fermentation of vitamin B₁₂ by the Pseudomonas denitrificans. In this study, the strain with the potential of high vitamin B₁₂ synthesis capability was acquired by the use of genetic engineering technology. And with the investigation of Q_o2 and cellular morphology, the oxygen supply strategy based on Q_o2 was proposed, which had great reference value to the fermentation of vitamin B₁₂ on large scale. The major results were detailed as follows:With the introduction of vgb gene, the yield of the vitamin B₁₂ was increased by 15% when compared with the control. Moreover, vgb gene also brought the enhancement of the growth rate of the strain. The metabolic flux analysis results revealed that the introduction of vgb gene had the effect of enhancing the PP metabolic pathway, which was important for the accumulation of precursor and reducing force for the growth of the cell. The increasement of the synthesis of glycine from betaine accelerated the generation of ALA and further benefited the yield of vitamin B₁₂. In addition, the overexpression of cobF-M and cobS-T genes which encode the enzymes that are related to the synthesis of cobyrinate from precorrin-2 elicited the increase of YP/X, which indicated the improvement of the amount of the biomass may be the key to optimize the yield of the vitamin B₁₂. The strain with the overexpression of cobS-T had better performance that implied the cobalt ion chelating process was the key rate-limiting step for vitamin B₁₂ synthesis.In this work, we also created the relationship between capacitance detected by viable biomass sensor online and the biomass concentration, and investigated the effect of Q_o2 on the morphological changes of Ratio-LW of P. denitrificans and vitamin B₁₂ biosynthesis rate and finally put forword the two stage oxygen supply strategies based on Q_o2-It can be concluded that in the node of the changes of the P. denitrificans morphology, the optimum capacitance was 10.45 PF/cm and when the process turned into the biosynthesis of the product, the optimum Q_o2 was controlled at 1.4 mmol/g/h and the Ratio-LW of P. denitrificans was 10:1. Compared with the oxygen supply strategies based on the OUR and DO, Q_o2 strategy proposed in this study is more useful to acquire constant high yield of vitamin B₁₂ and the final vitamin B₁₂ concentration achieved to 239.7mg/L.