Metabolic Engineering Of Bacillus Subtilis For Uridine Production

Uridine, as raw material for the synthesis of some antivirus and antitumor drugs,has extremely important medical care and has been widely applied in the pharmaceutical industry. Uridine production by microbial fermentation has many advantages, such as mild reaction condition, cheap raw material and simple operation.However, there is no industrial production strain in China. A higher uridine-producing strain, named as B..subtili.s F126 was obtained by atmospheric and room temperature plasma (ARTP) and high-throughput screening (HTS) method in our previous study.To improve uridine productivity, F126 strain was as the original strain for further genetic modification.The prs gene encoding 5-phosphoribosyl pyrophosphate (PRPP) synthetase was overexpressed in the F126 strain to construct F126-1 strain. The results by shake flasks fermentation showed that F126 and F126-1 strain could accumulate 5.72 g/L, and 8.76 g/L uridine, respectively. 5 L fed-batch fermentation results revealed that F126 and F126-1 strain were able to produce 20.16 g/L and 24.52 g/L uridine, and also accumulated 58.62 g/L and 56.62 g/L acetoin respectively.To reduce the accumulation of acetoin, alsS and aalsD gene was separately knocked out in the F126-1 srtain. The shake flasks fermentation results showed that F126-1 ?alsS and F126-1 ?alsD strain could accumulate 0.48 g/L and 6.43 g/L uridine.The F126-?alsD strain accumulated 18.32 g/L uridine and 18.56 g/L acetoin in 5 L fed-batch fermention, 25.29% and 67.22% decraesed in compare with F126-1 strain,respectively. Therefore, the elimination of acetoin synthesis had no positive effect on uridine generation. The F126-1 strain with overexpressed pycA gene, was able to accumulate 8.03 g/L uridine in shake flasks fermentation. The strain could produce 18.38 g/L uridine and 45.85 g/L acetoin in 5 L fed-batch fermention, 25.04% and 18.53% decreased in compare with F126-1 strain, respectively. Overexpression of pycA gene could reduce acetoin accumulation, but had no effect on uridine accumulation.The udk and argF gene was separately deleted in F126-1 strain. F126-1?udk and F126-1?argF strain could accumulate 9.07 g/L and 12.46 g/L uridine in shake flasks fermentation, 3.54% and 42.24% higher than that of F126-1 strain. F126-1?udk and F126-1?argF strain accumulated 23.68 g/L and 28.55 g/L uridine in 5 L fed-batch fermentation. The uridine yield of F126-1?argF was 16.55% higher than that of F126-1 strain. F126-1?udk and F126-1?argF strain also accumulated 52.56 g/L and 51.91 g/L acetoin respectively, 7.11% and 7.73% decreased in compare with F126-1 strain. Therefore, the deletion of argF gene could increase the uridine production and reduce acetoin accumulation.