Objective: To study the effects of co-overexpression of purF, purM, purN, purH and purD genes on adenosine production in Bacillus subtilis.
Methods: First, an extra purF gene under control of the P43 promoter was integrated into the B. subtilis chromosome at the native purF locus by single crossover, resulting in simultaneous expression of purF, purM, purN, purH and purD. Then the transcriptional levels of the five genes in the engineering strain were tested by Realtime Quantitative PCR. The activity of PRPP amidotransferase was also detected. Finally, cell growth, glucose consumption and adenosine production in engineering strain along with original strain were examined.
Results: The transcriptional analysis showed that purF and its downstream genes purM, purN, purH and purD were simultaneously upregulated at different level. The PRPP amidotransferase activity of engineering strain was about 2.4-fold that of original strain. Shake flask fermentation showed the improvement in adenosine yield and conversion ratio from glucose to adenosine (17.5% and 26.1%, respectively). Fed-batch fermentation by the engineering strain was conducted. Compared with the original strain, adenosine accumulation of engineering strain increased within the same fermentation time. However, the cell growth of engineering strain was retarded.
Conclusion: The co-overexpression of purF and its downstream genes purM, purN, purH and purD could enhance the adenosine yield in the culture broth. This paper could facilitate future research by providing theoretical evidence and method of metabolic engineering.