Study On Directed Evolution Of Bacillus Subtilis XGL And Optimization Of Adenosine Fermentation Process

Adenosine is required for life and has a wide range of applications in the field of medicine and food. Developed countries started earlier in adenosine production by fermentation-based method of and adenosine production has entered the stage of large-scale production. However, more research on adenosine fermentation was concentrated on shake flask-scale and lack of experimental datum in bioreactor. In this thesis, two important parameters dissolved oxygen (DO) and pH were investigated in adenosine fermentation process under the guidance of metabolic engineering theory. Furthermore, an engineering strain was constructed from Bacillus subtilis XGL (the original strain). This thesis would provide theoretical foundation and novel method genetic engineering strains construction and adenosine fermentation process optimization.The effect of DO level on adenosine production by Bacillus subtilis XGL was investigated in fed-batch fermentation process, and consequently a two-stage DO control strategy was developed, in which the DO level was controlled at 30%-40% in the first 20 h, and then to maintain a DO level at 10%-20%. Higher adenosine production (19.2 g/L) was achieved in shorter time (56 h), which was increased by 78.6%,66.7%,9.5%,18.6% and 32.2%, compared to that in conditions with DO uncontrolled or controlled at 0%-10%, 10%-20%,20%-30% and 30%-40%, respectively. On the basis of two-stage DO control strategy, fermentation was conducted at various pH values in order to investigate the effects on cell growth, glucose consumption and adenosine production. Results showed that two-stage DO combined pH-shift method was an optimal way for adenosine production, under which adenosine yield was further improved by 21.4%, reaching 23.3 g/L at 56 h.We obtained an engineered strain B. subtilis XGLF by single crossover in this study, which overexpressed one of key gene purF and its downstream genes purM, purN, purH, purD. Moreover, the purF gene driven by P43 promoter was integrated to the genomic DNA of parent strain via the temperature-sensitive plasmid pKS1. That lead to efficient increase of purine precursors supply to purine pathway. B. subtilis XGLF exhibited 17.5% and 26.1% improvement in adenosine yield and conversion ratio from glucose to adenosine respectively.Finally, adenylosuccinate synthase (ADSS) gene pur A was overexpressed in B. subtilis XGLF to investigate the impact on adenosine accumulation in the pur A-overexpression strain (B. subtilis XGLFA). However, similar improvement did not occur in the B. subtilis XGLFA because of the instability and the metabolic burden taken by plasmid.