Metabolic Engineering Of Escherichia Coli For Inosine Production

As a versatile purine nucleoside,inosine plays an important role in the synthesis of various energy carriers and genetic material in the body,and is therefore extensively used in pharmaceuticals and foods.Because of its low cost,low energy consumption and low environmental impact,microbial fermentation has been the attractive method for large-scale industrial production of inosine.With the increasing market demand for inosine,the production strains of inosine have been put forward higher requirements.In this study,systematic metabolic engineering of Escherichia coli MG1655 was implemented by directed chromosomal modifications using the method of CRISPR/Cas9 mediated genome editing to construct inosine engineered strain.The main research contents are as follows:Firstly,rih A,rih B,rih C,deo D,ppn P and gsk were knocked out to block the inosine degradation pathway to construct strain INO1-6.The results of fermentation showed that the growth of the strain was not significantly affected,which laid the foundation for the improving the efficiency of inosine synthesis.Further,to improve the carbon flux of de novo purine nucleotide synthesis and remove the feedback inhibition of PRPP amidotransferase,purine operon and pur F_baz^K316Qmutant from Bacillus amyloliquefaciens were introduced to construct strain INO2-6 and INO2-9.The results of fermentation showed that the titers of inosine were 155.2 mg/L and 308.3 mg/L,respectively.Further,to weaken the adenosine synthesis branch,pur A was replaced with pur A_bsu^P242Nmutant from Bacillus subtilis to construct strain INO3-4.The results of fermentation showed that under the condition of 0.3 g/L adenine addition,the titer of inosine was 1412.5 mg/L,which was increased by 19.6%compared with INO3-1.Further,to alleviate the transcriptional regulation of the native prs,pur R was knocked out to construct strain INO4-1.To eliminate the feedback inhibition of PRPP synthase,prs_eco^D128A mutant from Escherichia coli was overexpressed to construct strain INO4-2.The results of fermentation showed that the titers of inosine were 1.6 g/L and 3.1 g/L,which were increased by 14.3%and 93.8%compared with the control strains,respectively.Further,to enhance inosine excretion,pbu E from Bacillus amyloliquefaciens was overexpressed to construct strain INO5-2.To weaken the uptake of inosine,nup C was knocked out to construct strain INO5-3.The results of fermentation showed that the titers of inosine were 3.9 g/L and 4.5 g/L,which were increased by 25.8%and 15.4%compared with the control strains,respectively.Finally,the constructed strain INO5-3 produced 20.2 g/L inosine with an overall yield of 0.12 g/g glucose and productivity of 0.42 g/L/h after 48 h fed-batch fermentation in 5 L bioreactor.The inosine engineered strain is non-defective,stable and efficient,plasmid-free,and has a short fermentation cycle,which lays a solid foundation for subsequent industrial applications.