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 foods, pharmaceuticals and agriculture. 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. In this study, systematic metabolic engineering of Escherichia coli 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: (1) rihA, rihB, rihC, deoD and ppnP, which encoded the nucleoside degradation enzymes, were deleted to construct chassis strain INO1-1. Further, in order to improve the carbon flux of de novo purine nucleotide synthesis and remove the feedback inhibition of PRPP amidotransferase, purine operon from Bacillus amyloliquefaciens and purFbsuD293V,K316Q,S400W mutant from Bacillus subtilis were introduced, and the inosine titer increased to 308.3 mg/L. (2) The adenosine synthesis branch and the guanosine synthesis branch competed cellular resources, which limited the synthesis of inosine. To address this issue, purA and guaB were deleted to weaken the competition for metabolic flow of inosine synthesis branch. Shake flask results showed that blockage of the adenosine synthesis promoted inosine synthesis the most, while turned the strain into a nutrient-deficient. The balance between inosine production and cell growth in microbial cell factories is a long-standing challenge. To circumvent this problem, the native purA was replaced by the purAbsuP242N mutant from B. subtilis to weaken the adenosine synthesis branch, and the inosine titer increased to 1 412.5 mg/L. (3) As the critical precursor of the de novo purine nucleotide synthesis, the synthesis of PRPP is subjected to strict regulations. Therefore, purR was deleted to alleviate the transcriptional regulation of the native prs and prsecoD128A mutant was introduced to eliminate the feedback inhibition of PRPP synthase, which improved the supply of PRPP and elevated inosine titer to 3.1 g/L. (4) In order to further improve the titer of inosine, two copies of pbuE from B. amyloliquefaciens was introduced to strengthen the purine efflux pump, which promoted the inosine titer and cell growth at the same time. Then, nupC encoding nucleoside transport protein were deleted, and the extracellular inosine accumulation increased to 5.0 g/L. (5) The constructed strain INO4-5 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. In this study, the multiple metabolic modules of the complex inosine synthesis network in E. coli, including inosine degradation module, de novo purine nucleotide synthesis module, adenosine synthesis branch module, PRPP synthesis module, and inosine transport module, were systemically optimized to construct a superior inosine producer. Compared with other construction strategies, this study rationally solved the problem of nutrient-deficient commonly found in inosine production strains. The final strain can stably achieve high titers, yields, and productivities of inosine with a short fermentation time, which provides a valuable reference for the production of inosine.
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