Abstract: N-acetylneuraminic acid (NeuAc) has applications as nutritional chemicals and pharmaceutical intermediates in the fields of health care products and medicine. To increase NeuAcproduction in Bacillus subtilis, NeuAc-biosensor was used to regulate the expression of antibiotic resistance genes, and cell growth was correlated with NeuAc synthesis in the presence of antibiotic, which was further used for adaptive evolution to promote the efficiency of NeuAc synthesis. The results showed that high-yielding strains could grow under conditions of higher antibiotic concentration. The adaptive evolution was carried out by gradually increasing the concentration of antibiotics. When NeuAc-Biosensor was used to regulate spc and erm for dual resistance (spectinomycin and erythromycin) adaptive evolution, the false positive rate (the proportion of strains with no increase in NeuAc production) was 46.7%, which was significantly lower than that (73.3% and 67.7%) of the strains obtained by the evolution of using single antibiotic of spectinomycin or erythromycin, respectively. Fermentation experiments verified that the production of NeuAc in evolutionary strain reached (3.16±0.19) g/L, which was 31.7% higher than the original strain. In order to further solve the problem of plasmid loss in recombinant B. subtilis during fermentation, the essential gene folB (encoding dihydroneopterin aldolase) was inserted into the recombinant plasmid carrying the gene encoding the key enzyme of NeuAc synthesis pathway with deletion of folB in the genome. The plasmid loss rate dropped from 34.1% to 11.8%. In this study, the yield and stability of NeuAc production was improved by recombinant B. subtilis, which lays a foundation for the industrial production of NeuAc by recombinant B. subtilis.
钱蕾,刘延峰,李江华,等. 适应性进化和改造质粒稳定性促进枯草芽孢杆菌合成N-乙酰神经氨酸[J]. 食品与发酵工业, 2021, 47(5): 1-6.
QIAN Lei,LIU Yanfeng,LI Jianghua,et al. Regulating the synthesis of N-acetylneuraminic acid based on adaptive evolution and plasmid stability modification in Bacillus subtilis[J]. Food and Fermentation Industries, 2021, 47(5): 1-6.
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