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  

Regulating the synthesis of N-acetylneuraminic acid based on adaptive evolution and plasmid stability modification in Bacillus subtilis

作　　者：钱蕾 刘延峰 李江华[2] 刘龙[1,2] 堵国成[1,2] QIAN Lei;LIU Yanfeng;LI Jianghua;LIU Long;DU Guocheng(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;College of Bioengineering,Jiangnan University,Wuxi 214122,China)

机构地区：[1]工业生物技术教育部重点实验室(江南大学),江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2021年第5期1-6,共6页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2018YFA0900300);国家自然科学基金项目(31972854);江苏省重点研发计划(社会发展)项目(BE2019628)。

摘　　要：N-乙酰神经氨酸(N-acetylneuraminic acid,NeuAc)作为营养化学品和药物中间体在保健品和医药领域具有广泛的应用。为了提升重组枯草芽孢杆菌(Bacillus subtilis)合成NeuAc的产量,首先利用NeuAc生物传感器(NeuAc-Biosensor)调控抗生素抗性基因表达,在抗生素存在条件下将细胞生长与NeuAc合成相关联。进而通过增加抗生素浓度进行适应性进化,促进NeuAc合成效率提升。研究结果显示,当利用NeuAc-Biosensor分别调控壮观霉素抗性基因(spc)和红霉素抗性基因(erm)时,高产菌株能够在较高抗生素浓度条件下生长。通过在培养过程中逐步增加抗生素浓度开展适应性进化,结果表明采用壮观霉素和红霉素进行双抗性适应性进化时,进化获得的菌株中假阳性率(产量未提高的菌株比例)为46.7%,显著低于采用壮观霉素或红霉素单一抗生素进化获得的菌株中假阳性率(73.3%和66.7%)。通过适应性进化与发酵验证得到1株NeuAc产量为(3.16±0.19)g/L的菌株,产量比出发菌株提高了31.7%。为进一步解决发酵过程中重组B.subtilis质粒丢失的问题,通过将必需基因folB(编码二氢喋呤醛缩酶,dihydroneopterin aldolase)插入携带NeuAc合成途径关键酶编码基因重组质粒并且敲除基因组中的folB基因,质粒的丢失率由34.1%下降至11.8%。该研究提升了重组B.subtilis合成NeuAc的产量和稳定性,为重组B.subtilis发酵法生产NeuAc奠定了基础。N-acetylneuraminic acid(NeuAc)has applications as nutritional chemicals and pharmaceutical intermediates in the fields of health care products and medicine.To increase NeuAc production 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.

关 键 词：生物传感器 适应性进化 质粒稳定性 N-乙酰神经氨酸 枯草芽孢杆菌 

分 类 号：TS201.3[轻工技术与工程—食品科学]