费氏链霉菌脱氢酶NeoE的生物信息学分析及其对产新霉素的影响  

Bioinformatics analysis of Streptomyces fradiae dehydrogenase NeoE and its effect on neomycin production

作　　者：王芳[1] 周健 王珊 李闯 王洲[1] 刘艳[1] 薛正莲[1] Wang Fang;Zhou Jian;Wang Shan;Li Chuang;Wang Zhou;Liu Yan;Xue Zheng-lian(College of Biological and Chemical Engineering,Anhui Polytechnic University,Anhui Engineering Technology Research Center of Microbial Fermentation,Wuhu 241000)

机构地区：[1]安徽工程大学生物与食品工程学院,微生物发酵安徽省工程研究中心,芜湖241000

出　　处：《中国抗生素杂志》2022年第12期1260-1266,共7页Chinese Journal of Antibiotics

摘　　要：目的NeoE是费氏链霉菌合成新霉素途径中一种重要的NAD(P)+依赖性脱氢酶,本研究的目的是预测其生物学性质及其对新霉素合成能力的影响。方法由NCBI网站蛋白质数据库获得NeoE氨基酸序列,利用生物信息学网站及软件分析预测其特征。随后构建neoE过表达质粒,以接合转移方式得到高产重组菌株SF-neoE。最后经摇瓶发酵以及相关基因的转录分析探究NeoE对新霉素生物合成的影响。结果NeoE由340个氨基酸组成,理论相对分子质量约为35228.33 Da,理论pI值5.14,不存在信号肽和跨膜区,是一个疏水性的稳定胞内蛋白。对含空载质粒菌株和重组菌株摇瓶发酵与RT-qPCR分析,结果表明第48小时neoE的相对表达水平较对照菌株提高了2.1倍,且NeoE的过量表达使得新霉素效价提高42%。结论该结果为提高新霉素效价提供思路,并为后续定向改造NeoE探究其对费氏链霉菌生长代谢的影响奠定理论基础。Objective NeoE is an NAD(P)+-dependent dehydrogenase in the neomycin synthesis pathway of Streptomyces fradiae.In this study,the biological properties of NeoE and its effects on neomycin production are researched.Methods The amino acid sequence of NeoE was obtained from NCBI website,and its characteristics were predicted by several bioinformatic websites and software.The high-yielding recombinant strain,SE-neoE,was obtained via the construction of neoE overexpressing plasmids,followed by conjugative transfer.Finally,shake flask fermentation of SF-neoE and transcriptional analysis of related genes were carried out to explore the effect of NeoE on neomycin potency.Results NeoE consists of 340 amino acids,with a theoretical relative molecular mass of about 35228.33 Da and a theoretical pI value of 5.14.NeoE has no signal peptide or transmembrane region,and is a hydrophobic stable intracellular protein.The shake flask fermentation and RT-qPCR analysis of the plasmidcontaining strains and the recombinant strain showed that the relative expression level of neoE was increased by 2.1-fold compared to the plasmid-containing strains at the 48h.The overexpression of NeoE resulted in a 42%increase in neomycin potency.Conclusion This research may lay the foundation of the later in-depth study on NeoE,such as its directed modification,aiming to improve neomycin potency.

关 键 词：费氏链霉菌 新霉素 NeoE 生物信息学分析 过表达 

分 类 号：R978.1[医药卫生—药品]