氨肽酶PepN1在细胞外催化杀稻瘟菌素的成熟
Maturation of Blasticidin S is catalyzed by extracellular aminopeptidase N1
  
DOI:  10.13344/j.microbiol.china.180774
中文关键词:杀稻瘟菌素,灰色产色链霉菌,变铅青链霉菌,氨肽酶N,蛋白分泌系统
英文关键词:Blasticidin S, Streptomyces griseochromogenes, Streptomyces lividans, Aminopeptidase N, Protein secretion system
基金项目:国家自然科学基金(31470195)
作者单位E-mail
贺惠 上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200030  
禹贵阳 上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200030  
王先坤 上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200030  
邓子新 上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200030  
贺新义 上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200030 xyhe@sjtu.edu.cn 
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中文摘要:
      【背景】肽核苷类抗生素杀稻瘟菌素(Blasticidin S,BS)生物合成途径的最后一步是亮氨酰杀稻瘟菌素(Leucylblasticidin S,LBS)水解成熟为BS,BS原始产生菌灰色产色链霉菌和异源表达菌株变铅青链霉菌WJ2清洗过的细胞均能催化这一步反应。前期我们确定在这两种菌中都含有编码3个氨肽酶PepN同源蛋白的基因,其中编码产物PepN1主要负责水解LBS和亮氨酰脱甲基杀稻瘟菌素(Leucyldemethylblasticidin S,LDBS),且催化效率相当。但是,相比于原始产生菌只积累BS这一种组分,异源表达菌株变铅青链霉菌WJ2还积累了LBS和LDBS,这意味着原始产生菌与WJ2中PepN1的水解活性存在差异。【目的】研究PepN1在两个菌株中的表达和分泌对BS组分的影响。【方法】利用PepN1的多克隆抗体,通过蛋白免疫印迹法(Western blot)比较不同生长时间的原始产生菌和异源表达菌株在细胞内、细胞培养液中PepN1的表达水平。硫酸铵沉淀收集两种菌株培养液中的蛋白,通过Western blot检测PepN1的存在并在体外检测水解活性。【结果】原始产生菌第2?6天细胞内PepN1水平基本没有变化,而异源表达菌株自第4天起细胞内PepN1开始减弱,到第6天完全消失;另一方面,Western blot在原始产生菌细胞培养液中检测到PepN1,而在异源表达菌株中却没有检测到。细胞培养液水解LDBS的活性与Western blot检测到的PepN1表达水平相一致。【结论】BS原始产生菌能持续表达合成PepN1并将一部分PepN1分泌到细胞外,而异源表达菌株WJ2从第4天起则停止合成PepN1,第2?6天的细胞均不能将PepN1分泌到细胞外,这导致WJ2中积累亮氨酰化的产物。两个菌株清洗过的细胞均可以水解LDBS和LBS,推测在WJ2体内消失的PepN1分泌到细胞壁上但没有释放到溶液中,这部分PepN1可以在WJ2中将部分LDBS和LBS水解成对应的DBS和BS。
英文摘要:
      [Background] The final step of the peptide nucleoside antibiotic Blasticidin S (BS) biosynthesis pathway is the leucylblasticidin S (LBS) hydrolyzation to BS. The intact cells of BS native producer Streptomyces griseochromogenes and the heterologous producer Streptomyces lividans WJ2 can catalyze this step. Earlier work showed that both strains encode three PepN homologues, of which PepN1 from each strain is mainly responsible for the hydrolysis of LBS and leucyldemethylblasticidin S (LDBS) at comparable catalytic efficiency. However, comparing with the native producer that only synthesizes BS, WJ2 can also generate LBS and LDBS. This result indicates that the competence of PepN1 is different in these two producers. [Objective] Investigation of the effects of the expression level and localization of PepN1 on the productivity and components of BS and derivatives in two producers. [Methods] The concentration of PepN1 in the cell lysate and in the cell culture medium of two BS producers at different growth time were tracked and compared by Western blot using PepN1 polyclonal antibody. The protein in the culture medium of the two strains was collected by ammonium sulfate precipitation, and the activity of hydrolyzed LDBS was assayed in vitro. [Results] On one hand, the content of PepN1 in the native strain didn’t change from the 2nd to 6th day, whereas PepN1 started to diminish from the 4th day and totally disappeared on the 6th day in the heterologous strain. On the other hand, PepN1 was detected in the culture medium of native producer rather than the heterologous strain, which was consistent with the different activity of two strain’s cell culture medium in hydrolyzing LDBS. [Conclusion] The BS native producer can continuously express PepN1 and export part of PepN1 outside of the cell. On the contrary, the heterologous producer WJ2 cease to synthesize PepN1 from the 4th day and it can’t be detected in the culture medium, leading to the accumulation of leucylated intermediates. Given that intact cells of two strains are capable of hydrolyzing LDBS and LBS, PepN1 that diminished from the cell lysate might secrete to the cell wall, but not release into the medium, this part of activity accounts for the generation of DBS and BS in WJ2.
贺惠,禹贵阳,王先坤,邓子新,贺新义.氨肽酶PepN1在细胞外催化杀稻瘟菌素的成熟[J].微生物学通报,2019,46(2):223~232
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