刺糖多孢菌高产菌株和野生型菌株多杀菌素生物合成基因簇(spn)在发酵过程中的表达分析  

Expression Analysis of Spinosad Biosynthetic Gene Cluster(spn) in Hyperproducing and Wild-type Saccharopolyspora spinosa Strains During Fermentation

作　　者：黄颖[1,2,3] 赵晨[1] 杨博磊 陈园[1] 关雄[2] 宋渊[3] 张晓琳[1] 

机构地区：[1]国家粮食局科学研究院,北京100037 [2]福建农林大学生物农药与化学生物学教育部重点实验室,福州350002 [3]中国农业大学生物学院微生物学系,北京100193

出　　处：《农业生物技术学报》2014年第11期1337-1346,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31300092);粮食公益性行业科研专项(No.201313002-3)

摘　　要：多杀菌素(spinosad)作为一种高效、低毒生物杀虫剂,已在农药、兽药、卫生用药等领域得到应用。中国关于多杀菌素的研究起步较晚,进展缓慢,目前仍无法实现产业化生产。本研究旨在分析刺糖多孢菌(Saccharopolyspora spinosa)高产菌株ASAGF73与野生型菌株ATCC49460在发酵过程中多杀菌素合成基因簇(spinosyn biosynthetic gene cluster,spn)的表达变化及差异,初步判断影响多杀菌素合成的关键限速步骤。生物信息学分析表明,spn基因簇含有9个转录子,分别在各转录子中设计引物,进行RTPCR检测其表达情况。结果显示,突变株ASAGF73中多杀菌素的产量提高可能与spn基因簇中部分基因的高表达有关,其中编码聚酮合酶(polyketide synthase,PKS)的基因和参与大环内联桥形成的基因表达量远高于野生型菌株的。在ASAGF73中编码鼠李糖转移酶的spn G基因在发酵前期过量表达,而在后期大量合成多杀菌素时表达减弱,有可能成为多杀菌素合成的限速步骤。本研究初步阐明能够提高多杀菌素产量的分子机制,为构建多杀菌素高产基因工程菌提供参考。Spinosad is a kind of bio-insecticide showing advantages such as high-efficiency and low-toxicity, which is wildly used in pesticide, veterinary drug, sanitary field, etc. However, industrial production of spinosad still has not been implement in China, because of relatively late and undeveloped study on spinosad high-yield strain breeding and relative fermentation process. Previously, a spinosad high-yield strain, Saccharopolyspora spinosa ASAGF73, was obtained through multiple rounds mutagenesis. In this study, we focused on analysing spinosyn biosynthetic gene cluster（spn） expression profile and comparing the difference between wide- type strain ATCC49460 and high- yield ASAGF73 during fermentation process, and preliminarily elucidated the rate-limiting step for spinosad synthesis. Bioinformatic analysis revealed that spn gene cluster contained 9 transcripts, and their expressions were detected using RT-PCR with primers designed inside each transcript. The results showed that the high production of ASAGF73 strain could be due to the high expression of some transcripts in spn cluster, the expression of genes coding for polyketide synthase （PKS） and cross- bridging in ASAGF73 was higher than those in ATCC49460. spnG, which coded rhamnosyltransferase, showed high expression in ASAGF73 strain at the beginning of the fermentation but decreased to a quite low level later, which was not ideal for spinosad high production and might be the rate-limiting step for spinosad synthesis. The present study was the comprehensive and comparative expression analysis of spinosad biosynthetic gene cluster and genes for rhamnose biosynthesis in the 2 S. spinosa strains during fermentation, provided some clues to understand the molecular mechanisms which can lead to the increasing of spinosad production yield.

关 键 词：多杀菌素 刺糖多孢菌 多杀菌素合成基因簇(spn) 基因表达 

分 类 号：S482.3[农业科学—农药学]