阿维链霉菌中aveD基因阻断对阿维菌素合成的影响

EFFECT OF GENE DISRUPTION OF aveD ON AVERMECTINS PRODUCTION IN STREPTOMYCES AVERMITILIS

利用用于基因破坏的重组质粒pCZ2 (pKC1 1 39∷ 475bpaveD)对阿维菌素 (Avermectin)产生菌阿维链霉菌 (Streptomycesavermitilis) 76- 9的aveD基因进行插入失活 ,将获得的aveD破坏子进行摇瓶发酵和阿维菌素初步提取和HPLC检测 ,发现破坏子仅产生四个主要组分 ,但它们的保留时间分别比Bla、Blb、B2a和B2b的略长。进而将粗提液纯化并获得晶体 ,以UV、IR、NMR ( 1H NMR和13C NMR)和MS进行结构分析 ,并结合HPLC检验 ,证明它们属于C5 氧 阿维菌素B。说明阿维链霉菌的aveD基因破坏 ,不仅丧失了合成阿维菌素A组分的能力 ,也造成了其下游的aveF基因不能表达 ,因此只产生了C5 氧 阿维菌素B。

Recombinant plasmid pCZ2(pKC1139∷475bp aveD) was used for aveD gene disruption in Streptomyces avermitilis 76-9.The plasmid was inserted into the chromosome by homogenous recombination between partial aveD gene in the plasmid and aveD in the chromosome.Disruptants were confirmed by Southern blotting.Shaking flask experiments and HPLC analysis showed that the disruptant produced only four components, which were C5-oxo-avermectin B1a,B1b,B2a,B2b as identified by UV,IR,NMR,and MS.This revealed that both aveD and aveF were not expressed in the disruptant.This is consistent with that aveD and aveF are in a transcription unit.This paper also provided a new genetic method to obtain C5\|oxo\|avermectin B\|producing strain.