埃莎霉素Ⅰ组分高含量、高产量基因工程菌WSJ-IA及其原株的鉴定

摘要

[目的]利用调节基因acyB2激活异戊酰基转移酶(ist)基因表达的特点,将ist与调节基因acyB2在异戊酰螺旋霉素(埃莎霉素)Ⅰ产生菌菌株中共表达,获得埃莎霉素Ⅰ单组分的高含量及高产量菌株WSJ-IA.对其及原始螺旋霉素产生菌菌株Streptomyces spiramyceticus F21进行了初步鉴定.[方法]从形态学、培养和生理生化特征、细胞壁化学组成、16S rRNA基因序列、5个看家基因(atpD、gyrB、rpoB、recA和trpB)蛋白分析和系统发育树构建等方面对该菌株及其原株进行了鉴定.[结果]两株菌在形态培养特征、生理生化特征、细胞壁化学组成、16S rRNA基因序列和5个看家基因蛋白水平基本一致,在系统发育树分析中同处在一个分支中.而在16S rRNA基因序列和5个看家基因蛋白水平在系统发育上它们均与已知相近菌株处于不同的分支上,并且与不同基因的相近菌株各有不同,其中无一报道产生螺旋霉素.[结论]Streptomyces spiramyceticus F21可能是一个产生螺旋霉素的链霉菌新种,16S rRNA基因序列和5个看家基因蛋白序列分析可以作为埃莎霉素Ⅰ基因工程菌生产过程中进行鉴别的分子标志.%[Objective] A Streptomyces spiramyceticus WSJ-IA strain (herein designated as an isomycin I producer) with high proportion and high production of isovalerylspiramycin I was obtained by cloning and expression of acyBl (Regulatory gene) and ist (Isovaleryltransferase gene) into an isovalerylspiramycin I producting strain. Taxonomic studies were carried out to define the engineered isomycin I producer and its original parent strain Streptomyces spiramyceticus F21. [Methods] 16S rRNA gene sequencing and five housekeeping genes (atpD、 gyrB、 rpoB、 recA and trpB) coding proteins sequencing combining the traditional taxonomic studies, such as morphological, cultural, physiological and biochemical characteristics were performed for both strains. (Results] The Streptomyces spiramyceticus WSJ-IA and Streptomyces spiramyceticus F21 share highly similar phenotypes and 16S rRNA gene sequences as well as in the phenogenetic analysis of the five housekeeping gene protein sequences. While their 16S rRNA gene sequences and the five housekeeping gene protein sequences phyloge-netically were located in different clades comparing with all other strains so far described. And the closely related strains with each gene coding protein were also different,none of them was as spiramycin producer reported. [Conclusion] Our results suggested that Streptomyces spiramyceticus F21 was possible a new Streptomyces sp. of spiramycin producer. 16S rRNA gene sequence and five housekeeping genes (atpD? gyrB , rpoB., recA and trpB ) coding proteins sequences could be served as genetic markers in identifying the engineered strain's stability in long term of the commercial production.