Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, Tangjeong-myeon, Asansi, Chungnam, Republic of Korea.
J Appl Microbiol. 2010 Aug;109(2):398-407. doi: 10.1111/j.1365-2672.2010.04688.x. Epub 2010 Jan 24.
To characterize the function of both metK1-sp (sp1190) and metK2-sp (sp1566) in vitro and in vivo, and to study the regulation of doxorubicin production by overexpressing the metK.
We cloned two orfs into pET32a(+) respectively, and the formation of S-Adenosyl-l-methionine was clearly observed in the in vitro enzyme assays as functional MetKs. Reverse transcriptase polymerase chain reaction (PCR) analysis indicated that the transcripts for the metK1-sp were repressed as Streptomyces cells entered the decline phase, whereas that of the metK2-sp was induced, suggesting that these MetK proteins may be important for the growth and the regulation of secondary metabolites during the stationary growth phase, whether considered together or separately. Furthermore, we found that the introduction of high-copy-number plasmids containing the metK1-sp and metK2-sp resulted in 2.1- and 1.4-fold greater levels of doxorubicin production than the control transformants containing only the vector, respectively. We also attempted to disrupt the metK-sp and found that doxorubicin production from the metK1-sp-deleted mutant (Streptomyces peucetius/pNN1) was reduced when compared to the parent strain (S. peucetius var. caesius ATCC 27952).
The results of this study indicated that two metK are differentially expressed during cell growth, and that the expressions of the two metK genes are differentially regulated under the same conditions.
Streptomyces peucetius var. caesius contains two genes, metK1-sp and metK2-sp, which encode functional S-adenosyl-l-methionine synthetase (MetK). The degree of homology (90% identity) found between the two genes shows that metK1-sp and metK2-sp are duplicated genes. Although there is currently no evidence for the relationship of the duplicated metK genes involved in the regulation of doxorubicin production, metK1-sp and metK2-sp may play a role in controlling the stimulation of antibiotic production during secondary metabolism.
研究 metK1-sp（sp1190）和 metK2-sp（sp1566）在体外和体内的功能，并研究通过过表达 metK 对多柔比星产生的调节。
我们分别将两个 ORF 克隆到 pET32a(+)中，在体外酶测定中清楚地观察到 S-腺苷-L-甲硫氨酸的形成，这表明这两种 MetK 具有酶功能。反转录聚合酶链反应（PCR）分析表明，当链霉菌细胞进入衰退期时，metK1-sp 的转录被抑制，而 metK2-sp 的转录被诱导，这表明这些 MetK 蛋白可能在静止生长阶段的生长和次级代谢物的调节中很重要，无论是一起考虑还是分别考虑。此外，我们发现，引入含有 metK1-sp 和 metK2-sp 的高拷贝数质粒可使多柔比星的产量比仅含有载体的对照转化子分别增加 2.1 倍和 1.4 倍。我们还试图敲除 metK-sp，发现与亲本菌株（S. peucetius var. caesius ATCC 27952）相比，metK1-sp 缺失突变体（Streptomyces peucetius/pNN1）的多柔比星产量降低。
本研究结果表明，两种 metK 在细胞生长过程中差异表达，并且在相同条件下，两种 metK 基因的表达受到不同的调节。
产色链霉菌 var. caesius 含有两个基因 metK1-sp 和 metK2-sp，它们编码功能 S-腺苷-L-甲硫氨酸合成酶（MetK）。这两个基因之间发现的同源性（90%的同一性）表明 metK1-sp 和 metK2-sp 是重复基因。尽管目前没有证据表明涉及多柔比星产生调节的重复 metK 基因之间存在关系，但 metK1-sp 和 metK2-sp 可能在控制次级代谢中抗生素产生的刺激中发挥作用。
