摘要
【目的】由于谷氏菌素产生菌禾粟链霉菌的遗传操作效率较低,本研究在谷氏菌素生物合成基因簇成功异源表达的基础上,通过在异源宿主天蓝色链霉菌M1146中阻断谷氏菌素生物合成基因gouC和gouD,研究其在谷氏菌素生物合成中的作用,为谷氏菌素生物合成途径的阐明奠定基础。【方法】以含有谷氏菌素生物合成基因簇的黏粒D6-4H为基础,通过PCR-targeting的方法分别将gouC和gouD敲除得到重组质粒p GOUe-ΔC和p GOUe-ΔD。通过接合转移将这两个重组质粒分别导入天蓝色链霉菌M1146中,获得gouC和gouD的缺失突变株M1146-GOUe-ΔC和M1146-GOUe-ΔD,通过HPLC分析突变株的谷氏菌素中间产物积累情况,分离纯化后对其进行结构鉴定和生物活性检测。【结果】gouC和gouD的缺失均导致谷氏菌素不能合成,突变株发酵液中积累了不同的中间产物,生物活性分析发现这些中间产物均失去了对肿瘤细胞的抑制活性。【结论】gouC和gouD是谷氏菌素生物合成的重要基因,与谷氏菌素肽基部分的肌氨酸残基合成相关。本研究为阐明谷氏菌素的生物合成机制提供了更多的依据。
[Objective] To determine the functions of gou C and gou D in gougerotin biosynthesis, disruption of these two genes was performed. As gougerotin producing strain Streptomyces graminearus lacks efficient genetic manipulation system, the gene cluster for gougerotin biosynthesis was heterologously expressed in Streptomyces coelicolor M1146 to facilitate genetic manipulations of gou C and gou D. [Methods] By using fosmid D6-4H containing the complete gougerotin biosynthetic gene cluster, gou C and gou D were disrupted by PCR-targeting method to generate p GOUe-ΔC and p GOUe-ΔD. Both p GOUe-ΔC and p GOUe-ΔD were introduced into Streptomyces coelicolor M1146 by intergeneric conjugation, thus gou C and gou D disrpution mutants（M1146-GOUe-ΔC and M1146-GOUe-ΔD） were obtained. The gougerotin production of M1146-GOUe-ΔC and M1146-GOUe-ΔD were assayed by HPLC analysis. The intermediates accumulated in these mutants were purified and subjected to MS and NMR analyses for structure determinations. Bioassay of these intermediates against tumor cell line were also carried out. [Results] Disruption mutants of gou C and gou D failed to produce gougerotin and the mutants accumulated different gougerotin intermediates, which lost their ability to inhibit cancer cell proliferation.[Conclusion] gou C and gou D are key structual genes in the biosynthesis of gougerotin peptidyl moieties. This study will pave the way for the elucidation of gougerotin biosynthetic pathway.
出处
《微生物学报》
CAS
CSCD
北大核心
2016年第3期406-417,共12页
Acta Microbiologica Sinica
基金
国家“973计划”(2015CB150600)
国家自然科学基金(31270110,31571281)~~
关键词
禾粟链霉菌
谷氏菌素
生物合成基因簇
功能研究
产物鉴定
Streptomyces graminearus
gougerotin
gene cluster
functional characterization
compound identification
