变铅青链霉菌体内游离胞嘧啶的发现及检测

Discovery and detection of free cytosine in Streptomyces lividans

【背景】胞嘧啶(cytosine,C)是核酸分子4种基本碱基之一。胞嘧啶首先是以胞苷三磷酸(cytosine triphosphate,CTP)的形式合成,在核酸基础代谢中没有形成游离胞嘧啶的特定途径。杀稻瘟菌素(blasticidin S,BS)和谷氏菌素生物合成途径均以游离的胞嘧啶为前体,前者的生物合成基因簇中包含一个能水解胞苷单磷酸(cytidine monophosphate,CMP)生成胞嘧啶的水解酶BlsM,后者的生物合成基因簇及其产生菌的基因组中均没有这个水解酶对应的同源蛋白。【目的】检测不同细菌中是否普遍存在游离的胞嘧啶,探究是否存在能产生游离胞嘧啶的同工酶或新途径。【方法】在BS异源表达菌株Streptomyces lividans WJ2中敲除blsM,高效液相色谱(HPLC)检测突变株和WJ2发酵产物;液相色谱-质谱联用(LC-MS)检测10个经过分级纯化的微生物细胞裂解液上清中是否存在游离的胞嘧啶。【结果】突变株WJ2ΔblsM菌株仍能合成杀稻瘟菌素,但各组分产量与WJ2菌株相比均明显降低;除了变铅青链霉菌,在10株被检测的菌株中,金黄色葡萄球菌(Staphylococcusaureus)、地中海拟无枝酸菌(Amycolatopsismediterranei)、枯草芽孢杆菌(Bacillussubtilis)也检测到了含量较高的游离胞嘧啶。【结论】WJ2ΔblsM菌株仍具有产生BS的能力,说明野生型Streptomyceslividans菌株内存在一种未曾发现的游离胞嘧啶的产生方式,可以满足次级代谢的需求。另外,不同微生物中游离胞嘧啶的含量不同。

[Background]Cytosine is one of the four basic bases of nucleic acids.Cytosine is firstly synthesized in the form of cytosine triphosphate.There is no specific primary pathway for the formation of free cytosine in nucleic acid primary metabolism.The biosynthetic pathways of blasticidin S and gougerotin both utilize free cytosine as the precursor.The biosynthetic gene cluster of blasticidin S contains a hydrolase BlsM that can hydrolyze cytidine monophosphate into cytosine,while gougerotin producer doesn’t encodes a homolog to BlsM.[Objective]To detect the existence of free cytosine in different bacteria,and to explore whether there is an isoenzyme or a new pathway to produce free cytosine.[Methods]blsM was knocked out in blasticidin S heterologous producer Streptomyces lividans WJ2,its fermentation products of the mutant strain and WJ2 were detected by high performance liquid chromatography.Free cytosine in the supernatant of 10 fractionated cell lysates was measured by LC-MS.[Results]The mutant WJ2ΔblsM strain still synthesizes blasticidin S,but the yield of each component is significantly lower than that of WJ2.In addition to Streptomyces lividans,free cytosine was detected in Staphylococcus aureus,Amycolatopsis mediterranei and Bacillus subtilis.[Conclusion]WJ2ΔblsM still produces blasticidin S,indicating that wild-type Streptomyces lividans has an uncovered pathway for synthesis of free cytosine to meet blasticidin S synthesis.The content of free cytosine varies in different microorganisms.