螺旋霉素生物合成基因簇中N,N-二甲基转移酶基因功能的鉴定

Identification of N, N-dimethyltransferase gene function in spiramycin biosynthesis gene cluster

目的 确定螺旋霉素(SP)生物合成途径中编码两个N, N-二甲基转移酶的基因bsm9和bsm22的功能,并在基因阻断变株中分离纯化相应的SP前体物。方法 利用抗性基因插入或CRISPR-Cas基因编辑的方法阻断bsm9和bsm22基因,并对其进行基因回复实验。利用薄层色谱(TLC)和高效液相色谱-质谱(HPLC-MS)联用分析变株Δbsm9和Δbsm22的发酵产物。利用半制备液相色谱的方法分离纯化缺少甲基化修饰的SP前体物,经核磁共振波谱(NMR)数据分析确定其化学结构。结果 成功构建了两个基因的阻断株Δbsm9和Δbsm22。Δbsm9变株的发酵液没有抗菌活性的物质产生,经HPLC-MS分析也未发现相应的SP前体物。而Δbsm22变株的发酵产物的抗菌活性下降50%以上,从Δbsm22变株发酵产物中分离出福洛胺糖缺少两个甲基的SP前体物。结论 bsm22基因负责SP生物合成中福洛胺糖上的N, N-二甲基的催化,而bsm9基因可能参与麦洛胺糖的N, N-二甲基化修饰,且两者在功能上不能互补。

Objective To confirm the function of the two N, N-dimethyltransferase genes bsm9 and bsm22 in the spiramycin(SP) biosynthetic pathway, and to isolate and purify the corresponding SP precursor from these gene disruption mutants.Methods Antibiotic resistance gene insertion or CRISPR-Cas gene editing were used to bsm9 and bsm22 gene disruption and the gene complementation was also carried out in the mutants, respectively. Thin-layer chromatography(TLC) and high-performance liquid chromatography-mass spectrometry(HPLC-MS) were employed for analyze the fermentation products of the mutants Δbsm9and Δbsm22. The demethylated SP precursors were purified by semi-preparative liquid chromatography, and its chemical structure was elucidated by nuclear magnetic resonance spectroscopy(NMR).Results Two gene disruption strains Δbsm9 and Δbsm22 were successfully constructed. The fermentation broth of the Δbsm9mutant showed little antibacterial activity, and the corresponding SP precursors were not found by HPLC-MS analysis. However, the antibacterial activity of the fermentation product of the Δbsm22 decreased by more than 50%. The fermentation product isolated from the Δbsm22 was an SP precursor with forosamine devoid of N, N-dimethylation.Conclusion The bsm22 gene is responsible for the N, N-dimethylation of forosamine in SP biosynthesis. The bsm9 gene is probably involved in the N, N-dimethylation of mycaminose. No complementation was observed between the bsm9 and bsm22.