醌那霉素生物合成中间产物的一锅酶促体系研究

One-pot enzymatic total synthesis of intermediates in the biosynthesis of kinamycin

摘要:

【目的】 通过一锅酶法在体外无细胞条件下重构醌那霉素的生物合成途径,实现从原料辅酶A、乙酰-辅酶A和丙二酸到醌那霉素重要中间体的高效转化。【方法】 纯化得到AlpAB、RavC等8个醌那霉素合成相关蛋白和MCAT、MatB等2个辅助蛋白;利用一锅酶法进行体外反应,并用高效液相色谱(high performance liquid chromatography, HPLC)检测反应产物;利用该体系探究Ⅱ型硫酯酶AlpS在合成通路中的功能及作用底物;利用单一变量法对体系温度、pH、最小聚酮合酶(minimal polyketide synthase, minimal PKS)浓度和Ⅱ型硫酯酶AlpS浓度等条件进行优化。【结果】 体系所需的聚酮合酶和辅助蛋白均获得可溶性表达;利用一锅酶法成功合成了醌那霉素的早期重要中间体SEK15、UWM6、rabelomycin、prejadomycin和dehydrorabelomycin;加入AlpS蛋白后以上5种产物产量均有不同程度的提高,其中prejadomycin和dehydrorabelomycin提高较为显著;优化后的反应最适条件为:温度30℃、pH 7.0、最小聚酮合酶(AlpA、AlpB和RavC)浓度各2.8 μmol/L、AlpS 7.2 μmol/L;prejadomycin的产量提高到302 mg/L。【结论】 本研究成功利用一锅酶法在无细胞条件下重构了醌那霉素的早期生物合成途径,合成了醌那霉素的重要中间体SEK15、UWM6、rabelomycin、prejadomycin和dehydrorabelomycin。研究进一步验证了硫酯酶AlpS的链释放功能并推测其作用底物。

关键词:醌那霉素;一锅酶法;体外重构;II型聚酮化合物

Abstract:

[Objective] To reconstitute the partial biosynthetic pathway of the type II polyketide kinamycin in a cell-free system starting from CoA, acetyl-CoA and malonate.[Methods] The polyketide synthases (PKSs) and two supplemental proteins (MCAT and MatB) were purified and quantitated. The one-pot enzymatic synthesis of kinamycin intermediates was accomplished, and the products were analyzed by high performance liquid chromatography (HPLC). The system was used to explore the function and exact substrate of the stand-alone thioesterase, AlpS, in the biosynthetic pathway. The parameters including temperature, pH, and the concentrations of minimal PKSs and AlpS were optimized by the single-variable method. [Results] Eight PKSs and two supplemental proteins (MCAT and MatB) were expressed and purified. The intermediates and shunt products in kinamycin biosynthesis were produced in vitro, including SEK15, UWM6, rabelomycin, prejadomycin, and dehydrorabelomycin. The yields of these products, especially prejadomycin and dehydrorabelomycin, were all increased when AlpS was added. The optimal conditions were 30℃, pH 7.0, 2.8 μmol/L minimal PKSs (AlpA, AlpB, and RavC, respectively), and 7.2 μmol/L AlpS, under which the yield of prejadomycin was increased to 302 mg/L. [Conclusion] A one-pot enzymatic synthesis system with high yields of kinamycin-related products including SEK15, UWM6, rabelomycin, prejadomycin, and dehydrorabelomycin was successfully constructed. Furthermore, the chain-release function and substrate specificity of AlpS in the synthetic pathway were investigated.

Key words:kinamycin;one-pot enzymatic synthesis system;in vitro biosynthesis;type II polyketide