Al-Hilfi Aimen, Walker Kevin D
Department of Chemistry, Michigan State University, East Lansing, MI, 48824, United States.
Department of Chemistry, Michigan State University, East Lansing, MI, 48824, United States; Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, United States.
Arch Biochem Biophys. 2022 Apr 15;719:109165. doi: 10.1016/j.abb.2022.109165. Epub 2022 Feb 25.
A 10-O-deacetylbaccatin III 10-O-acetyltransferase biocatalyst from Taxus plants was expressed in bacteria whole-cells that were fed 10-O-deacetylbaccatin III and cyclopropane carboxylic acid. Product analysis by qualitative LC/ESI-MS suggested that the C10-acylated products baccatin III, 10-O-n-propionyl-10-O-deacetylbaccatin III, and 10-O-cyclopropanecarbonyl-10-O-deacetylbaccatin III were made in vivo. The results implied that the cells provided non-natural cyclopropanecarbonyl CoA, from a broad-specificity CoA ligase, and natural products, acetyl CoA and n-propionyl CoA, from reserves in the bacteria for use by acyltransferase to acylate 10-O-deacetylbaccatin III in vivo. The 10-acyl-10-O-deacetylbaccatin III are precursors used to synthesize new-generation paclitaxel analogs SB-T-1214 and SB-T-121303, which are effective against cancer cells resistant to paclitaxel and its drug derivatives. The k and K of the acyltransferase for cyclopropanecarbonyl CoA (0.83 s, 0.15 M) and n-propionyl CoA (1.2 s, 0.15 M) guided scale-up efforts. The 10-acyl-10-O-deacetylbaccatin III analogs (∼45 mg each) were made in vitro by the acyltransferase when incubated with the commercial taxane 10-O-deacetylbaccatin III and synthesized cyclopropanecarbonyl or n-propionyl CoA. The structures of the 10-acyl products were verified by NMR analyses that confirmed C10 acylation of the taxane substrate. LC/ESI-MS/MS analysis also supported the identities of the biocatalyzed products. This effort provides a biocatalysis framework to produce new-generation taxane precursors.
从红豆杉属植物中提取的一种10-O-去乙酰浆果赤霉素III 10-O-乙酰转移酶生物催化剂在细菌全细胞中表达，该细菌全细胞以10-O-去乙酰浆果赤霉素III和环丙烷羧酸为原料。通过定性液相色谱/电喷雾电离质谱进行的产物分析表明，C10-酰化产物浆果赤霉素III、10-O-正丙酰基-10-O-去乙酰浆果赤霉素III和10-O-环丙烷羰基-10-O-去乙酰浆果赤霉素III是在体内合成的。结果表明，细胞提供了由广泛特异性辅酶A连接酶产生的非天然环丙烷羰基辅酶A，以及细菌储备中的天然产物乙酰辅酶A和正丙酰辅酶A，供酰基转移酶在体内将10-O-去乙酰浆果赤霉素III酰化。10-酰基-10-O-去乙酰浆果赤霉素III是用于合成新一代紫杉醇类似物SB-T-1214和SB-T-121303的前体，这些类似物对耐紫杉醇及其药物衍生物的癌细胞有效。酰基转移酶对环丙烷羰基辅酶A（0.83 s，0.15 M）和正丙酰辅酶A（1.2 s，0.15 M）的k和K指导了放大生产的努力。当与商业紫杉烷10-O-去乙酰浆果赤霉素III和合成的环丙烷羰基或正丙酰辅酶A一起孵育时，酰基转移酶在体外制备了10-酰基-10-O-去乙酰浆果赤霉素III类似物（各约45 mg）。通过核磁共振分析验证了10-酰基产物的结构，证实了紫杉烷底物的C10酰化。液相色谱/电喷雾电离串联质谱分析也支持了生物催化产物的身份。这项工作提供了一个生物催化框架来生产新一代紫杉烷前体。
