Abstract:
The diterpene ent-copalol is an important precursor to the synthesis of andrographolide and is found only in green chiretta (Andrographis paniculata). De novo biosynthesis of ent-copalol has not been reported, because the catalytic activity of ent-copalyl diphosphate synthase (CPS) is very low in microorganisms. In order to achieve the biosynthesis of ent-copalol, Saccharomyces cerevisiae was selected as the chassis strain, because its endogenous mevalonate pathway and dephosphorylases could provide natural promotion for the synthesis of ent-copalol. The strain capable of synthesizing diterpene geranylgeranyl pyrophosphate was constructed by strengthening the mevalonate pathway genes and weakening the competing pathway. Five full-length ApCPSs were screened by transcriptome sequencing of A. paniculata and ApCPS2 had the best activity and produced ent-CPP exclusively. The peak area of ent-copalol was increased after the ApCPS2 saturation mutation and its configuration was determined by NMR and ESI-MS detection. By appropriately optimizing acetyl-CoA supply and fusion-expressing key enzymes, 35.6 mg/L ent-copalol was generated. In this study, de novo biosynthesis and identification of ent-copalol were achieved and the highest titer ever reported. It provides a platform strain for the further pathway analysis of andrographolide and derivatives and provides a reference for the synthesis of other pharmaceutical intermediates.
摘要:
二萜-copalol是合成穿心莲内酯的重要前体,仅在绿色chiretta(穿心莲)中发现。从头生物合成的恩替-copalol尚未报道,因为在微生物中,ent-copalyl二磷酸合酶(CPS)的催化活性非常低。为了实现恩替卡醇的生物合成,选择酿酒酵母作为底盘菌株,因为其内源性甲羟戊酸途径和去磷酸酶可以为ent-copalol的合成提供自然促进。通过增强甲羟戊酸途径基因和减弱竞争途径,构建了能够合成二萜香叶基香叶基焦磷酸的菌株。通过A.paniculata的转录组测序筛选出五个全长ApCPSs,ApCPS2具有最佳活性并仅产生ent-CPP。ApCPS2饱和突变后,恩替卡醇的峰面积增加,其构型通过NMR和ESI-MS检测确定。通过适当优化乙酰辅酶A供应和融合表达关键酶,产生35.6mg/L的恩替卡洛尔。在这项研究中,从头生物合成和鉴定的恩替-copalol实现了有史以来的最高滴度。为穿心莲内酯及其衍生物的进一步途径分析提供了平台菌株,为其他药物中间体的合成提供了参考。