Screening of ent-copalyl diphosphate synthase and metabolic engineering to achieve de novo biosynthesis of ent-copalol in Saccharomyces cerevisiae  

作　　者：Shan Li Shuangshuang Luo Xinran Yin Xingying Zhao Xuyang Wang Song Gao Sha Xu Jian Lu Jingwen Zhou 

机构地区：[1]Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology,School of Biotechnology,Jiangnan University,1800 Lihu Road,Wuxi,Jiangsu,214122,China [2]School of Biotechnology,Jiangnan University,1800 Lihu Road,Wuxi,Jiangsu,214122,China [3]Science Center for Future Foods,Jiangnan University,1800 Lihu Rd,Wuxi,Jiangsu,214122,China

出　　处：《Synthetic and Systems Biotechnology》2024年第4期784-792,共9页合成和系统生物技术(英文)

基　　金：supported by the National Natural Science Foundation of China(22278188);the National Natural Science Foundation of China(22208123);the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study(Grant No.SN-ZJU-SIAS-0013);the National First-class Discipline Program of Light Industry Technology and Engineering(QGJC20230102).

摘　　要：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.

关 键 词：Andrographis paniculata Ent-copalyl diphosphate synthase ent-CPP Diterpene synthase ACETYL-COA Quantification 

分 类 号：O62[理学—有机化学]