Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin Ⅲ to baccatin Ⅲ  

作　　者：Hao Wang Bo-Yong Zhang Ting Gong Tian-Jiao Chen Jing-Jing Chen Jin-Ling Yang Ping Zhu 

机构地区：[1]State Key Laboratory of Bioactive Substance and Function of Natural Medicines,NHC Key Laboratory of Biosynthesis of Natural Products,CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs,Institute of Materia Medica,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China

出　　处：《Acta Pharmaceutica Sinica B》2021年第10期3322-3334,共13页药学学报（英文版）

基　　金：supported by the National Key Research and Development Program of China(grant Nos.2018YFA0901900 and 2020YFA0908003);the Drug Innovation Major Project(grant No.2018ZX09711001-006-001,China);the National Natural Science Foundation of China(grant No.81573325);the CAMS Innovation Fund for Medical Sciences(CIFMS,(grant No.2017-I2M-2-004,2019-I2M-1-005,China);PUMC Disciplinary Development of Synthetic Biology(201920100801,China)。

摘　　要：10-DeacetylbaccatinⅢ(10-DAB)C10 acetylation is an indispensable procedure for Taxol semi-synthesis,which often requires harsh conditions.10-DeacetylbaccatinⅢ-10-β-O-acetyltransferase(DBAT)catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor.Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell.Then,we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatinⅢ.We found that baccatinⅢcould be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+under alkaline condition.Thus,we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatinⅢ.The fermentation conditions were optimized and the baccatinⅢtiters reached 2,3 and 4.6 g/L,respectively,in a 3-L bioreactor culture when 2,3 and 6 g/L of 10-DAB were supplied.Our study provides an environmentfriendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis.The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.

关 键 词：Microbial cell factory Biosynthetic pathway of acetyl-CoA Bioconversion of 10-deacetylbaccatinⅢto baccatinⅢ 10-DeacetylbaccatinⅢ-10-β-Oacetyltransferase Deacetylation of baccatinⅢ High-cell-density fermentation Taxol semi-synthesis 

分 类 号：R284.3[医药卫生—中药学]