Huang Jin-Quan, Li Dong-Mei, Tian Xiu, Lin Jia-Ling, Yang Lei, Xu Jing-Jing, Fang Xin
, Yunnan University, Kunming 650091, P. R. China.
, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology/CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, P. R. China.
J Agric Food Chem. 2021 Feb 24;69(7):2168-2178. doi: 10.1021/acs.jafc.0c07462. Epub 2021 Feb 10.
Amorpha-4,11-diene synthase (ADS) is the first committed enzyme in the biosynthesis of artemisinin. Artemisinin production by biobased fermentation is considered a reliable alternative pathway. Heterologously expressed ADS has been established to generate several minor products, including structural analogues of amorpha-4,11-diene, but their fate in fermentation is still unknown. Here, using chiral analysis, we found that ADS produces one of the analogues, amorpha-4-en-11-ol, as a pair of epimers. Labeling experiments revealed that ADS mutants yielded amorphene-type sesquiterpenes, indicating the co-occurrence of initial 1,6 and 1,10 cyclization of farnesyl diphosphate in a single enzyme. Interestingly, the immediate downstream oxidase CYP71AV1 had very low affinity to the side products of the recombinant ADS, including amorpha-4-en-7-ol, which is structurally similar to amorpha-4,11-diene. Our data uncover the complex catalytic mechanism of recombinant ADS and reveal a potential negative effect of the side products of recombinant ADS on the production of the artemisinin precursor in microbes.
紫穗槐-4,11-二烯合酶（ADS）是青蒿素生物合成中的首个关键酶。通过生物基发酵生产青蒿素被认为是一条可靠的替代途径。已证实异源表达的ADS会产生几种次要产物，包括紫穗槐-4,11-二烯的结构类似物，但其在发酵中的去向仍不明确。在此，通过手性分析，我们发现ADS产生了其中一种类似物——紫穗槐-4-烯-11-醇，它以一对差向异构体的形式存在。标记实验表明，ADS突变体产生了紫穗槐烯型倍半萜，这表明在单一酶中同时发生了法呢基二磷酸的初始1,6和1,10环化反应。有趣的是，紧邻下游的氧化酶CYP71AV1对重组ADS的副产物，包括与紫穗槐-4,11-二烯结构相似的紫穗槐-4-烯-7-醇，亲和力非常低。我们的数据揭示了重组ADS复杂的催化机制，并揭示了重组ADS副产物对微生物中青蒿素前体生产的潜在负面影响。
