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Side Products of Recombinant Amorpha-4,11-diene Synthase and Their Effect on Microbial Artemisinin Production

2021; American Chemical Society; Volume: 69; Issue: 7 Linguagem: Inglês

10.1021/acs.jafc.0c07462

1520-5118

Jinquan Huang, Dongmei Li, Xiu Tian, Jia‐Ling Lin, Lei Yang, Jing-Jing Xu, Xin Fang,

Microbial Natural Products and Biosynthesis

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.