Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
ACS Chem Biol. 2023 Feb 17;18(2):419-430. doi: 10.1021/acschembio.2c00873. Epub 2023 Feb 3.
Systematic screening of morphine pathway intermediates in engineered yeast revealed key biosynthetic enzymes displaying potent feedback inhibition: 3'-hydroxy--methylcoclaurine 4'-methyltransferase (4'OMT), which yields ()-reticuline, and the coupled salutaridinol-7--acetyltransferase (SalAT) and thebaine synthase (THS2) enzyme system that produces thebaine. The addition of deuterated reticuline-d1 to a yeast strain able to convert ()-norcoclaurine to ()-reticuline showed reduced product accumulation in response to the feeding of all four successive pathway intermediates. Similarly, the addition of deuterated thebaine-d3 to a yeast strain able to convert salutaridine to thebaine showed reduced product accumulation from exogenous salutaridine or salutaridinol. In vitro analysis showed that reticuline is a noncompetitive inhibitor of 4'OMT, whereas thebaine exerts mixed inhibition on SalAT/THS2. In a yeast strain capable of de novo morphine biosynthesis, the addition of reticuline and thebaine resulted in the accumulation of several pathway intermediates. In contrast, morphine had no effect, suggesting that circumventing the interaction of reticuline and thebaine with 4'OMT and SalAT/THS2, respectively, could substantially increase opiate alkaloid titers in engineered yeast.
系统筛选吗啡途径中间产物在工程酵母中揭示了关键的生物合成酶，表现出强烈的反馈抑制：3'-羟基-β-甲基可可碱 4'-甲基转移酶（4'OMT），它产生（）-reticuline，以及偶联的salutaridinol-7-O-乙酰转移酶（SalAT）和thebaine 合酶（THS2）酶系统，产生 thebaine。向能够将（）-norcoclaurine 转化为（）-reticuline 的酵母菌株中添加氘代 reticuline-d1，在添加所有四个连续途径中间产物时，产物积累减少。同样，向能够将 salutaridine 转化为 thebaine 的酵母菌株中添加氘代 thebaine-d3，从外源 salutaridine 或 salutaridinol 转化而来的产物积累减少。体外分析表明，reticuline 是非竞争性 4'OMT 的抑制剂，而 thebaine 对 SalAT/THS2 表现出混合抑制作用。在能够从头合成吗啡的酵母菌株中，添加 reticuline 和 thebaine 导致途径中间产物的积累。相比之下，吗啡没有影响，表明绕过 reticuline 和 thebaine 分别与 4'OMT 和 SalAT/THS2 的相互作用，可以大大增加工程酵母中鸦片生物碱的含量。
