Dastmalchi Mehran, Chang Limei, Torres Miguel A, Ng Kenneth K S, Facchini Peter J
Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada.
Plant J. 2018 May 19. doi: 10.1111/tpj.13975.
Codeinone reductase (COR) catalyzes the reversible NADPH-dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3-O-demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O-demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR-B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1-1.4 and COR-B) were used to determine sequence-function relationships that influence product selectivity. Biochemical characterization and site-directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.
可待因酮还原酶（COR）催化可待因酮可逆的、依赖NADPH的还原反应生成可待因，这是罂粟（Papaver somniferum）中吗啡生物合成的倒数第二步。它还能不可逆地将新皮酮（由可待因酮在水溶液中自发异构化形成）还原为新皮啡。在涉及3 - O - 去甲基类似物的平行途径中，COR将吗啡酮转化为吗啡，将新吗啡酮转化为新吗啡。与新皮啡类似，COR生成新吗啡的过程是不可逆的。新皮啡是可待因O - 去甲基酶（CODM）的次要底物，可生成吗啡。在植物中，新皮啡水平较低，且未检测到新吗啡。CODM沉默会导致上游代谢物如可待因和蒂巴因的积累，但不会导致新皮啡相对浓度升高，这表明植物中存在限制新皮啡产生的机制。在经过基因工程改造以生产阿片生物碱的酵母（Saccharomyces cerevisiae）中，COR的催化特性导致新皮啡和新吗啡作为主要产物积累。从罂粟化学型Bea's Choice中分离出一种同工型（COR - B），其催化活性高于先前表征的COR同工型，并且在体外和工程酵母中主要生成新皮啡。使用五种催化特性不同的COR同工型（COR1.1 - 1.4和COR - B）来确定影响产物选择性的序列 - 功能关系。对天然COR同工型的生化表征和定点诱变确定了四个影响蛋白质稳定性、反应速度、产物选择性和产量的残基（V25、K41、F129和W279）。提高COR性能并具备引导途径通量的能力对于促进工程微生物中阿片生物碱的商业化生产是必要的。
