Crocetin, a high-value apocarotenoid in saffron, is widely applied to the fields of food and medicine. However, the existing method of obtaining crocetin through large-scale cultivation is far from meeting the market demand. Microbial synthesis of crocetin is a potential alternative to traditional resources, and it is found that carotenoid cleavage dioxygenase (CCD) is the critical enzyme to synthesize crocetin. So, in this study, we used “hybrid-tunnel” engineering to obtain variants of Crocus sativus-derived CsCCD2, essential for zeaxanthin conversion into crocetin, with a broader substrate specificity and higher catalytic efficiency. Variants including S323A, with a lower charge bias and a larger tunnel size than the wild-type, showed a 5-fold higher crocetin titer in yeast-based fermentations. S323A could also convert the β-carotene substrate to crocetin dialdehyde and exhibited a 12.83-fold greater catalytic efficiency (k_cat/K_m) toward zeaxanthin than the wild-type in vitro. This strategy enabled the production of 107 mg/L crocetin in 5 L fed-batch fermentation, higher than that previously reported. Our findings demonstrate that engineering access tunnels to expand the substrate profile by in silico protein design represents a viable strategy to refine the catalytic properties of enzymes across a range of applications.

中文翻译：

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CsCCD2 通道设计用于在藏红花素生产中获得更广泛的底物轮廓

番红花素是藏红花中的一种高价值的类胡萝卜素，广泛应用于食品和医药领域。但是，现有的通过大规模种植获得藏红花素的方法远远不能满足市场需求。微生物合成藏红花素是传统资源的潜在替代品，发现类胡萝卜素裂解双加氧酶（CCD）是合成藏红花素的关键酶。因此，在这项研究中，我们使用“混合隧道”工程来获得番红花衍生的Cs 的变体CCD2 是玉米黄质转化为藏红花素所必需的，具有更广泛的底物特异性和更高的催化效率。与野生型相比，包括 S323A 在内的变体具有更低的电荷偏置和更大的隧道尺寸，在基于酵母的发酵中表现出高 5 倍的藏红花素滴度。S323A 还可以将 β-胡萝卜素底物转化为番红花素二醛，并且在体外对玉米黄质的催化效率 ( k _cat / K _m ) 是野生型的 12.83 倍。该策略能够在 5 L 补料分批发酵中生产 107 mg/L 的藏红花素，高于之前报道的产量。我们的研究结果表明，工程通道可以通过计算机技术扩展基板轮廓 蛋白质设计代表了一种可行的策略，可以在一系列应用中改进酶的催化特性。