CsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production,Journal of Agricultural and Food Chemistry

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CsCCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2021-09-23 , DOI: 10.1021/acs.jafc.1c04588
Nan Liang _{1,

2} , Ming-Dong Yao _{1,

2} , Ying Wang _{1,

2} , Jia Liu ₃ , Lu Feng ₃ , Zhi-Ming Wang ₄ , Xiang-Yu Li ₄ , Wen-Hai Xiao _{1,

2,

5} , Ying-Jin Yuan _{1,

2}

Affiliation

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：

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。

更新日期：2021-10-06

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