Carotenoids, a variety of natural products, have significant pharmaceutical and commercial potential. Phytoene dehydrogenase (CrtI) is the rate-limit enzyme for carotenoid synthesis, whose catalysis specificity results in various carotenoids. However, the structural characteristics of CrtI for controlling the catalysis specificity on dehydrogenation steps are still unclear, which limited the development of CrtI function. Here we confirmed two mutation sites H136 and H453 in the mutant library of CrtI from Blakeslea trispora, which markedly regulated catalytic specificity. Interestingly, the sequence alignment features at H136 and H453 were consistent with the phylogenetic analysis of CrtI families. Subsequently, the functions of saturated mutants at H136 and H453 were clustered by principal component analysis (PCA) and k-means. According to the clustering results, diversiform mutants with specific dehydrogenation function provided important application value for carotenoid product customization. Meanwhile, this study also enriched the theory of enzyme evolution and guided the functional development of enzymes.

中文翻译：

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探索植物脱氢酶CrtI在类胡萝卜素合成中的催化特异性。

类胡萝卜素是多种天然产物，具有巨大的制药和商业潜力。苯丙二烯脱氢酶（CrtI）是类胡萝卜素合成的限速酶，其催化特异性导致多种类胡萝卜素。然而，用于控制脱氢步骤的催化特异性的CrtI的结构特征仍然不清楚，这限制了CrtI功能的发展。在这里我们在来自Blakeslea trispora的CrtI突变体文库中确认了两个突变位点H136和H453，这明显调节了催化特异性。有趣的是，H136和H453的序列比对特征与CrtI家族的系统发育分析一致。随后，通过主成分分析（PCA）和k均值对H136和H453处的饱和突变体功能进行了聚类。根据聚类结果，具有特定脱氢功能的多样化突变体为类胡萝卜素产品定制提供重要的应用价值。同时，本研究还丰富了酶进化的理论，指导了酶的功能发展。