Main conclusion

The identification of a functional cinnamoyl-CoA reductase enzyme from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis offers the potential for enhancing trans-cinnamaldehyde production through genetic engineering.

A significant accumulation of trans-cinnamaldehyde has been found in the bark tissues of C. cassia, used in traditional Chinese medicine. trans-Cinnamaldehyde exhibits various pharmacological properties such as anti-inflammatory, analgesic, and protection of the stomach and the digestive tract. However, further elucidation and characterization of the biosynthetic pathway for trans-cinnamaldehyde is required. In this study, we conducted an integrated analysis of trans-cinnamaldehyde accumulation profiles and transcriptomic data from five different C. cassia tissues to identify the genes involved in its biosynthesis. The transcriptome data we obtained included nearly all genes associated with the trans-cinnamaldehyde pathway, with the majority demonstrating high abundance in branch barks and trunk barks. We successfully cloned four C. cassia cinnamoyl-CoA reductases (CcCCRs), a key gene in trans-cinnamaldehyde biosynthesis. We found that the recombinant CcCCR1 protein was the only one that more efficiently converted cinnamoyl-CoA into trans-cinnamaldehyde. CcCCR1 exhibited approximately 14.7-fold higher catalytic efficiency (k_cat/K_m) compared to the Arabidopsis thaliana cinnamoyl-CoA reductase 1 (AtCCR1); therefore, it can be utilized for engineering higher trans-cinnamaldehyde production as previously reported. Molecular docking studies and mutagenesis experiments also validated the superior catalytic activity of CcCCR1 compared to AtCCR1. These findings provide valuable insights for the functional characterization of enzyme-coding genes and hold potential for future engineering of trans-cinnamaldehyde biosynthetic pathways.

中文翻译：

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肉桂中参与反式肉桂醛生物合成的肉桂酰辅酶 A 还原酶的鉴定

主要结论

从肉桂中鉴定出参与反式肉桂醛生物合成的功能性肉桂酰辅酶A还原酶，为通过基因工程增强反式肉桂醛生产提供了潜力。

在中药决明子的树皮组织中发现了大量反式肉桂醛的积累。反式肉桂醛具有多种药理特性，例如抗炎、镇痛、保护胃和消化道。然而，需要进一步阐明和表征反式肉桂醛的生物合成途径。在这项研究中，我们对来自五种不同肉桂组织的反式肉桂醛积累谱和转录组数据进行了综合分析，以鉴定参与其生物合成的基因。我们获得的转录组数据几乎包括与反式肉桂醛途径相关的所有基因，其中大多数在树枝树皮和树干树皮中表现出高丰度。我们成功克隆了四种肉桂酰辅酶A还原酶（CcCCRs ），这是反式肉桂醛生物合成的关键基因。我们发现重组 CcCCR1 蛋白是唯一能更有效地将肉桂酰辅酶 A 转化为反式肉桂醛的蛋白。与拟南芥肉桂酰辅酶 A 还原酶 1 (AtCCR1)相比，CcCCR1 的催化效率 ( k _cat / K _m )高约 14.7 倍；因此，如之前报道的，它可用于设计更高的反式肉桂醛产量。分子对接研究和诱变实验也验证了 CcCCR1 与 AtCCR1 相比具有优越的催化活性。这些发现为酶编码基因的功能表征提供了有价值的见解，并为未来反式肉桂醛生物合成途径的工程设计提供了潜力。