Comparative analysis of CYP93E proteins for improved microbial synthesis of plant triterpenoids
Comparative analysis of CYP93E proteins for improved microbial synthesis of plant triterpenoids
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DOI：
10.1016/j.phytochem.2014.10.002
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发表时间：
2014-12-01
期刊：
影响因子：
3.8
通讯作者：
Pollier, Jacob
中科院分区：
文献类型：
--
作者：
Moses, Tessa;Thevelein, Johan M.;Pollier, Jacob
细胞色素p450依赖的单加氧酶（p450）属于CYP93E亚家族，在三萜皂苷的生物合成过程中催化三萜骨架的C-24氧化，三萜皂苷是具有生物活性的植物天然产物。在我们尝试在酿酒酵母中产生植物三萜的过程中，我们观察到紫花苜蓿CYP93E2在体内的催化效率很差。为了克服这一生物合成瓶颈，我们筛选了公开可用的CYP93E亚家族成员的植物基因组和转录组数据。从豆科植物中鉴定了6个CYP93E同源物，并对其进行了功能鉴定。尽管高度的氨基酸保守，CYP93E同源物在酵母菌的酶效率上表现出很大的差异。其中，菜豆CYP93E9活性最高，可将体内积累产生的底物β -amyrin转化为齐墩-12-烯-3 β、24-二醇和可能的3 -羟基齐墩-12-烯-24-酸，催化效率是truncatula CYP93E2的61倍。综上所述，我们已经将CYP93E的功能同源物列表扩展到总共9个蛋白，并表明它们在异源宿主中表达时的催化效率存在很大变化。虽然这里展示的是参与三萜皂苷生物合成的CYP93E家族，但这一现象无疑可以扩展到参与天然产物合成的其他酶家族。因此，同源酶的筛选可能成为合成生物学家设计优质生产底盘的宝贵工具。(c) 2014 Elsevier Ltd.版权所有。
Cytochrome P450-dependent monooxygenases (P450s) belonging to the CYP93E subfamily catalyze the C-24 oxidation of the triterpene backbone during the biosynthesis of triterpenoid saponins, which are bioactive plant natural products. In our attempts to produce plant triterpenoids in the yeast Saccharomyces cerevisiae, we observed a poor in vivo catalytic efficiency of the Medicago truncatula CYP93E2. To overcome this biosynthetic bottleneck, we screened publicly available plant genome and transcriptome data for CYP93E subfamily members. Six CYP93E orthologs, exclusively from leguminous plant species, were identified and functionally characterized in S. cerevisiae. Despite the high degree of amino acid conservation, the CYP93E orthologs showed large variations in enzymatic efficiency in yeast. The CYP93E9 from Phaseolus vulgaris showed the highest activity and converted similar to 80% of the accumulating in vivo produced substrate beta-amyrin to the products olean-12-ene-3 beta,24-diol and probable 3 beta-hydroxy olean-12-en-24-oic acid, with a catalytic efficiency that was 61 times higher than that of the M. truncatula CYP93E2. In conclusion, we have expanded the list of functional CYP93E orthologs to a total of nine proteins and show that there are large variations in their catalytic efficiencies when expressed in a heterologous host. Although demonstrated here for the CYP93E family involved in triterpenoid saponin biosynthesis, this phenomenon is undoubtedly extendable to other enzyme families involved in natural product synthesis. Hence, screening for homologous enzymes may become a valuable synthetic biologist's tool for engineering superior production chassis. (c) 2014 Elsevier Ltd. All rights reserved.