芽胞角苔苯丙氨酸解氨酶基因的克隆及功能分析

Cloning and functional analysis of the phenylalanine ammonia-lyase gene from Anthoceros angustus

摘要:

苔藓植物芽胞角苔(Anthoceros angustus Steph.)中含有丰富的迷迭香酸等酚酸类化合物。苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)是植物苯丙烷代谢途径的入口酶，对迷迭香酸的生物合成具有重要作用。为研究芽胞角苔PAL对迷迭香酸合成的重要作用，从芽胞角苔中克隆到2个苯丙氨酸解氨酶基因AanPAL1和AanPAL2，分别编码755个和753个氨基酸，均包含PAL保守结构域和核心活性氨基酸残基Ala-Ser-Gly。在系统进化树上与苔藓、蕨类植物PALs聚在一个分支，与小立碗藓的进化距离最近。实时荧光定量PCR分析表明，AanPAL1和AanPAL2基因的表达受外源茉莉酸甲酯的诱导。高效液相色谱分析显示，迷迭香酸的含量在茉莉酸甲酯处理后也显著增加。利用大肠杆菌进行异源表达，纯化后进行功能表征发现，AanPALs可高效催化l-苯丙氨酸(l-phenylalanine, l-Phe)生成反式肉桂酸，最适温度和pH分别为50℃、pH 8.0。AanPAL1和AanPAL2的K_m、k_cat分别为0.062 mmol/L、4.35 s^–1和0.198 mmol/L、14.48 s^–1；比酶活分别为2.61 U/mg和8.76 U/mg。两者热稳定性较差，但pH稳定性较好。全细胞催化结果显示，AanPAL2在10 h内可催化1 g/L的l-Phe转化成反式肉桂酸。本研究结果为深入分析AanPAL在芽胞角苔迷迭香酸生物合成中的调控作用奠定了基础，同时为生物合成肉桂酸提供了重要的候选酶基因。

Abstract:

Anthoceros angustus Steph. is rich in phenolic acids such as rosmarinic acid (RA). Phenylalanine ammonia-lyase (PAL) is an entry enzyme in the phenylpropanoid pathway of plants, playing an important role in the biosynthesis of RA. To investigate the important role of PAL in rosmarinic acid synthesis, two PAL genes (designated as AanPAL1 and AanPAL2) were cloned from A.angustus, encoding 755 and 753 amino acid residues, respectively. The AanPAL deduced amino acid sequences contain the conserved domains of PAL and the core active amino acid residues Ala-Ser-Gly. The phylogenetic analysis indicated that AanPAL1 and AanPAL2 were clustered with PALs from bryophytes and ferns and had the shortest evolutionary distance with the PALs from Physcomitrella patens. Quantitative real-time PCR results showed that the expression of AanPAL1 and AanPAL2 was induced by exogenous methyl jasmonate (MeJA). HPLC results showed that the MeJA treatment significantly increased the accumulation of RA. AanPAL1 and AanPAL2 were expressed in Escherichia coli and purified by histidine-tag affinity chromatography. The recombinant proteins catalyzed the conversion of l-phenylalanine to generate trans-cinnamic acid with high efficiency, with the best performance at 50 °C and pH 8.0. The K_m and k_cat of AanPAL1 were 0.062 mmol/L and 4.35 s^–1, and those of AanPAL2 were 0.198 mmol/L and 14.48 s^–1, respectively. The specific activities of AanPAL1 and AanPAL2 were 2.61 U/mg and 8.76 U/mg, respectively. The two enzymes had relatively poor thermostability but good pH stability. The high activity of AanPAL2 was further confirmed via whole-cell catalysis with recombinant E.coli, which could convert 1 g/L l-phenylalanine into trans-cinnamic acid with a yield of 100% within 10 h. These results give insights into the regulatory role of AanPAL in the biosynthesis of RA in A.angustus and provide candidate enzymes for the biosynthesis of cinnamic acid.