Cloning and functional analysis of the phenylalanine ammonia-lyase gene from Anthoceros angustus.
- Author:
Haina YU
1
;
Jian MO
1
;
Jiayi YANG
1
;
Xiaochun QIN
1
Author Information
- Publication Type:Journal Article
- Keywords: Anthoceros angustus; expression analysis; gene cloning; kinetics analysis of enzymes; phenylalanine ammonia-lyase; rosmarinic acid; whole-cell catalysis
- MeSH: Phenylalanine Ammonia-Lyase/metabolism*; Cloning, Molecular; Cinnamates/metabolism*; Recombinant Proteins/metabolism*; Rosmarinic Acid; Depsides/metabolism*; Escherichia coli/metabolism*; Amino Acid Sequence; Plant Proteins/metabolism*; Phylogeny; Acetates/pharmacology*; Cyclopentanes; Oxylipins
- From: Chinese Journal of Biotechnology 2025;41(7):2855-2870
- CountryChina
- Language:Chinese
- 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 ℃ and pH 8.0. The Km and kcat 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.
