An Intronless β-amyrin Synthase Gene is More Efficient in Oleanolic Acid Accumulation than its Paralog in Gentiana straminea.
Paralogous members of the oxidosqualene cyclase (OSC) family encode a diversity of enzymes that are important in triterpenoid biosynthesis. This report describes the isolation of the Gentiana straminea gene GsAS2 that encodes a β-amyrin synthase (βAS) enzyme. Unlike its previously isolated paralog GsAS1, GsAS2 lacks introns. Its predicted protein product was is a 759 residue polypeptide that shares high homology with other known β-amyrin synthases (βASs). Heterologously expressed GsAS2 generates more β-amyrin in yeast than does GsAS1. Constitutive over-expression of GsAS2 resulted in a 5.7 fold increase in oleanolic acid accumulation, while over-expression of GsAS1 led to a 3 fold increase. Additionally, RNAi-directed suppression of GsAS2 and GsAS1 in G. straminea decreased oleonolic acid levels by 65.9% and 21% respectively, indicating that GsAS2 plays a more important role than GsAS1 in oleanolic acid biosynthesis in G. straminea. We uses a docking model to explore the catalytic mechanism of GsAS1/2 and predicted that GsAS2, with its Y560, have higher efficiency than GsAS1 and mutated versions of GsAS2 in β-amyrin produce. When the key residue in GsAS2 was mutagenized, it produced about 41.29% and 71.15% less β-amyrin than native, while the key residue in GsAS1 was mutagenized to that in GsAS2, the mutant produced 38.02% more β-amyrin than native GsAS1.
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.|Department of Information Engineering, Laiwu Vocational and Technical College, Laiwu 271100, China.
Yanling
Y
Liu
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.|Key Laboratory for Applied Microbiology of Shandong Province, Ecology Institute of Shandong Academy of Sciences, Jinan 250014, China.
Zhongjuan
Z
Zhao
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Fragrant Hill, Beijing 100093, China.
Zheyong
Z
Xue
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Long
L
Wang
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Yunfei
Y
Cai
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Peng
P
Wang
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Tiandi
T
Wei
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Jing
J
Gong
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Zhenhua
Z
Liu
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Juan
J
Li
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Shuo
S
Li
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China.
Fengning
F
Xiang
D055162:Biocatalysis; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D030012:Gentiana / Q000201:enzymology* / Q000235:genetics*; D019751:Intramolecular Transferases / Q000235:genetics* / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009828:Oleanolic Acid / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D050505:Mutant Proteins; D009828:Oleanolic Acid; D019751:Intramolecular Transferases; C039924:2,3-oxidosqualene-beta-amyrin-cyclase; C036380:beta-amyrin