1.Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata.
Jincui YE ; Ping ZHANG ; Jieyin SUN ; Chaotan GUO ; Guoshen CHEN ; Ikuro ABE ; Hiroshi NOGUCHI
Acta Pharmaceutica Sinica 2011;46(10):1273-8
A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).
2.Cloning, expression and functional identification of a type III polyketide synthase gene from Huperzia serrata.
Jin-cui YE ; Ping ZHANG ; Jie-yin SUN ; Chao-tan GUO ; Guo-shen CHEN ; Ikuro ABE ; Hiroshi NOGUCHI
Acta Pharmaceutica Sinica 2011;46(10):1273-1278
A cDNA encoding novel type III polyketide synthase (PKS) was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata (Thunb.) Trev. by RT-PCR using degenerated primers based on the conserved sequences of known CHSs, and named as H. serrata PKS2. The terminal sequences of cDNA were obtained by the 3'- and 5'-RACE method. The full-length cDNA of H. serrata PKS2 contained a 1212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids. The deduced amino acid sequence of H. serrata PKS2 showed 50%-66% identities to those of other chalcone synthase super family enzymes of plant origin. The recombinant H. serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 1, 3-dihydroxy-N-methylacridone. Interestingly, H. serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).
Acyltransferases
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genetics
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isolation & purification
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metabolism
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Amino Acid Sequence
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Cloning, Molecular
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DNA, Complementary
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genetics
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DNA, Plant
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genetics
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Escherichia coli
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genetics
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metabolism
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Gene Expression Regulation, Plant
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Huperzia
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enzymology
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genetics
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Molecular Sequence Data
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Plant Leaves
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enzymology
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genetics
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Plants, Medicinal
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enzymology
;
genetics
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Recombinant Proteins
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genetics
;
metabolism
;
Sequence Alignment
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Substrate Specificity
3.Extensive expansion of the chemical diversity of fusidane-type antibiotics using a stochastic combinational strategy.
Xiaojun SONG ; Jianming LV ; Zhiqin CAO ; Huiyun HUANG ; Guodong CHEN ; Takayoshi AWAKAWA ; Dan HU ; Hao GAO ; Ikuro ABE ; Xinsheng YAO
Acta Pharmaceutica Sinica B 2021;11(6):1676-1685
Fusidane-type antibiotics, represented by helvolic acid, fusidic acid and cephalosporin P
4.Biosynthesis of clinically used antibiotic fusidic acid and identification of two short-chain dehydrogenase/reductases with converse stereoselectivity.
Zhiqin CAO ; Shaoyang LI ; Jianming LV ; Hao GAO ; Guodong CHEN ; Takayoshi AWAKAWA ; Ikuro ABE ; Xinsheng YAO ; Dan HU
Acta Pharmaceutica Sinica B 2019;9(2):433-442
Fusidic acid is the only fusidane-type antibiotic that has been clinically used. However, biosynthesis of this important molecule in fungi is poorly understood. We have recently elucidated the biosynthesis of fusidane-type antibiotic helvolic acid, which provides us with clues to identify a possible gene cluster for fusidic acid ( cluster). This gene cluster consists of eight genes, among which six are conserved in the helvolic acid gene cluster except and . Introduction of the two genes into the NSAR1 expressing the conserved six genes led to the production of fusidic acid. A stepwise introduction of and revealed that the two genes worked independently without a strict reaction order. Notably, we identified two short-chain dehydrogenase/reductase genes and in the cluster, which showed converse stereoselectivity in 3-ketoreduction. This is the first report on the biosynthesis and heterologous expression of fusidic acid.