Construction and expression of a dual vector for chemo-enzymatic synthesis of plant indole alkaloids in Escherichia coli.
A dual vector (pQE-70-STR1-SG) containing coding regions of strictosidine synthase (STR1, EC 4.3.3.2) and strictosidine glucosidase (SG, EC 3.2.1.105) from the Indian medicinal plant Rauvolfia serpentina was constructed. Functional expression of the vector in Escherichia coli cells (M15 strain) was proven by isolation of prepurified enzyme extracts, which show both STR1 and SG activities. Incubation of the enzyme in the presence of tryptamine and secologanin delivered the indole alkaloid cathenamine, demonstrating functional co-expression of both STR1- and SG-cDNAs. Cathenamine reduction by sodium borohydride leading to tetrahydroalstonine revealed the chemo-enzymatic indole alkaloid synthesis.
Stockigt
Joachim
J
College of Pharmaceutical Sciences, Institute of Materia Medica, Zhejiang University, Hangzhou 310058, P.R. China. joesto2000@yahoo.com
Hammes
Bodo
B
Ruppert
Martin
M
D019759:Carbon-Nitrogen Lyases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism*; D026121:Indole Alkaloids / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011926:Rauwolfia / Q000201:enzymology* / Q000235:genetics; D046948:Secologanin Tryptamine Alkaloids / Q000737:chemistry
D018076:DNA, Complementary; D026121:Indole Alkaloids; D010940:Plant Proteins; D046948:Secologanin Tryptamine Alkaloids; C551413:cathenamine; C005709:raubasine; D005959:Glucosidases; C456847:strictosidine glucosidase; D019759:Carbon-Nitrogen Lyases; C022684:strictosidine synthetase