Biocatalytic synthesis of tritium ((3)H)-labelled taxa-4(5),11(12)-diene, the pathway committing precursor of the taxoid diterpenoids.
Availability of isotope-labelled metabolites often proves to be an essential prerequisite for the successful identification of a biosynthetic pathway. Also for the metabolic engineering isotope-labelled, either radioactive or non-radioactive, biosynthetic intermediates represent valuable tools for the assessment of metabolic flux, identification of unexpected biosynthetic side reactions, or for the confirmation of the functionality of the engineered reaction step. Most often the required compounds are neither available commercially nor prepared by chemical means within an acceptable time span and effort. Biocatalytic synthesis of early pathway intermediates may offer an attractive solution for this problem. For the metabolic engineering of taxol biosynthesis in a heterologous host, like yeast, isotope-labelled taxanes represent useful tools for the establishment and functional assessment of the introduced biosynthetic steps. Using Taxus chinensis taxadiene synthase expressed in the heterologous organism Pichia pastoris, we describe a method for the biocatalytic synthesis of tritium ((3)H)-labelled taxa-4(5),11(12)-diene, which represents the pathway committing biosynthetic precursor for all taxoid diterpenoids.
Schmeer
Hans
H
Institute of Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
Jennewein
Stefan
S
D000475:Alkenes / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D015153:Blotting, Western; D003001:Cloning, Molecular; D004224:Diterpenes / Q000302:isolation & purification / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D007535:Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017239:Paclitaxel / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D013194:Staining and Labeling; D043823:Taxoids / Q000378:metabolism*; D020946:Taxus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014316:Tritium
D000475:Alkenes; D004224:Diterpenes; D043823:Taxoids; C093125:taxa-4(5),11(12)diene; D014316:Tritium; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase; D017239:Paclitaxel