Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.
A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
Geerlings
A
A
Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Redondo
F J
FJ
Contin
A
A
Memelink
J
J
van der Heijden
R
R
Verpoorte
R
R
D001709:Biotechnology / Q000379:methods; D019759:Carbon-Nitrogen Lyases / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D005638:Fruit / Q000737:chemistry / Q000378:metabolism*; D005818:Genetic Engineering; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism; D026121:Indole Alkaloids / Q000378:metabolism*; D057888:Iridoid Glucosides; D039823:Iridoids; D011714:Pyrans / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D019076:Transgenes; D014363:Tryptamines / Q000378:metabolism*
D003470:Culture Media; D026121:Indole Alkaloids; D057888:Iridoid Glucosides; D039823:Iridoids; D011714:Pyrans; D014363:Tryptamines; C021086:secologanin; C030820:tryptamine; D005959:Glucosidases; D019759:Carbon-Nitrogen Lyases; C022684:strictosidine synthetase