One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces cerevisiae cells.
For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as "enzyme bags" and incubated at 30 degrees C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 microg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26 degrees C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.
Katsuyama
Yohei
Y
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan.
Miyahisa
Ikuo
I
Funa
Nobutaka
N
Horinouchi
Sueharu
S
D000103:Acetyl-CoA Carboxylase / Q000235:genetics; D000217:Acyltransferases / Q000235:genetics; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics; D003373:Coumaric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D044950:Flavanones / Q000096:biosynthesis; D015870:Gene Expression; D019833:Genistein / Q000378:metabolism*; D006035:Glycyrrhiza / Q000201:enzymology / Q000235:genetics; D019753:Intramolecular Lyases / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000031:analogs & derivatives / Q000494:pharmacology; D010105:Oxygenases / Q000235:genetics; D010650:Phenylalanine Ammonia-Lyase / Q000235:genetics; D011401:Promoter Regions, Genetic; D011422:Propionates; D029908:Pueraria / Q000201:enzymology / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D003373:Coumaric Acids; D044950:Flavanones; D011422:Propionates; D007544:Isopropyl Thiogalactoside; D014443:Tyrosine; C070641:isopropyl-beta-galactopyranoside; D019833:Genistein; D010105:Oxygenases; C048489:isoflavone synthase; D000217:Acyltransferases; C020141:flavanone synthetase; D010650:Phenylalanine Ammonia-Lyase; D019753:Intramolecular Lyases; C019865:chalcone isomerase; D003066:Coenzyme A Ligases; C021393:hydroxycinnamate-CoA ligase; D000103:Acetyl-CoA Carboxylase; C005273:naringenin; C495469:p-coumaric acid