One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces cerevisiae cells

• DOI: 10.1007/s00253-006-0568-2
• Publication Date: 2006-09-07T14:37:53Z
• Authors: Yohei Katsuyama; Ikuo Miyahisa; Nobutaka Funa; Sueharu Horinouchi
• Keywords: Isopropyl Thiogalactoside; 0301 basic medicine; Coumaric Acids; Gene Expression; Streptomyces coelicolor; Saccharomyces cerevisiae; Genistein; 3. Good health; Corynebacterium glutamicum; Pueraria; 03 medical and health sciences; Coenzyme A Ligases; Flavanones; Escherichia coli; Glycyrrhiza; Oxygenases; Cloning, Molecular; Propionates; Intramolecular Lyases; Promoter Regions, Genetic; Acyltransferases; Acetyl-CoA Carboxylase; Phenylalanine Ammonia-Lyase

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.