@article {Willits:2004p8030,
	title = {Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites},
	journal = {Phytochemistry},
	volume = {65},
	number = {1},
	year = {2004},
	month = {Jan},
	pages = {31{\textendash}41},
	abstract = {A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro. Additionally, using mixed cultures of E. coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed. This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach.},
	keywords = {Amino Acid Sequence, Arabidopsis, Cloning: Molecular, Consensus Sequence, Escherichia coli, Fermentation, Flavonoids, Glucosyltransferases, Mentha piperita, Methyltransferases, Molecular Sequence Data, Onions, Phylogeny, Recombinant Proteins, Sequence Alignment, Sequence Homology: Amino Acid, Stereoisomerism, Substrate Specificity},
	author = {Michael G Willits and Ma{\"{\i}t{\'e} Giovanni and Rogerio T N Prata and Catherine M Kramer and Vincenzo De Luca and John C Steffens and Gerson Graser}
}
@article {Gang:2002p8027,
	title = {Characterization of phenylpropene O-methyltransferases from sweet basil: facile change of substrate specificity and convergent evolution within a plant O-methyltransferase family},
	journal = {Plant Cell},
	volume = {14},
	number = {2},
	year = {2002},
	month = {Feb},
	pages = {505{\textendash}19},
	abstract = {Some basil varieties are able to convert the phenylpropenes chavicol and eugenol to methylchavicol and methyleugenol, respectively. Chavicol O-methyltransferase (CVOMT) and eugenol O-methyltransferase (EOMT) cDNAs were isolated from the sweet basil variety EMX-1 using a biochemical genomics approach. These cDNAs encode proteins that are 90\% identical to each other and very similar to several isoflavone O-methyltransferases such as IOMT, which catalyzes the 4{\textquoteright}-O-methylation of 2,7,4{\textquoteright}-trihydroxyisoflavanone. On the other hand, CVOMT1 and EOMT1 are related only distantly to (iso)eugenol OMT from Clarkia breweri, indicating that the eugenol O-methylating enzymes in basil and C. breweri evolved independently. Transcripts for CVOMT1 and EOMT1 were highly expressed in the peltate glandular trichomes on the surface of the young basil leaves. The CVOMT1 and EOMT1 cDNAs were expressed in Escherichia coli, and active proteins were produced. CVOMT1 catalyzed the O-methylation of chavicol, and EOMT1 also catalyzed the O-methylation of chavicol with equal efficiency to that of CVOMT1, but it was much more efficient in O-methylating eugenol. Molecular modeling, based on the crystal structure of IOMT, suggested that a single amino acid difference was responsible for the difference in substrate discrimination between CVOMT1 and EOMT1. This prediction was confirmed by site-directed mutagenesis, in which the appropriate mutants of CVOMT1 (F260S) and EOMT1 (S261F) were produced that exhibited the opposite substrate preference relative to the respective native enzyme.},
	keywords = {Amino Acid Sequence, Anisoles, DNA: Complementary, Escherichia coli, Eugenol, Evolution: Molecular, Gene Expression, Gene Expression Regulation: Enzymologic, Gene Expression Regulation: Plant, Isoflavones, Methyltransferases, Molecular Sequence Data, Molecular Structure, Mutagenesis: Site-Directed, Mutation, Ocimum basilicum, Phylogeny, Recombinant Proteins, Sequence Homology: Amino Acid, Substrate Specificity},
	author = {David R Gang and Noa Lavid and Chloe Zubieta and Feng Chen and Till Beuerle and Efraim Lewinsohn and Joseph P Noel and Eran Pichersky}
}
@article {Hirotani:2000p8040,
	title = {Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis},
	journal = {Planta},
	volume = {210},
	number = {6},
	year = {2000},
	month = {May},
	pages = {1006{\textendash}13},
	abstract = {A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53,094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4{\textquoteright}-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments.},
	keywords = {Amino Acid Sequence, Blotting: Northern, Chromatography: High Pressure Liquid, Cloning: Molecular, Escherichia coli, Flavonoids, Glucosyltransferases, Lamiaceae, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Phylogeny, Plant Roots, Recombinant Proteins},
	author = {M Hirotani and R Kuroda and H Suzuki and T Yoshikawa}
}
