title: Comparative analysis of CYP93E proteins for improved microbial synthesis of plant triterpenoids.
abstract: Cytochrome P450-dependent monooxygenases (P450s) belonging to the CYP93E subfamily catalyze the C-24 oxidation of the triterpene backbone during the biosynthesis of triterpenoid saponins, which are bioactive plant natural products. In our attempts to produce plant triterpenoids in the yeast Saccharomyces cerevisiae, we observed a poor in vivo catalytic efficiency of the Medicago truncatula CYP93E2. To overcome this biosynthetic bottleneck, we screened publicly available plant genome and transcriptome data for CYP93E subfamily members. Six CYP93E orthologs, exclusively from leguminous plant species, were identified and functionally characterized in S. cerevisiae. Despite the high degree of amino acid conservation, the CYP93E orthologs showed large variations in enzymatic efficiency in yeast. The CYP93E9 from Phaseolus vulgaris showed the highest activity and converted ∼80% of the accumulating in vivo produced substrate β-amyrin to the products olean-12-ene-3β,24-diol and probable 3β-hydroxy olean-12-en-24-oic acid, with a catalytic efficiency that was 61 times higher than that of the M. truncatula CYP93E2. In conclusion, we have expanded the list of functional CYP93E orthologs to a total of nine proteins and show that there are large variations in their catalytic efficiencies when expressed in a heterologous host. Although demonstrated here for the CYP93E family involved in triterpenoid saponin biosynthesis, this phenomenon is undoubtedly extendable to other enzyme families involved in natural product synthesis. Hence, screening for homologous enzymes may become a valuable synthetic biologist's tool for engineering superior production chassis.
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; Department of Molecular Microbiology, VIB, 3001 Leuven-Heverlee, Belgium; Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, 3001 Leuven-Heverlee, Belgium.
authors.forename: Tessa
authors.initials: T
authors.lastname: Moses
authors.affiliation: Department of Molecular Microbiology, VIB, 3001 Leuven-Heverlee, Belgium; Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, 3001 Leuven-Heverlee, Belgium.
authors.forename: Johan M
authors.initials: JM
authors.lastname: Thevelein
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium. Electronic address: alain.goossens@psb-vib.ugent.be.
authors.forename: Alain
authors.initials: A
authors.lastname: Goossens
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Gent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium.
authors.forename: Jacob
authors.initials: J
authors.lastname: Pollier
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D046913:Medicago truncatula / Q000201:enzymology; D009828:Oleanolic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012503:Saponins / Q000737:chemistry / Q000378:metabolism*; D014315:Triterpenes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012503:Saponins; D014315:Triterpenes; D009828:Oleanolic Acid; D003577:Cytochrome P-450 Enzyme System; C036380:beta-amyrin
keywords: 3β-Hydroxy olean-12-en-24-oic acid; Fabaceae; Olean-12-ene-3β,24-diol; Saccharomyces cerevisiae; Saponins; Synthetic biology