Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

Abstract

Biochemical analyses of metabolic enzymes are performed using in vitro assays with enzymes and substrates. Enzyme samples are generally prepared from heterologous cell expression systems, which are also used as tools to obtain substrates that are not commercially available or cannot be easily isolated from natural sources. Cytochrome P450 (CYP) comprises a widely distributed family of monooxygenases that are commonly used for biosynthesis of natural products. Since CYP activity requires an electron transport system with membrane bound CYP reductase (CPR), it has been believed that CYP is not easily expressed in Escherichia coli, despite multiple advantages as a heterologous system compared with other systems that employ eukaryotic yeast and insect cells. In this study, we demonstrated simple and efficient methods for functional expression of CYPs in E. coli using commercially available vectors in which the transmembrane-domain truncated CYP was co-expressed with CPR as a discrete polypeptide, and we also used them to identify CYP81E11, CYP81E12, and CYP81E18 of soybean as isoflavone 2′-hydroxylase. Culture conditions were optimized for the bioconversion of I2′H, and the highest production was 161 mg l −1 of medium under optimized conditions. Subsequently, six other CYPs that are involved in flavonoid biosynthesis were tested for their applicability in the E. coli expression system. Establishment of the present method may facilitate functional expression of CYPs for preparation of CYP products, including substrates for enzymatic reactions, valuable natural products, and their unnatural derivatives.

Quantitative Phosphoproteomic and Metabolomic Analyses Reveal GmMYB173 Optimizes Flavonoid Metabolism in Soybean under Salt Stress

“…It is now generally accepted that the process leading to the production of dihydroxy B-ring-substituted flavonoids and their derivatives plays positive roles in soybean salt tolerance, whereas the formation of monohydroxy B-ring/-substituted flavonoids and their derivatives have negative contributions to soybean resistance to oxidative stresses (67)(68)(69). Consistently we found at the metabolome level, the contents of endogenous dihydroxy B-ring-substituted flavonoids, such as cyanidin 3-arabinoside chloride, cyanidin 3-(6Љ-succinylglucoside), quercetin 3-(6Љ-methylglucuronide), luteolin 3Јmethyl ether 7-glucuronosyl-(1-Ͼ2)-glucuronide and quercetin 3, 7, 3Ј-tri-O-sulfate, were found to be significantly up-regulated; the monohydroxy B-ring/-substituted flavonoids, such as daidzin, genistein and formononetin, were found to be down-regulated in soybean roots in response to salt stress (Table I).…”

The Missing Link in Leguminous Pterocarpan Biosynthesis is a Dirigent Domain-Containing Protein with Isoflavanol Dehydratase Activity

“…Daidzein and formononetin were purchased from LC Laboratories and Toronto Research Chemicals, respectively. 2′-Hydroxyformononetin and 2′-hydroxydaidzein were prepared from formononetin and daidzein by bioconversion using E. coli expressing L. japonicus I2′H (CYP81E6) and soybean I2′H (CYP81E18) ( Uchida et al 2015 ), respectively. (±)-Medicarpin and (−)-medicarpin were obtained from our laboratory stocks ( Akashi et al 2006 ).…”

“…The function of I2′H was identified through heterologous expression in yeast cells of cDNA clones isolated from G. echinata (CYP81E1), L. japonicus (CYP81E6) and barrel medic ( Medicago truncatula ; CYP81E7) ( Akashi et al 1998 , Shimada et al 2000 , Liu et al 2003 ). Soybean I2′Hs (CYP81E11, CYP81E12 and CYP81E18) have been expressed and characterized using an Escherichia coli expression system ( Uchida et al 2015 ). Isoflavone reductase (IFR), which catalyzes the second reaction step, was purified from soybean cultured cells ( Fischer et al 1990 ), and biochemical analyses have been performed on proteins expressed from cDNA clones from alfalfa ( Medicago sativa ), chickpea ( Cicer arietinum ) and pea ( P. sativum ) ( Paiva et al 1991 , Tiemann et al 1991 , Paiva et al 1994 ).…”

Molecular Characterization of Soybean Pterocarpan 2-Dimethylallyltransferase in Glyceollin Biosynthesis: Local Gene and Whole-Genome Duplications of Prenyltransferase Genes Led to the Structural Diversity of Soybean Prenylated Isoflavonoids

“…(−)-Glycinol and glyceollins I and III were prepared as described previously (Akashi et al 2009). 2,7,4′-Trihydroxyisoflavanone was prepared using Escherichia coli expressing the cDNA for CYP93C2 (Uchida et al 2015). …”