Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae

Abstract

Cytochrome P450s are heme-thiolate oxygenases involved in a wide number of reactions such as epoxidation, hydroxylation, and demethylation. Heterologously expressed eukaryotic P450s are potentially useful biocatalysts for stereospecific oxygenation reactions under mild conditions. Numerous factors, such as intracellular pH, cytochrome P450, cytochrome P450 reductase, NADPH, and oxygen concentration all influence the in vivo activity. To systematically examine these factors, we selected ferulate 5-hydroxylase (F5H), a plant P450, with the Saccharomyces cerevisiae WAT11 strain as an expression host. Two media compositions and two cultivation procedures were investigated to optimize the in vivo activity of F5H. We modified a previously published selective growth medium (Pompon et al. [1996] Methods Enzymol 272:51-64) that increased the specific growth rate and cell yield of the host strain. A cultivation procedure with separate growth and induction stages that each contained selective media resulted in a 45% increase of whole cell F5H specific activity. In a medium designed for simultaneous growth and induction, we observed a 2.6-fold higher specific F5H activity, but substantially lower cell yield. Surprisingly, in this medium the higher specific F5H activity did not correlate with a higher P450 concentration. The effects of addition of the first committed heme precursor, delta-aminolevulinic acid, and Fe(III) at the beginning of induction period were also studied for our two-stage procedure. A small, but significant (P < 0.05) increase in whole cell F5H activity was observed following ALA addition.

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta

“…We clearly demonstrated that an exogenous addition of heme precursor, 5-aminolevulinic acid, to liquid culture medium, can elevate protein concentration of several PpCYPs (Fig. 2); this supports the Wnding of earlier studies using a plant P450 (Jiang and Morgan 2004). Each transformant was therefore grown in culture medium supplemented with 5-aminolevulinic acid and was applied to spectroscopic analysis.…”

Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon

“…The vectors were transformed into the yeast strain using a LiCl method according to the manufacturer's instructions provided with the pYES2 vector (Invitrogen). P450 expression was induced according to the modified two-stage cultivation method using modified SLI medium (Jiang and Morgan, 2004). For construction of the expression vector, original nucleotide sequences of E. phyllopogon P450 genes were used because no significant improvements were observed by recoding the N-terminal part of the P450 genes according to the procedure described by Hehn et al (2002).…”

Metabolic Engineering of the Phenylpropanoid Pathway in Saccharomyces cerevisiae

“…In addition, yeast has similar intracellular compartments to those of plant cells. Furthermore, several cytochrome P450 (CYP) enzymes are involved in flavonoid biosynthesis, and yeast has been shown by several groups to be an excellent host for in vivo CYP activity (16,19,28,33,36). One reason for this is the presence of an endoplasmic reticulum, which is where CYP and CYP reductase are targeted in plants.…”