title: 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.
authors.affiliation: School of Chemical Engineering, FRNY Hall, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Hanxiao
authors.initials: H
authors.lastname: Jiang
authors.forename: John A
authors.initials: JA
authors.lastname: Morgan
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C097486:CYP84A1 protein, Arabidopsis; D009251:NADPH-Ferrihemoprotein Reductase