Whole cell-dependent biosynthesis of drug metabolites using genetically engineered budding yeast

Abstract

Xenobiotic phase I and II reactions generally render a compound more water soluble and pharmacologically inactive, thereby eliminating the need for further evaluation. However, if the metabolite forms a toxic compound such as acylglucuronide, additional safety assessment may be needed. Glucuronidation is the most common pathway for detoxification and elimination of hydrophobic xenobiotics in mammals. Thus, development of an efficient in vitro synthesis of glucuronides from parent drugs often becomes critical during studies of drug metabolism undertaken in the development of a new pharmaceutical product. To produce glucuronides as drug metabolites, we have developed coexpression systems for mammalian cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and UDP-glucose dehydrogenase in Saccharomyces cerevisiae cells, and combination between each of the human P450s and UGTs was achieved. Glucuronide formation in yeast cells was performed in reaction medium containing 8 % glucose, and most of the glucuronides were readily recovered from the cell medium. In addition, we have expressed human sulfotransferase (SULT) with P450s in S. cerevisiae cells, and successfully obtained sulfo-conjugates from the cell medium. Coexpression of P450 electron transfer and glucuronidation or sulfo-conjugate production systems allow us to obtain the phase I metabolites and phase II metabolites from the parent compound. In conclusion, our yeast expression systems of xenobiotic-metabolizing enzymes have made it possible to produce xenobiotic phase I and phase II metabolites on the milligram to gram scale.