Glucuronide Production by Whole-Cell Biotransformation Using Genetically Engineered Fission YeastSchizosaccharomyces pombe

Abstract

ABSTRACT:Drug metabolites generated by UDP glycosyltransferases (UGTs) are needed for drug development and toxicity studies, especially in the context of safety testing of metabolites during drug development. Because chemical metabolite synthesis can be arduous, various biological approaches have been developed; however, no whole-cell biotransformation with recombinant microbes that express human UGTs was yet achieved. In this study we expressed human UDP glucose-6-dehydrogenase together with several human or rat UGT isoforms in the fission yeast Schizosaccharomyces pombe and generated strains that catalyze the whole-cell glucuronidation of standard substrates. Moreover, we established two methods to obtain stable isotope-labeled glucuronide metabolites: the first uses a labeled aglycon, whereas the second uses 13 C 6 -glucose as a metabolic precursor of isotope-labeled UDP-glucuronic acid and yields a 6-fold labeled glucuronide. The system described here should lead to a significant facilitation in the production of both labeled and unlabeled drug glucuronides for industry and academia.

Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae

“…Bureik and co-workers developed a glucuronide production system using whole-cell biotransformation with the fission yeast, Schizosaccharomyces pombe (S. pombe) 19,20,34,35 . Both S. cerevisiae and S. pombe are classified as yeasts, which are thought to have branched off during their evolution some 300-400 million years ago 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 24 numbers of genes, there are marked differences between them in chromosome number and growth characteristics.…”

Yeast as a Humanized Model Organism for Biotransformation-Related Toxicity

“…Well-known drugs showing glucuronidation-related toxicity are the carboxylic NSAIDs, for which metabolism by UGTs is the main cause of protein-adduct formation leading to immune reactions [11]. Nine human UGTs belonging to the UGT1A, -2A and -2B families have been expressed in fission yeast S. pombe for production of glucuronides [136]. Expression of the UGTs alone did not produce a growth phenotype, however, co-expression with the cofactor-providing UDP-glucose dehydrogenase decreased biomass yield by 30-50% in the absence of a xenobiotic substrate [136].…”

“…Nine human UGTs belonging to the UGT1A, -2A and -2B families have been expressed in fission yeast S. pombe for production of glucuronides [136]. Expression of the UGTs alone did not produce a growth phenotype, however, co-expression with the cofactor-providing UDP-glucose dehydrogenase decreased biomass yield by 30-50% in the absence of a xenobiotic substrate [136]. A first spin-off is the production of ibuprofen-acyl glucosides using human UGT2B7 and demonstrating pronounced differences between two UGT2B7 isoforms [106].…”

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…However, synthesis of the phase II metabolites can be cumbersome and stereochemically demanding and hence go beyond possibilities of most laboratories [34]. Versatile alternative to chemical synthesis is enzyme-assisted in vitro production of these metabolites using liver homogenates, liver microscale cultures, cell culture lines or microbial systems where each of these methods has its specific drawbacks [61,62]. Raloxifene, which is metabolized to two distinct monoglucuronides and one diglucuronide, is an illustrative example for in-house production of authentic standards.…”

“…Stable isotope labeled metabolites can be obtained by microsomal incubation of labeled drug, of course if it is available and not too expensive [4]. The alternative approach is to use a labeled UDP-glucuronic acid as cofactor in bioproduction of metabolites [62].…”