Cofactor regeneration by a soluble pyridine nucleotide transhydrogenase for biological production of hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudom onas fluorescens to a cell-free system for the regeneration of the nicotina mide cofactors NAD and NADP in the biological production of the important s emisynthetic opiate drug hydromorphone, The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NA DP(+)-dependent morphine dehydrogenase and an NADH-dependent morphinone red uctase. By applying a soluble pyridine nucleotide transhydrogenase, which c an transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catal ytic amounts of cofactors occurs, resulting in high yields of hydromorphone . The ratio of morphine dehydrogenase, morphinone reductase, and soluble py ridine nucleotide transhydrogenase is critical for diminishing the producti on of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabol ic engineering and biocatalysis.