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

Abstract

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP(+)-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production 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 metabolic engineering and biocatalysis.

FIG. 1

Transformation of morphine by MDH and MR from P. putida M10.

FIG. 2

Cofactor cycling by STH in the biological production of hydromorphone.

FIG. 3

Cell-free biotransformation of morphine at various ratios of MDH to MR in the presence and absence of STH. The transformations were performed without (A, C, and E) or with (B, D, and F) 1.25 U of STH per ml. MDH and MR were used as the ratios 1:1 (1.25 U of each per ml) (A and B), 5:1 (6.25 U of MDH per ml and 1.25 U of MR per ml (C and D), 1:5 (1.25 U of MDH per ml and 6.25 U of MR per ml (E and F). The cofactors NADPH and NAD⁺ were supplied at a concentration of 0.2 mM.

FIG. 4

Cell-free biotransformation of morphine in the presence of increasing amounts of STH. The transformations were performed with 1.25 U of MDH per ml and 6.25 U of MR per ml and various amounts of STH as follows: none (A), 0.0625 U/ml (B), 0.125 U/ml (C), and 0.625 U/ml (D). With 1.25 and 6.25 U of STH per ml, the result was similar to that shown in panel D. The cofactors used were 2 mM NAD⁺, 0.15 mM NADH, 0.25 mM NADP⁺, and 0.2 mM NADPH.

FIG. 5

Whole-cell biotransformation of morphine with E. coli JM109/pMORAB5 (A), E. coli JM109/pMORAB5/pPFSTH4 (B), E. coli JM109/pMORB3-AC80S · K244M (C), and E. coli JM109/pMORB3-AC80S · K244M/pPFSTH4 (D). Figures shown are the averages of duplicate measurements, and error bars represent standard deviations.

FIG. 6

Reuse of cells. The graph shows hydromorphone yields obtained from consecutive incubations of recombinant E. coli JM109 with 20 mM morphine. Biotransformations were carried out as described in the text. Cells were harvested once the biotransformation had reached completion (12 to 22 h) and washed with 50 mM Tris-HCl (pH 8.0) prior to the start of the next biotransformation. Figures shown are the averages of duplicate measurements, and error bars represent the standard deviations.