Engineering novel biocatalytic routes for production of semisynthetic opiate drugs.

A strain of Pseudomonas putida was isolated by selective enrichment with morphine that was capable of utilising morphine as a primary source of carbon and energy for growth. Experiments with whole cells showed that both morphine and codeine, but not thebaine, could be utilised. A novel NADP-dependent dehydrogenase, morphine dehydrogenase, was purified from crude cell extracts and was shown to be capable of oxidising morphine and codeine to morphinone and codeinone, respectively. This NADP-dependent morphine dehydrogenase was not observed in any other species of pseudomonads examined and was quite distinct from the β-hydroxysteroid dehydrogenase found in Pseudomonas testosteroni, which had previously been shown to have activity against morphine.

Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10

Structure determination of microbiologically hydroxylated Δ4-3-oxosteroids by NMR spectroscopy and its applications

The morphine alkaloid hydromorphone (dihydromorphinone) was identified as an intermediary metabolite in the degradation of morphine by Pseudomonas putida M10. A constitutive NADH-dependent morphinone reductase capable of catalyzing the reduction of the 7,8-unsaturated bond of morphinone and codeinone, yielding hydromorphone and hydrocodone, respectively, was shown to be present in cell extracts. The structures have been identified by 1H nuclear magnetic resonance and mass spectrometry. Morphinone reductase has been partially purified by anion-exchange and gel filtration chromatography. This enzyme has potential applications as a biocatalyst for the synthesis of the highly potent analgesic hydromorphone and the antitussive hydrocodone.

Biological synthesis of the analgesic hydromorphone, an intermediate in the metabolism of morphine, by Pseudomonas putida M10.

14-Hydroxycodeine and norcodeine were rigorously identified as products arising from codeine oxidation by Streptomyces griseus ATCC 10137. Both products were routinely detected in extracted culture filtrates after growth of cells in the presence of codeine for 1 week. Under these conditions, about 4 mol% of the codeine starting material was consumed, with norcodeine and 14-hydroxycodeine representing the only identifiable transformation products (molar ratio, 4:1, respectively). Extraction of a series of culture filtrates and purification of the pooled metabolites by thin-layer and high-pressure liquid chromatography led to the isolation of both biological products, the structures of which were verified by high-resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. The identities of both biological products were further confirmed by comparison of their spectral properties with those of authentic standards. This is the first report providing structural evidence for the biological formation of 14-hydroxycodeine from codeine and of codeine oxidation by S. griseus.

Identification of transformation products arising from bacterial oxidation of codeine by Streptomyces griseus.

Mikrobiologische Verwandlung von Thebain in das 14-Hydroxycodeinon mittels einiger Pilze

Bei der Inkubation von Progesteron mit Helminthosporium sativum lieβen sich zwei Monohydroxy-und drei Dihydroxy-progesterone gewinnen. Drei davon erwiesen sich als identisch mit den bekannten Verbindungen, namlich 15α-und 15β-Hydroxyprogesteron, und 7β, 15β-Dihydroxyprogesteron. Fur die ubrigen Hydroxylierungsprodukte wurde nach dem chemischen Verhalten die Struktur von 7α, 15β-Dihydroxy-bzw. von 14α, 15β-Dihydroxyprogesteron erteilt.

Untersuchungen auf dem Gebiet der mikrobiologischen Umsetzung. XI. Hydroxylierung von Progesteron in der 7α-, 7β-, 14α-, 15α- oder 15β-Stellung durch Helminthosporium sativum.

Inkubation von Progesteron (I) mit Kulturen von Syncephalastrum racemosum COHN ergab neben den bekannten Produkten, namlich 15β-Hydroxyprogesteron (II), 6β, 11α-Dihydroxyprogesteron (VI), 7β, 15β-Dihydroxyprogesteron (IV) und 7β, 14α, 15β-Trihydroxyprogesteron (VII), noch unbekannte Produkte, namlich 6β, 15β-Dihydroxyprogesteron (V) und 15β-Hydroxy-5α-pregnan-3, 6-20-trion (III).

Untersuchungen auf dem Gebiet der mikrobiologischen Umsetzung. XX. Hydroxylierung von Progesteron mittels Syncepharastrum racemosum COHN (5).

Das Dihydroxyprogesteron, welches von diesen Autoren durch Einwirkung von Syncephalastrum racemosum auf Progesteron (I) einst gewonnen wurde und damals nach dem Strukturzeichen von 7α, 15β-Dihydroxyprogesteron genannt wurde, ergibt sich diesmal bei der Umsetzung von (I) sowie von 7β-Hydroxyprogesteron (II) mit Diplodia tubericola. Aus diesen Tatsachen sowie aus den Ergebnissen der vorherigen Mitteilungen ist ersichtlich, dass das richtige Strukturzeichen des Dihydroxyprogesterons 7β, 15β-Dihydroxyprogesteron (III) ist.

Mikrobiologische Hydroxylierung der Steroide. X. Uber 7β, 15β-Dihydroxypregn-4-en-3, 20-dion.

On the strain which was used in the previous report (2, 3) and described as Syncephalastrum sp, taxonomical studies were made and it was decided as Syncephalastrum racemosum (Cohn) SchroeterThis fungus formed 7α, 15β-dihydroxyprogesterone from progesterone in a short period of incubation, while 7α, 14α, 15β-trihydroxyprogesterone in a ong period By this strain, 6β, 11α-dihydroxyprogesterone and 11- epihydrocortisone were also formed from 11α-hydroxyprogesterone and compound S, respectively However, no oxidation products could be found from 6β, 11α-dihydroxyprogesterone and hydrocortisone

Tokuji Tanaka

Papers

Mikrobiologische Verwandlung von Thebain in das 14-Hydroxycodeinon mittels einiger Pilze

Untersuchungen auf dem Gebiet der mikrobiologischen Umsetzung. XI. Hydroxylierung von Progesteron in der 7α-, 7β-, 14α-, 15α- oder 15β-Stellung durch Helminthosporium sativum.

Untersuchungen auf dem Gebiet der mikrobiologischen Umsetzung. XX. Hydroxylierung von Progesteron mittels Syncepharastrum racemosum COHN (5).

Mikrobiologische Hydroxylierung der Steroide. X. Uber 7β, 15β-Dihydroxypregn-4-en-3, 20-dion.

Microbiological hydroxylation of steroid ix. hydroxylation of steroid by syncephalastrum racemosum. (2)