Pseudomonas Putida M10 Research Articles

Progress in Efforts To Harness Yeast for Making Opioid Drugs

Saccharomyces cerevisiae, more commonly known as baker's yeast, is being developed as a means for producing opioid-based drugs, with the long-term goal of making those drugs from glucose instead of extracting them from poppy plants, according to Christina Smolke at Stanford University in Stanford, Calif., and her collaborators. They recently reported progress toward that goal after inserting genes from the poppy plant, Papaver somniferum, as well as other genes from Pseudomonas putida M10, which grows on poppy straw waste, into yeast cells—enabling them to produce opioids from intermediates that poppy plants make relatively early in that metabolic pathway and to do so with improved efficiency. Details appear October 2014 in Nature Chemical Biology (doi:10.1038/nchembio.1613).

A microbial biomanufacturing platform for natural and semisynthetic opioids.

Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone, and oxycodone. A new biosynthetic branch to neopine and neomorphine was discovered, which diverted pathway flux from morphine and other target products. Strain titer and specificity was optimized by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy, and performing high-density fermentation, resulting in total opioid titers up to 131 mg/L. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.

Transformations of codeine to important semisynthetic opiate derivatives by Pseudomonas putida m10.

A biotransformation mixture which contained codeine and washed cells of Pseudomonas putida M10 gave rise to a number of transformation products that are of clinical importance which included hydrocodone, dihydrocodeine and 14beta-hydroxycodeine. Incubations with the same organism and codeinone gave rise to 14beta-hydroxycodeinone and 14beta-hydroxycodeine. Cell-free extracts and membrane fractions of P. putida M10 were shown to catalyse the 14beta-hydroxylation of codeinone. In addition, the potent analgesic oxycodone was shown to be produced from 14beta-hydroxycodeinone.

Transformations of codeine to important semisynthetic opiate derivatives by Pseudomonas putida m10

A biotransformation mixture which contained codeine and washed cells of Pseudomonas putida M10 gave rise to a number of transformation products that are of clinical importance which included hydrocodone, dihydrocodeine and 14β-hydroxycodeine. Incubations with the same organism and codeinone gave rise to 14β-hydroxycodeinone and 14β-hydroxycodeine. Cell-free extracts and membrane fractions of P. putida M10 were shown to catalyse the 14β-hydroxylation of codeinone. In addition, the potent analgesic oxycodone was shown to be produced from 14β-hydroxycodeinone.

Crystallization and preliminary diffraction studies of morphinone reductase, a flavoprotein involved in the degradation of morphine alkaloids.

Morphinone reductase from Pseudomonas putida M10, a flavoprotein involved in the degradation of morphine alkaloids, was purified from an overexpressing strain of Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.5 A. The I-centred orthorhombic cell has a monomer in the asymmetric unit. Preliminary molecular replacement calculations have been performed using Old Yellow Enzyme as the search model.

Bacterial morphinone reductase is related to Old Yellow Enzyme

Morphinone reductase, produced by Pseudomonas putida M10, catalyses the NADH-dependent saturation of the carbon-carbon double bond of morphinone and codeinone, and is believed to be involved in the metabolism of morphine and codeine. The structural gene encoding morphinone reductase, designated morB, was cloned from Ps. putida M10 genomic DNA by the use of degenerate oligonucleotide probes based on elements of the amino acid sequence of the purified enzyme. Sequence analysis and structural characteristics indicated that morphinone reductase is related to the flavoprotein alpha/beta-barrel oxidoreductases, and is particularly similar to Old Yellow Enzyme of Saccharomyces spp. and the related oestrogen-binding protein of Candida albicans. Expressed sequence tags from several plant species show high homology to these enzymes, suggesting the presence of a family of enzymes conserved in plants and fungi. Although related bacterial proteins are known, morphinone reductase appears to be more similar to the eukaryotic proteins. Morphinone reductase was overexpressed in Escherichia coli, and has potential applications for the industrial preparation of semisynthetic opiates.

Transformations of morphine alkaloids by Pseudomonas putida M10.

The oxidation of morphine by washed-cell incubations of Pseudomonas putida M10 gave rise to a large number of transformation products including hydromorphone (dihydromorphinone), 14 beta-hydroxymorphine, 14 beta-hydroxymorphinone, and dihydromorphine. Similarly, in incubations with oxymorphone (14 beta-hydroxydihydromorphinone) as substrate, the major transformation product was identified as oxymorphol (14 beta-hydroxydihydromorphine). The identities of all these biological products were confirmed by mass spectrometry and 1H nuclear magnetic resonance spectroscopy. This is the first report describing structural evidence for the biological synthesis of 14 beta-hydroxymorphine and 14 beta-hydroxymorphinone. These products have applications as intermediates in the synthesis of semisynthetic opiate drugs.

Biological production of semisynthetic opiates using genetically engineered bacteria.

Semisynthetic derivatives of morphine and related alkaloids are in widespread clinical use. Due to the complexity of these molecules, however, chemical transformations are difficult to achieve in high yields. We recently identified the powerful analgesic hydromorphone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Here we describe the construction of recombinant strains of Escherichia coli that express morphine dehydrogenase and morphinone reductase. These strains are capable of efficiently transforming the naturally occurring alkaloids morphine and codeine to hydromorphone and the antitussive hydrocodone, respectively. Our results demonstrate the potential for recombinant DNA technology to provide biological routes for the synthesis of known and novel semisynthetic opiate drugs.

Purification and characterization of morphinone reductase from Pseudomonas putida M10.

The NADH-dependent morphinone reductase from Pseudomonas putida M10 catalyses the reduction of morphinone and codeinone to hydromorphone and hydrocodone respectively. Morphinone reductase was purified from crude cell extracts to apparent homogeneity in a single affinity-chromatography step using Mimetic Yellow 2. The purified enzyme was a dimeric flavoprotein with two identical subunits of M(r) 41,100, binding non-covalently one molecule of FMN per subunit. The N-terminal sequence was PDTSFSNPGLFTPLQ. Morphinone reductase was active against morphinone, codeinone, neopinone and 2-cyclohexen-1-one, but not against morphine, codeine or isocodeine. The apparent Km values for codeinone and 2-cyclohexen-1-one were 0.26 mM and 5.5 mM respectively. The steroids progesterone and cortisone were potent competitive inhibitors; the apparent K1 for cortisone was 35 microM. The pH optimum for codeinone reduction was 8.0 in phosphate buffer. No reverse reaction could be detected, and NADPH could not be used as a reducing substrate in place of NADH. Morphinone reductase activity was strongly inhibited by 0.01 mM CuSO4 and p-hydroxymercuribenzoate, suggesting the presence of a vital thiol group. Steady-state kinetic studies suggested a Ping Pong (substituted enzyme) kinetic mechanism; however, product-inhibition patterns were inconsistent with a classical Ping Pong mechanism. Morphinone reductase may, like several other flavoprotein dehydrogenases, operate by a hybrid two-site Ping Pong mechanism.

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

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.

Nucleotide sequence and over-expression of morphine dehydrogenase, a plasmid-encoded gene from Pseudomonas putida M10.

Pseudomonas putida M10 was originally isolated from factory waste liquors by selection for growth on morphine. The NADP(+)-dependent morphine dehydrogenase that initiates morphine catabolism is encoded by a large plasmid of 165 kb. Treatment of P. putida M10 with ethidium bromide led to the isolation of a putative plasmid-free strain that was incapable of growth on morphine. The structural gene for morphine dehydrogenase, morA, has been located on the plasmid by oligonucleotide hybridization, by coupled transcription-translation of cloned restriction fragments and by nucleotide sequence analysis and is contained within a 1.7 kb SphI fragment that has been cloned into Escherichia coli. The cloned dehydrogenase enzyme is expressed at high levels in E. coli resulting in a 65-fold increase in morphine dehydrogenase activity in cell-free extracts compared with P. putida M10. Morphine dehydrogenase was rapidly purified to homogeneity, as judged by SDS/PAGE, by a one-step affinity chromatography procedure on Mimetic Orange 3 A6XL. The properties of the purified enzyme were identical with those previously reported for P. putida M10 morphine dehydrogenase. The morA gene was sequenced and the deduced amino acid sequence confirmed by N-terminal amino acid sequencing of the over-expressed protein. The predicted amino acid sequence of morA, deduced from the nucleotide sequence, indicated that morphine dehydrogenase did not belong to the non-metal-requiring short-chain class of dehydrogenases, but was more closely related to the aldo-ketoreductases.

Microbial degradation of the morphine alkaloids. Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10

The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine. The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine. The Km values for morphine and codeine were 0.46 mM and 0.044 mM respectively. The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1,10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme.

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

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 beta-hydroxysteroid dehydrogenase found in Pseudomonas testosteroni, which had previously been shown to have activity against morphine.