Optimization of yeast-based production of medicinal protoberberine alkaloids.

BACKGROUND: Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac-norlaudanosoline to the protoberberine alkaloid (S)-canadine. RESULTS: This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale. CONCLUSIONS: By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.

Oxidative modification of neurofilament-L and neuronal cell death induced by the catechol neurotoxin, tetrahydropapaveroline.

Tetrahydropapaveroline (THP), which is an endogenous neurotoxin, has been suspected to be associated with dopaminergic neurotoxicity of l-DOPA. In this study, we examined oxidative modification of neurofilament-L (NF-L) and neuronal cell death induced by THP. When disassembled NF-L was incubated with THP, protein aggregation was increased in a time- and THP dose-dependent manner. The formation of carbonyl compounds and dityrosine were observed in the THP-mediated NF-L aggregates. Radical scavengers reduced THP-mediated NF-L modification. These results suggest that the modification of NF-L by THP may be due to oxidative damage resulting from the generation of reactive oxygen species (ROS). When THP exposed NF-L was subjected to amino acid analysis, glutamate, proline and lysine residues were found to be particularly sensitive. We also investigated the effects of copper ions on THP-mediated NF-L modification. At a low concentration of THP, copper ions enhanced the modification of NF-L. Treatment of C6 astrocyte cells with THP led to a concentration-dependent reduction in cell viability. When these cells were treated with 100µM THP, the levels of ROS increased 3.5-fold compared with control cells. Furthermore, treatment of cells with THP increased NF-L aggregate formation, suggesting the involvement of NF-L modification in THP-induced cell damage.

Urinary excretion of morphine and biosynthetic precursors in mice.

It has been firmly established that humans excrete a small but steady amount of the isoquinoline alkaloid morphine in their urine. It is unclear whether it is of dietary or endogenous origin. There is no doubt that a simple isoquinoline alkaloid, tetrahydropapaveroline (THP), is found in human and rodent brain as well as in human urine. This suggests a potential biogenetic relationship between both alkaloids. Unlabeled THP or [1,3,4-D(3)]-THP was injected intraperitoneally into mice and the urine was analyzed. This potential precursor was extensively metabolized (96%). Among the metabolites found was the phenol-coupled product salutaridine, the known morphine precursor in the opium poppy plant. Synthetic [7D]-salutaridinol, the biosynthetic reduction product of salutaridine, injected intraperitoneally into live animals led to the formation of [7D]-thebaine, which was excreted in urine. [N-CD(3)]-thebaine was also administered and yielded [N-CD(3)]-morphine and the congeners [N-CD(3)]-codeine and [N-CD(3)]-oripavine in urine. These results show for the first time that live animals have the biosynthetic capability to convert a normal constituent of rodents, THP, to morphine. Morphine and its precursors are normally not found in tissues or organs, presumably due to metabolic breakdown. Hence, only that portion of the isoquinoline alkaloids excreted in urine unmetabolized can be detected. Analysis of urine by high resolution-mass spectrometry proved to be a powerful method for tracking endogenous morphine and its biosynthetic precursors.

Mammalian morphine: de novo formation of morphine in human cells.

BACKGROUND: Morphine, the major alkaloid of opium of Papaver somniferum, is one of the strongest analgesic compounds known. "Endogenous morphine" has been long isolated and authenticated by mass spectrometry in trace amounts from animal and human specific tissue or fluids. The most widely accepted explanation presently is that morphine detected in human and animal tissue is of exogenous sources (e.g. dietary origin). MATERIAL/METHODS: The biosynthetic experiments were performed with human neurobalstoma cells (SH-SY5Y) and human pancreas carcinoma (DAN-G) cells. The application experiments were done in the presence of isotopically labeled potential precursors such as (18)O(2) and [ring-(13)C(6)]-L-tyrosine. Benzylisoquinoline alkaloids present in mammalian cells were identified by GC-MS/MS. RESULTS: Growth of the SH-SY5Y cells in the presence of (18)O2 led to [(18)O(2)]-labeled morphine and [(18)O(2)]-labeled reticuline, each had a molecular mass four mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule. DAN-G cells cultured in an (18)O(2) atmosphere produced (S)-[ (18)O(2)]-norlaudanosoline and (S)-[ (18)O(2)]-reticuline, each labeled with (18)O atoms at only two of the four possible positions. Feeding of [ring-(13)C(6)]-tyramine, (S)-[1-(13)C, N-(13)CH(3)]-reticuline and [N-CD(3)]-thebaine to SH-SY5Y cells led each to the labeling of morphine, as established by GC-MS/MS. CONCLUSIONS: Taken together, morphine, reticuline and norlaudanosoline are unequivocally biosynthesized by cultured human cells. The precursors of morphine in the human cell lines were conclusively shown to be oxygen, tyramine, reticuline and thebaine.

Endogenous formation of morphine in human cells.

Morphine is a plant (opium poppy)-derived alkaloid and one of the strongest known analgesic compounds. Studies from several laboratories have suggested that animal and human tissue or fluids contain trace amounts of morphine. Its origin in mammals has been believed to be of dietary origin. Here, we address the question of whether morphine is of endogenous origin or derived from exogenous sources. Benzylisoquinoline alkaloids present in human neuroblastoma cells (SH-SY5Y) and human pancreas carcinoma cells (DAN-G) were identified by GC/tandem MS (MS/MS) as norlaudanosoline (DAN-G), reticuline (DAN-G and SH-SY5Y), and morphine (10 nM, SH-SY5Y). The stereochemistry of reticuline was determined to be 1-(S). Growth of the SH-SY5Y cell line in the presence of (18)O(2) led to the [(18)O]-labeled morphine that had the molecular weight 4 mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule of morphine. Growth of DAN-G cells in an (18)O(2) atmosphere yielded norlaudanosoline and (S)-reticuline, both labeled at only two of the four oxygen atoms. This result clearly demonstrates that all three alkaloids are of biosynthetic origin and suggests that norlaudanosoline and (S)-reticuline are endogenous precursors of morphine. Feeding of [ring-(13)C(6)]-tyramine, [1-(13)C, N-(13)CH(3)]-(S)-reticuline and [N-CD(3)]-thebaine to the neuroblastoma cells led each to the position-specific labeling of morphine, as established by GC/MS/MS. Without doubt, human cells can produce the alkaloid morphine. The studies presented here serve as a platform for the exploration of the function of "endogenous morphine" in the neurosciences and immunosciences.

Ectopic deposition of melanin pigments as detoxifying mechanism: a paradigm for basal nuclei pigmentation.

Melanins are UV shielding pigments found in skin and other light exposed tissues. However, a kind of melanin, named neuromelanin (NM), is found in those deep brain loci that degenerate in Parkinson's disease (PD), where no such a function may be imagined. The NM synthetic pathway, different from the one of eumelanin based on tyrosinase, is still obscure as well as its physiological function. Here we show that under conditions of excess of toxic quinone concentration, nonmelanocytic cell strains (i.e., primary keratinocytes) may accumulate a dark cytoplasmatic pigment that proved to be a melanin. The ability of pigment deposition, possibly driven by peroxidases, is restricted to diploid cells and increases cell survival acting as a sink for potentially hazardous quinones. We suggest that in the basal nuclei, exposed to high level of catecholaminergic neurotransmitters, NM deposition is a relevant antioxidant mechanism by trapping quinones and semiquinones, thus protecting neurons from accumulating damage over many years. In this perspective, just as a hypothesis, we may imagine that PD neuron degeneration is the consequence of a reduced/abrogated ability to produce neuromelanin.

Tyrosinase protects human melanocytes from ROS-generating compounds.

The effects of two tetrahydroisoquinolines (TIQs), tetrahydropapaveroline (THP) and salsolinol (SAL), on human primary melanocytes were studied. These compounds are naturally occurring alkaloids deriving from the condensation of dopamine with aldehydes. The effects on the viability were studied by treating the cells with variable concentration of THP or SAL; both TIQs were well tolerated up to roughly 30 micro M. At higher concentrations, THP became overtly toxic while SAL showed no cytotoxic effect up to 100 micro M. TIQs treatment determined an impairment of intracellular activity of antioxidant enzymes, like SOD, DT-diaphorase, and glutathione peroxidase. A decrease of alpha-ketoglutarate dehydrogenase activity was also evidenced following TIQs treatment; a very strong diminution was found in THP-treated cells, whose viability was highly decreased. Both TIQs increased tyrosinase-specific mRNA transcription followed, in the case of SAL, by an activation of tyrosinase. In the presence of tyrosinase inhibitors TIQs treatment resulted in a sharp cytotoxic effect even at concentrations normally well tolerated. Taken together these data suggest that tyrosinase represents an outstanding protective mechanism against ROS-generating compounds, once primary detoxifying mechanisms are impaired or not available.

Chromatographic separation and spectrometric identification of the oxidation products from a tetrahydro-isoquinoline alkaloid.

The oxidation chemistry of 3',4'-deoxynorlaudanosoline carboxylic acid, a tetrahydroisoquinoline alkaloid, has been studied by electrochemical approaches. Four reaction products were isolated by semi-preparative high performance liquid chromatography and identified structurally by nuclear magnetic resonance, mass spectrometry, ultraviolet-visible spectrophotometry and electrochemistry studies. An oxidation mechanism was proposed.

Enantio-selective occurrence of (S)-tetrahydropapaveroline in human brain.

Tetrahydropapaveroline is an endogenous complex alkaloid derived from dopamine through the oxidation by monoamine oxidase. This alkaloid is considered to be involved in the pathogenesis of alcoholism and to act as a false neurotransmitter. Recently the (S) enantiomer was proposed to be a precursor of morphine biosynthesis in the opium poppy. In this paper stereo-chemical characteristic of tetrahydropapaveroline in human brains was examined. In all four control human brains examined, only the (S)-tetrahydropapaveroline was detected. The concentrations were 0.12-0.22 pmol/g wet weight of brain tissue, and the presence of alcohol in blood did not affect the concentration. The results suggest that (S)-tetrahydropapaveroline may be enantio-selectively synthesized in human brain and it may be an intermediate of the de novo synthesis of morphine analogues.

Studies of polymerized sodium N-undecylenyl-L-valinate in chiral micellar electrokinetic capillary chromatography of neutral, acidic, and basic compounds.

The polymerized surfactant poly(sodium N-undecylenyl amino L-valinate) [poly(L-SUV)] has been used in micellar electrokinetic capillary chromatography for the chiral separation of various acidic and basic drugs, as well as neutral compounds. Under the conditions studied, poly(L-SUV) was shown to be a very versatile anionic chiral selector in the pH range of 5.6-11. The micelle was used for the enantioseparation of coumarinic anticoagulant drugs with various buffers under moderately acidic conditions. Neutral and alkaline buffer conditions were used to successfully separate the neutral atropisomers (+/-)-1,1'-bi-2-naphthol, (+/-)-1,1'-binaphthyl-2,2'-diamine, and Tröger's base. Chiral separation of the cationic paveroline drugs, laudanosine, norlaudanosoline, and laudanosoline, was influenced by pH and the use of coated capillaries. The acquired data focused on optimizing the migration times, capacity and separation factors, and electrophoretic mobilities of the various racemic mixtures.

Study of the antinociceptive effect of tetrahydro-papaveroline derivatives. Interaction with opioids.

The antinociceptive effect of racemic tetrahydropapaveroline (THP), of its two R(+)- and S(-) enantiomers, of 1-2-dehydro-THP and of 1-carboxy-THP was assessed using different pain tests in mice. None of these drugs possessed a significant activity in the hot-plate and tail-flick tests. However, after i.p. injection, they reduced the number of abdominal writhes induced by phenylbenzoquinone, with ED50 values of 51 +/- 7, 73 +/- 9 and 79 +/- 7 mg/kg for the most potent compounds: 1,2-dehydro-THP, +/- THP and -THP, respectively. This activity was not antagonized by naloxone (1 mg/kg, s.c.). However combination of inactive doses of these three compounds (32 mg/kg, i.p.) and of morphine (0.5 mg/kg, s.c.) led to a significant antinociceptive effect (83 to 85% of reduction of the number of writhes). This synergistic potentiation confirmed with the combination of +/- THP (16 mg/kg, i.p.) and morphine (0.5 mg/kg, s.c.) was totally inhibited by naloxone (1 mg/kg, s.c.). These results, although excluding a direct agonistic effect of THP derivatives on opiate receptors, suggest an indirect interaction of these drugs with the endogenous opioid system.