Psychoactive Plant Database - Neuroactive Phytochemical Collection

1. ACS Chem Biol. 2023 Feb 17;18(2):419-430. doi: 10.1021/acschembio.2c00873.
Epub  2023 Feb 3.

Strong Feedback Inhibition of Key Enzymes in the Morphine Biosynthetic Pathway 
from Opium Poppy Detectable in Engineered Yeast.

Ozber N(1), Yu L(1), Hagel JM(1), Facchini PJ(1).

Author information:
(1)Department of Biological Sciences, University of Calgary, Calgary, Alberta 
T2N 1N4, Canada.

Systematic screening of morphine pathway intermediates in engineered yeast 
revealed key biosynthetic enzymes displaying potent feedback inhibition: 
3'-hydroxy-N-methylcoclaurine 4'-methyltransferase (4'OMT), which yields 
(S)-reticuline, and the coupled salutaridinol-7-O-acetyltransferase (SalAT) and 
thebaine synthase (THS2) enzyme system that produces thebaine. The addition of 
deuterated reticuline-d1 to a yeast strain able to convert (S)-norcoclaurine to 
(S)-reticuline showed reduced product accumulation in response to the feeding of 
all four successive pathway intermediates. Similarly, the addition of deuterated 
thebaine-d3 to a yeast strain able to convert salutaridine to thebaine showed 
reduced product accumulation from exogenous salutaridine or salutaridinol. In 
vitro analysis showed that reticuline is a noncompetitive inhibitor of 4'OMT, 
whereas thebaine exerts mixed inhibition on SalAT/THS2. In a yeast strain 
capable of de novo morphine biosynthesis, the addition of reticuline and 
thebaine resulted in the accumulation of several pathway intermediates. In 
contrast, morphine had no effect, suggesting that circumventing the interaction 
of reticuline and thebaine with 4'OMT and SalAT/THS2, respectively, could 
substantially increase opiate alkaloid titers in engineered yeast.

DOI: 10.1021/acschembio.2c00873
PMID: 36735832 [Indexed for MEDLINE]


2. Nat Commun. 2016 Feb 5;7:10390. doi: 10.1038/ncomms10390.

Total biosynthesis of opiates by stepwise fermentation using engineered 
Escherichia coli.

Nakagawa A(1), Matsumura E(1), Koyanagi T(1), Katayama T(2), Kawano N(3), 
Yoshimatsu K(3), Yamamoto K(1), Kumagai H(1), Sato F(4), Minami H(1).

Author information:
(1)Applied Microbiology laboratory, Research Institute for Bioresources and 
Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Ishikawa 
921-8836, Japan.
(2)Department of Applied Molecular Biology, Graduate School of Biostudies, Kyoto 
University, Oiwake-cho, Kitashirakawa, Kyoto 606-8502, Japan.
(3)Breeding and Physiology Laboratory, Tsukuba Research Center for Medicinal 
Plant Resources, National Institutes of Biomedical Innovation, Health and 
Nutrition, 1-2 Hachimandai, Tsukuba 305-0843, Japan.
(4)Department of Plant Gene and Totipotency, Division of Integrated Life 
Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, 
Kitashirakawa, Kyoto 606-8502, Japan.

Opiates such as morphine and codeine are mainly obtained by extraction from 
opium poppies. Fermentative opiate production in microbes has also been 
investigated, and complete biosynthesis of opiates from a simple carbon source 
has recently been accomplished in yeast. Here we demonstrate that Escherichia 
coli serves as an efficient, robust and flexible platform for total opiate 
synthesis. Thebaine, the most important raw material in opioid preparations, is 
produced by stepwise culture of four engineered strains at yields of 
2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently 
developed yeast systems. This improvement is presumably due to strong activity 
of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. 
Furthermore, by adding two genes to the thebaine production system, we 
demonstrate the biosynthesis of hydrocodone, a clinically important opioid. 
Improvements in opiate production in this E. coli system represent a major step 
towards the development of alternative opiate production systems.

DOI: 10.1038/ncomms10390
PMCID: PMC4748248
PMID: 26847395 [Indexed for MEDLINE]


3. World J Microbiol Biotechnol. 2013 Nov;29(11):2125-31. doi: 
10.1007/s11274-013-1377-2. Epub 2013 May 17.

Enhanced morphinan alkaloid production in hairy root cultures of Papaver 
bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via 
Agrobacterium rhizogenes mediated transformation.

Sharafi A(1), Hashemi Sohi H, Mousavi A, Azadi P, Dehsara B, Hosseini Khalifani 
B.

Author information:
(1)National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, 
Iran.

Papaver bracteatum is an important medicinal plant valued for its high content 
of thebaine and an alternative to P. somniferum for benzylisoquinoline alkaloid 
production. Salutaridinol 7-o-acetyltransferase (SalAT) is a key gene in 
morphinan alkaloids biosynthesis pathway. Over expression of SalAT gene was used 
for metabolic engineering in P. bracteatum hairy root cultures. Transcript level 
of the salutaridinol 7-o-acetyltransferase gene in transgenic hairy root lines 
increased up to 154 and 128 % in comparison with hairy roots without SalAT over 
expression and wild type roots, respectively. High performance liquid 
chromatography analysis showed that the transgenic hairy roots relatively 
improved levels of thebaine (1.28 % dry weight), codeine (0.02 % dry weight) and 
morphine (0.03 % dry weight) compared to those hairy roots without SalAT over 
expression. This suggests that P. bracteatum hairy roots expressing the SalAT 
gene could be potentially used for the production of valuable morphinan 
alkaloids.

DOI: 10.1007/s11274-013-1377-2
PMID: 23681746 [Indexed for MEDLINE]


4. Forensic Sci Int. 2012 Oct 10;222(1-3):387-93. doi: 
10.1016/j.forsciint.2012.08.002. Epub 2012 Aug 24.

Genetic and chemical components analysis of Papaver setigerum naturalized in 
Korea.

Choe S(1), Lee E, Jin GN, Lee YH, Kim SY, Choi H, Chung H, Hwang BY, Kim S.

Author information:
(1)Narcotics Analysis Division, National Forensic Service, Seoul, Republic of 
Korea.

Of the 110 species of genus Papaver, only Papaver somniferum and P. setigerum 
are controlled poppies in Korea. All poppy samples share similar morphology 
therefore it is important to check if they contain controlled substances such as 
morphine and codeine for forensic purpose. Since the alkaloid content of Papaver 
plants varies according to their growing stage, chemical components analysis 
alone is not enough to identify exact species. In 2010, hundreds of poppy plants 
suspected to be P. somniferum were found in Jeju Island, South Korea. They had a 
slightly different but overall similar appearance to P. somniferum. Using GC-MS 
analysis, codeine, rhoeadine, papaverine, protopine, noscapine, setigeridine and 
trace amounts of morphine were detected in these samples. Although their 
chemical components were different from what has been described in literatures 
for P. setigerum, they could be assumed to be P. setigerum based on their 
morphological features and GC-MS results. Also, chromosome numbers using their 
seeds showed 2n=44 and the numbers were in accordance with those of P. 
setigerum. Nucleotide substitution or insertion/deletion of ITS (internal 
transcribed spacer), 18S rRNA (ribosomal RNA), rbcL (large subunit of ribulose 
1,5-bisphosphate carboxylase), trnL-trnF IGS (intergenic spacer), trnL intron 
and psbA-trnH were assessed as universal genetic markers for P. setigerum. Also, 
genetic analysis using six target genes involved in the biosynthesis of 
benzylisoquinoline alkaloids, including TYDC (tyrosine/dopa decarboxylase), SAT 
(salutaridinol-7-O-acetyltransferase), BBE (berberine bridge enzyme), COR 
(codeinone reductase), CYP80B1 ((S)-N-methylcoclaurine 3'-hydroxylase) and NCS 
(norcoclaurine synthase) were tested as Papaver-specific genetic markers by the 
existence of their PCR products. From the results, the sequences of the 6 
universal genetic markers and 6 Papaver-specific genetic markers for P. 
setigerum were identified and then Genbank accession numbers of them were 
registered in NCBI. Also, the trnL intron and psbA-trnH nucleic acid sequences 
of the 7 Papaver species were identified and registered.

Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

DOI: 10.1016/j.forsciint.2012.08.002
PMID: 22921420 [Indexed for MEDLINE]


5. Phytochemistry. 2009 Mar;70(5):579-89. doi: 10.1016/j.phytochem.2009.03.002. 
Epub 2009 Apr 7.

RNAi suppression of the morphine biosynthetic gene salAT and evidence of 
association of pathway enzymes.

Kempe K(1), Higashi Y, Frick S, Sabarna K, Kutchan TM.

Author information:
(1)Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 
63132, USA.

Papaver somniferum L. was transformed with an RNAi construct designed to reduce 
transcript levels of the gene encoding the morphine biosynthetic enzyme, 
salutaridinol 7-O-acetyltransferase (SalAT). RNA interference of salAT led to 
accumulation of the intermediate compounds, salutaridine and salutaridinol, in a 
ratio ranging from 2:1 to 56:1. Along the morphine biosynthetic pathway, 
salutaridine is stereospecifically reduced by salutaridine reductase (SalR) to 
salutaridinol, which is subsequently acetylated by SalAT. SalAT transcript was 
shown by quantitative PCR to be diminished, while salR transcript levels 
remained unaffected. Yeast two-hybrid and co-immunoprecipitation analyses 
indicated an interaction between SalR and SalAT, which suggested the occurrence 
of an enzyme complex and provided an explanation for the unexpected accumulation 
of salutaridine. Decreased concentrations of thebaine and codeine in latex were 
also observed, while the morphine levels remained constant compared to 
concentrations found in untransformed control plants.

DOI: 10.1016/j.phytochem.2009.03.002
PMID: 19359021 [Indexed for MEDLINE]