Psychoactive Plant Database - Neuroactive Phytochemical Collection

1. Food Sci Biotechnol. 2022 May 20;31(6):635-655. doi:
10.1007/s10068-022-01082-3.  eCollection 2022 Jun.

The biological feasibility and social context of gene-edited, caffeine-free 
coffee.

Leibrock NV(1), Santegoets J(2), Mooijman PJW(3), Yusuf F(2), Zuijdgeest XCL(4), 
Zutt EA(2)(3), Jacobs JGM(5), Schaart JG(6).

Author information:
(1)Programme Molecular Life Sciences, Wageningen University and Research, 
Grumbachtalweg 129, 66121 Saarbrücken, Germany.
(2)Programme Plant Sciences, Wageningen University and Research, Wageningen, The 
Netherlands.
(3)Programme Plant Biotechnology, Wageningen University and Research, 
Wageningen, The Netherlands.
(4)Programme Biology, Wageningen University and Research, Wageningen, The 
Netherlands.
(5)Philosophy, Wageningen University and Research, Wageningen, The Netherlands.
(6)Plant Breeding, Wageningen University and Research, Wageningen, The 
Netherlands.

Coffee, especially the species Coffea arabica and Coffea canephora, is one of 
the world's most consumed beverages. The consumer demand for caffeine-free 
coffee is currently being met through chemical decaffeination processes. 
However, this method leads to loss of beverage quality. In this review, the 
feasibility of using gene editing to produce caffeine-free coffee plants is 
reviewed. The genes XMT (7-methylxanthosine methyltransferase) and DXMT 
(3,7-dimethylxanthine methyltransferase) were identified as candidate target 
genes for knocking out caffeine production in coffee plants. The possible effect 
of the knock-out of the candidate genes was assessed. Using Agrobacterium 
tumefaciens-mediated introduction of the CRISPR-Cas system to Knock out XMT or 
DXMT would lead to blocking caffeine biosynthesis. The use of CRISPR-Cas to 
genetically edit consumer products is not yet widely accepted, which may lead to 
societal hurdles for introducing gene-edited caffeine-free coffee cultivars onto 
the market. However, increased acceptance of CRISPR-Cas/gene editing on products 
with a clear benefit for consumers offers better prospects for gene editing 
efforts for caffeine-free coffee.

© The Author(s) 2022.

DOI: 10.1007/s10068-022-01082-3
PMCID: PMC9133285
PMID: 35646415

Conflict of interest statement: Conflict of interestOn behalf of all authors, 
the corresponding author states that there is no conflict of interest.


2. J Chem Inf Model. 2016 Sep 26;56(9):1755-61. doi: 10.1021/acs.jcim.6b00153.
Epub  2016 Aug 15.

Understanding the Catalytic Mechanism of Xanthosine Methyltransferase in 
Caffeine Biosynthesis from QM/MM Molecular Dynamics and Free Energy Simulations.

Qian P(1)(2)(3), Guo HB(1), Yue Y(1)(2), Wang L(3), Yang X(4), Guo H(1)(2).

Author information:
(1)Department of Biochemistry and Cellular and Molecular Biology, University of 
Tennessee , Knoxville, Tennessee 37996, United States.
(2)UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory , Oak 
Ridge, Tennessee 37830, United States.
(3)Chemistry and Material Science Faculty, Shandong Agricultural University , 
Tai'an 271018, Shandong, People's Republic of China.
(4)Biosciences Division, Oak Ridge National Laboratory , Oak Ridge, Tennessee 
37831, United States.

Erratum in
    J Chem Inf Model. 2016 Nov 28;56(11):2280. doi: 10.1021/acs.jcim.6b00634.

S-Adenosyl-l-methionine (SAM) dependent xanthosine methyltransferase (XMT) is 
the key enzyme that catalyzes the first methyl transfer in the caffeine 
biosynthesis pathway to produce the intermediate 7-methylxanthosine (7mXR). 
Although XMT has been a subject of extensive discussions, the catalytic 
mechanism and nature of the substrate involved in the catalysis are still 
unclear. In this paper, quantum mechanical/molecular mechanical (QM/MM) 
molecular dynamics (MD) and free energy (potential of mean force or PMF) 
simulations are undertaken to determine the catalytic mechanism of the 
XMT-catalyzed reaction. Both xanthosine and its monoanionic form with N3 
deprotonated are used as the substrates for the methylation. It is found that 
while the methyl group can be transferred to the monoanionic form of xanthosine 
with a reasonable free energy barrier (about 17 kcal/mol), that is not the case 
for the neutral xanthosine. The results suggest that the substrate for the first 
methylation step in the caffeine biosynthesis pathway is likely to be the 
monoanionic form of xanthosine rather than the neutral form as widely adopted. 
This conclusion is supported by the pKa value on N3 of xanthosine both measured 
in aqueous phase and calculated in the enzymatic environment. The structural and 
dynamics information from both the X-ray structure and MD simulations is also 
consistent with the monoanionic xanthosine scenario. The implications of this 
conclusion for caffeine biosynthesis are discussed.

DOI: 10.1021/acs.jcim.6b00153
PMID: 27482605 [Indexed for MEDLINE]


3. PLoS One. 2014 Aug 18;9(8):e105368. doi: 10.1371/journal.pone.0105368. 
eCollection 2014.

Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine 
production.

Jin L(1), Bhuiya MW(2), Li M(1), Liu X(1), Han J(2), Deng W(1), Wang M(1), Yu 
O(3), Zhang Z(1).

Author information:
(1)Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, 
Anhui Agricultural University, Hefei, PR China.
(2)Conagen Inc., St. Louis, Missouri, United States of America.
(3)Donald Danforth Plant Science Center, St. Louis, Missouri, United States of 
America.

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are 
the major purine alkaloids in plants, e.g., tea (Camellia sinensis) and coffee 
(Coffea arabica). Caffeine is a major component of coffee and is used widely in 
food and beverage industries. Most of the enzymes involved in the caffeine 
biosynthetic pathway have been reported previously. Here, we demonstrated the 
biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica 
xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase 
(TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this 
production platform for making other purine-based alkaloids. To increase the 
catalytic activity of TCS in an effort to increase theobromine production, we 
identified four amino acid residues based on structural analyses of 3D-model of 
TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in 
theobromine accumulation and simultaneously decreased in caffeine production. 
The application and further optimization of this biosynthetic platform are 
discussed.

DOI: 10.1371/journal.pone.0105368
PMCID: PMC4136831
PMID: 25133732 [Indexed for MEDLINE]

Conflict of interest statement: Competing Interests: The authors have the 
following interests. M. W. Bhuiya and J. X. Han are employed by Conagen Inc. 
This does not alter the authors’ adherence to PLOS ONE policies on sharing data 
and materials.


4. Handb Exp Pharmacol. 2011;(200):11-31. doi: 10.1007/978-3-642-13443-2_2.

Distribution, biosynthesis and catabolism of methylxanthines in plants.

Ashihara H(1), Kato M, Crozier A.

Author information:
(1)Department of Biological Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, 
Tokyo, Japan. ashihara.hiroshi@ocha.ac.jp

Methylxanthines and methyluric acids are purine alkaloids that are synthesized 
in quantity in a limited number of plant species, including tea, coffee and 
cacao. This review summarizes the pathways, enzymes and related genes of 
caffeine biosynthesis. The main biosynthetic pathway is a sequence consisting of 
xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine. 
Catabolism of caffeine starts with its conversion to theophylline. Typically, 
this reaction is very slow in caffeine-accumulating plants. Finally, the 
ecological roles of caffeine and the production of decaffeinated coffee plants 
are discussed.

DOI: 10.1007/978-3-642-13443-2_2
PMID: 20859792 [Indexed for MEDLINE]


5. Z Naturforsch C J Biosci. 2010 Mar-Apr;65(3-4):257-65. doi: 
10.1515/znc-2010-3-414.

Essential region for 3-N methylation in N-methyltransferases involved in 
caffeine biosynthesis.

Mizuno K(1), Kurosawa S, Yoshizawa Y, Kato M.

Author information:
(1)Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, 
Akita 010-0195, Japan. koumno@akita-pu.ac.jp

The caffeine biosynthetic pathway is composed of three methylation steps, and 
N-methyltransferase catalyzing each step has high substrate specificity. Since 
the amino acid sequences among coffee 7-methylxanthosine synthase (CmXRS1), 
theobromine synthase, and caffeine synthase are highly homologous to each other, 
these substrate specificities seem to be determined in a very restricted region. 
The analysis of site-directed mutants for CmXRS1 that naturally acts at the 
initial step, i.e., 7-N methylation of xanthosine, revealed that the activity of 
3-N methylation needs a histidine residue at corresponding position 161 in the 
CmXRS1 sequence. We succeeded in producing the mutant enzyme which can catalyze 
the first and second methylation steps in caffeine biosynthesis.

DOI: 10.1515/znc-2010-3-414
PMID: 20469646 [Indexed for MEDLINE]