1. 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.
2. 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]
3. Plant Physiol. 2007 Jun;144(2):879-89. doi: 10.1104/pp.106.094854. Epub 2007
Apr 13.
The structure of two N-methyltransferases from the caffeine biosynthetic
pathway.
McCarthy AA(1), McCarthy JG.
Author information:
(1)European Molecular Biology Laboratory, Grenoble 38042, France.
andrewmc@embl.fr
Caffeine (1,3,7-trimethylxanthine) is a secondary metabolite produced by certain
plant species and an important component of coffee (Coffea arabica and Coffea
canephora) and tea (Camellia sinensis). Here we describe the structures of two
S-adenosyl-l-methionine-dependent N-methyltransferases that mediate caffeine
biosynthesis in C. canephora 'robusta', xanthosine (XR) methyltransferase (XMT),
and 1,7-dimethylxanthine methyltransferase (DXMT). Both were cocrystallized with
the demethylated cofactor, S-adenosyl-L-cysteine, and substrate, either
xanthosine or theobromine. Our structures reveal several elements that appear
critical for substrate selectivity. Serine-316 in XMT appears central to the
recognition of XR. Likewise, a change from glutamine-161 in XMT to histidine-160
in DXMT is likely to have catalytic consequences. A phenylalanine-266 to
isoleucine-266 change in DXMT is also likely to be crucial for the
discrimination between mono and dimethyl transferases in coffee. These key
residues are probably functionally important and will guide future studies with
implications for the biosynthesis of caffeine and its derivatives in plants.
DOI: 10.1104/pp.106.094854
PMCID: PMC1914188
PMID: 17434991 [Indexed for MEDLINE]
4. Front Biosci. 2004 May 1;9:1833-42. doi: 10.2741/1364.
Caffeine synthase and related methyltransferases in plants.
Misako K(1), Kouichi M.
Author information:
(1)Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka,
Bunkyo-ku, Tokyo112-8610, Japan. mkato@cc.ocha.ac.jp
Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high
concentrations in tea and coffee and it is also found in a number of beverages
such as coca cola. It is necessary to elucidate the caffeine biosynthetic
pathway and to clone the genes related to the production of caffeine not only to
determine the metabolism of the purine alkaloid but also to control the content
of caffeine in tea and coffee. The available data support the operation of a
xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine
pathway as the major route to caffeine. Since the caffeine biosynthetic pathway
contains three S-adenosyl-L-methionine (SAM) dependent methylation steps,
N-methyltransferases play important roles. This review focuses on the enzymes
and genes involved in the methylation of purine ring. Caffeine synthase, the
SAM-dependent methyltransferase involved in the last two steps of caffeine
biosynthesis, was originally purified from young tea leaves (Camellia sinensis).
The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a
protein of 369 amino acids. Subsequently, the homologous genes that encode
caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The
recombinant proteins are classified into the three types on the basis of their
substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and
caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic
enzymes derived from C. arabica exhibit more than 80% homology with those of the
clones and but show only 40% homology with TCS1 derived from C. sinensis. In
addition, they share 40% homology with the amino acid sequences of salicylic
carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic
acid carboxyl methyltransferase which belong to a family of motif B'
methyltransferases which are novel plant methyltransferases with motif B'
instead of motif B as the conserved region.
DOI: 10.2741/1364
PMID: 14977590 [Indexed for MEDLINE]
5. FEBS Lett. 2003 Jul 17;547(1-3):56-60. doi: 10.1016/s0014-5793(03)00670-7.
The first committed step reaction of caffeine biosynthesis: 7-methylxanthosine
synthase is closely homologous to caffeine synthases in coffee (Coffea arabica
L.).
Mizuno K(1), Kato M, Irino F, Yoneyama N, Fujimura T, Ashihara H.
Author information:
(1)Faculty of Bioresource Sciences, Akita Prefectural University, Akita City,
010-0195, Akita, Japan. koumno@akita-pu.ac.jp
In coffee and tea plants, caffeine is synthesized from xanthosine via a pathway
that has three methylation steps. We identified and characterized the gene
encoding the enzyme for the first methylation step of caffeine biosynthesis. The
full-length cDNA of coffee tentative caffeine synthase 1, CtCS1, previously
isolated by the rapid amplification of cDNA ends was translated with an
Escherichia coli expression system and the resultant recombinant protein was
purified using Ni-NTA column. The protein renamed CmXRS1 has 7-methylxanthine
synthase (xanthosine:S-adenosyl-L-methionine methyltransferase) activity. CmXRS1
was specific for xanthosine and xanthosine 5'-monophosphate (XMP) could not be
used as a substrate. The K(m) value for xanthosine was 73.7 microM. CmXRS1 is
homologous to coffee genes encoding enzymes for the second and third methylation
steps of caffeine biosynthesis.
DOI: 10.1016/s0014-5793(03)00670-7
PMID: 12860386 [Indexed for MEDLINE]