REENGINEERING
CAFFINE
PROJECT PROPOSAL BY JACQUELYN
CAFFEINE
Stimulant blocks adenosine receptors (A1 and
A2A) in the brain.
Adenosine normally promotes relaxation &
sleepiness.
Bind adenosine receptors ⟶ prevent adenosine
from slowing neural activity --. increased
alertness & reduced fatigue
PROBLEMS
Withdrawal Symptoms
Crash
Heacache,
caffeine wears off ⟶
irrritability, fatigue,
fatigue & decreased
difficulty
alertness
concentrating
Addiction Others
Depndence due to increased heart rate,
brain adaption anxiety, sleep
Changes adenosine disruption
receptor sensitivity
PROJECT OBJECTIVE
Optimize caffeine molecular
variants with slower
metabolism and gentler
stimulation to reduce crashes
and withdrawal.
BACKGROUND RESEARCH
Enzymes used:
CaXMT1 (xanthosine methyltransferase) from coffee
TCS1 (caffeine synthase) from tea
Method: Clone & functionally express plant eynzymes in yeast
supplied with exogenous xanthosine (precursor)
Result: yeast produced caffine at low yield (0.39 mg/L)
Challenges: limited precursor availability, potential enzyme insuffiencies in yeast
MECHANISM
1. Pick the Microbe 2. Insert Enzyme 3. Adjust promoters
& optimize codons
Copy the Recipe from
(the “factory”) Balance the Recipe
Coffee Plants
4. Persecutors & 5. Efflux Pumps 6 . Te s t & M e a s u r e
helpers
Add extra Build an export
Ta s t e t e s t !
ingredients system
PROPOSED METHODS
Apply site-directed
mutagenesis targeting
amino acid residues
Ex: mutating residues that
interact with the nitrogen
atoms of the purine ring
possible effect:
alternative site -->
different receptor
affinities and slower
metabolism
PROPOSED METHODS
Use directed evolution to
mutate residues in the
SAM-binding site to
accept cofactor analogs
or alter catalysis speed.
caffeine analogs with
modified methyl groups
(e.g., fluoromethyl) or
slower methylation rates
possible effect: gentler
stimulation & reduced
side effects.
THANK YOU
Citations:
Jin, Lu, Mohammad Wadud Bhuiya , Mengmeng Li, et al . “Metabolic Engineering of
S a c c h a r o m y c e s C e r e v i s i a e f o r C a f f e i n e a n d T h e o b r o m i n e P r o d u c t i o n .” P L O S O N E 9 , n o . 8
( 2 0 1 4 ) : e 1 0 5 3 6 8 . h t t p s : // d o i . o r g / 1 0 . 1 3 7 1 / j o u r n a l . p o n e . 0 1 0 5 3 6 8 .
A s h i h a r a , H i r o s h i & S u z u k i , Ta k e o . ( 2 0 0 4 ) . D i s t r i b u t i o n a n d b i o s y n t h e s i s o f c a f f e i n e i n p l a n t s .
F r o n t i e r s i n B i o s c i e n c e . 9 . 1 0 . 2 7 4 1 / 1 3 6 7.