By

AMBRISH SINGH AGASTYA

M.Tech Biotech

Under the guidance of

Dr. S.MEENAKSHISUNDARAM
Introduction
S-Adenosyl L-Methionine (SAM, SAMe, SAM-e) is
a common co-substrate involved in methyl group
transfers.
IUPAC name: (2S)-2-Amino-4-[[(2S,3S,4R,5R)-5-(6-
aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl-
methylsulfonio]butanoate
Molecular formula: C15H22N6O5S+
Molar mass 398.44 g/mol
Uses of SAMe
Used as Antidepresent
Liver protective agent and used to reverse hyper
bilirubinemia
Used in osteoarthritis
It is used to protect against neuronal death caused by
lack of oxygen. It regenerate nerves and provokes the re-
methylation of nerve fibers.
Used to treat fibromyalgia and Alzheimer’s diseases.
Used as diet supplement in USA and Europe.
Need for producing SAMe through
microbial methods…
 As a commercial product, SAMe has great potential. It is
used as an anti depressant, analgesic for arthritic pains,
nutritional supplement and SAMe prevents oxidative
damage and cognitive impairment.
 The current method of producing SAMe through chemical
means is inefficient as a large portion of the product is in the
form of the unusable D stereoisomer.
 If SAMe is produced through microbial means, this problem
is overcome as cells naturally produce amino acids in the L
form thereby increasing efficiency and lowering DSP costs
Why Pichia pastoris?
It can be easily manipulated at the molecular genetic
level.
It can express recombinant protein at very high level,
intracellular / extracellular (recombinant protein levels
of approx 12g/L or nearly 35% of total protein)
Pichia pastoris can grow in cheap and simple minimal
media at very high cell densities –upto 600-800 ODs.
As a eukaryote, correctly folded recombinant proteins
with all post translational modifications and efficient
secretion are produced.
Particular strain of SMD 1168 is protease deficient
Objectives of project
Observing the growth kinetics of Pichia pastoris in BSM
and modified medium.
Studying the product expression at shake flask and reactor
levels.
Optimizing the bioprocess condition and medium so as to
get maximum SAMe production.
Growth kinetics In BSM
Observation
LAG PHASE- From 0th hr to 32nd hr.

LOG PHASE-Start from 32nd hr to 60th hr.

STATIONARY PHASE-From 60th hr to 136th hr.

 1 2 3 4
Confermation of SAM2 gene in Recombinant
Pichia pastoris SMD 1168/pGAPZB/SAM2
Expression study in BSM
SAM
synthetase 42
kD
Groth kinetic in BSM in
bioreactor
SAMe production kinetic in
bioreactor
Trying nitrogen source
Different nitrogen source were tried and there
effect on growth profile and SAMe production
profile was checked
Nitrogen source used were as follow:
Ammonium formate .5% and 2.5%
Ammonium acetate .5% and 2.5%
Diammonium hydrogen phosphate .25% and
1.2%
Growth kinetic with different
nitrogen sources and
concentration
SAMe production kinetics with 1.5%
Diammonium hydrogen phosphate
 So, what we got
By inferring all the above result we come to
conclusion that to increase the SAMe production by
improving biomass production we can use different
nitrogen source with appropriate condition
In our work Diammonium
hydrogen phosphate has shown very promising result
in shake flask and it could be one of the key
component of the optimized medium for SAMe
production where as its concentration optimization in
shake flask and scale up to bioreactor is yet to be
done
 Other media component as carbon
source, precursors , stimulator and process
condition as pH, Temperature e.t.c. would be
optimized in further work also the emphasis
will be given to develop more efficient SAMe
extraction protocol .