Mori Grant
Research Report:
Research Title: Dynamic
Simulation and Enzymological Analysis for Fermentation Strategy of Escherichia
Coli
Md. Aminul Hoque1
1. Institute for Advanced Biosciences (IAB),
Baba-cho, Tsuruoka,
Introduction
Dynamic responses against pulse addition
of glucose during glucose-limited continuous culture have been investigated for
the wild type Escherichia coli, Saccharomyces cerevisiae, Bacillus
subtilis and other microorganisms. It is quite important to
investigate the metabolism at transient state to make clear the metabolic
regulation mechanism. In the present study, we first developed a new rapid
sampling device that enables us to take the sample from the reactor every 1
second or less. Using this device, we investigated the metabolic responses
against pulse addition of glucose during glucose-limited continuous culture and
the responses against NH3 pulse addition during NH3-limited
continuous culture by measuring the intracellular metabolite concentrations.
Moreover, in the present research, we investigated the metabolism of a single
gene knockout mutant such as pykA mutant. Previously, we investigated
the metabolism of the pykF mutant E.coli. Although many
researchers have reported the effect of pykF knockout on the metabolism,
little research is done on pykA mutant. Although the impact of pykA
gene knockout is less than that of pykF gene knockout, this type of
mutation often occur in various organisms, and it is worth investigating the
metabolic changes.
Materials and Methods
Strains and culture
condition
The strains used
are Escherichia coli BW25113 and its pykA mutant JWK1843 which
were constructed by deletion of the corresponding gene pykA from E.coli derivative
BW25113 using the method of Datsenko and Wanner by Mori et al. (http://www.ttck.keio.ac.jp/IAB/english/research/index.htm).
The medium used in the present experiment
was as follows (per liter): 5.0 g of glucose, 3.96g of (NH4)2SO4,
6.81 g of Na2HPO4, 2.99 g of KH2PO4,
0.58 g of NaCl, 0.614g of MgSO4; 7H2O, 1.0 mg of thiamine
HCl, and 10 mL of trace element solution containing (per liter) 1 g of FeCl2,
0.55g of CaCl2. 2H2O, 0.1g of MnCl2, 4H2O,
0.17g of ZnCl2, 0.043 CuCl2. 2H2O, 0.06g CoCl2.
6H2O and 0.06g of Na2MoO4. 2H2O.
Cultivations
were made at 37C in a 2-L bioreactor (BMJ-02PI, ABLE Co.,
For the NH3-limited culture, the culture medium was
the same as above except using 0.2 g/L of NH4Cl and 0.24 g/L of (NH4)2SO4
instead of using 3.96 g/L of (NH4)2SO4.
Brief Research Results
The dynamics of the
intracellular metabolite concentrations were investigated for the wild-type and
pykA gene knock-out mutant Escherichia coli in responses to the
glucose pulse addition during glucose-limited continuous culture and in
responses to the ammonia pulse addition during ammonia-limited continuous
culture. For this, we developed a new automated rapid sampling device which
enables us to take samples rapidly within a second. The intracellular
concentrations of G6P, F6P, 2PG, PEP, OAA, 6PG, Ribu5P, E4P and NADPH were
higher for pykA mutant as compared with the wild type under both limited
continuous cultures, and the concentrations of PYR, ATP and acetate were much
lower for pykA mutant than those of the wild type. These phenomena
reflected the fact that the accumulation of PEP caused the increased flux from
PEP to OAA and that the accumulated PEP inhibited pfk which caused the
accumulation of G6P and F6P, which intern increased the flux toward pentose
phosphate (PP) pathway and increased the PP pathway metabolite concentrations.
Oxygen uptake rate (OUR) was a little higher for pykA mutant as compared
with that of its wild type, while CO2 production rate (CER) shows
the reverse trend. OUR and CER were much less for NH3 limited
condition than that of NH3 rich condition. The intracellular
concentrations of PEP, ATP, and PYR decreased rapidly within several seconds,
whereas the concentrations of G6P, F6P FBP, 6PG, ADP, NADH, and NADPH increased
after glucose pulse addition during glucose limited condition for both wild
type and pykA knock-out mutant. Initial decrease in PEP concentration
was considered to be due to PTS system. The intracellular concentration of
NADPH decreased after NH3 pulse addition under NH3-
limited condition for both strains, which is due to the fact that NADPH is
utilized through glutamate production under NH3 addition.
Paper and Presenttaions
During my last
year research period I have submitted one of my research papers entitled g Dynamic
responses of the Intracellular Metabolite Concentrations of the Wild Type and pykA
mutant Escherichia coli against Pulse Addition of Glucose or NH3
under Those Limiting Continuous Culturesh in Biochemical Engineering
Journal for publication.
I have attended
several national and international conferences and presented my research
successfully. 50th NIBB conferences was one of them that held at
Aichi prefecture,
During my research period I
needed some necessary materials for my reseach and I bought them using
Morigrant. I also used the money for my conference travels and related. It is
needless to say that Mori Grant helped me very much in my research and study.
(Md. Aminul Hoque)