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), Keio University (TTCK),

Baba-cho, Tsuruoka, Yamagata 997-0035, Japan

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 37°C in a 2-L bioreactor (BMJ-02PI, ABLE Co., Tokyo, Japan) with a working volume of 1 liter. The culture medium was continuously fed to the bioreactor at the dilution rate of 0.1 ±0.005 h⁻¹, and the working volume was kept constant by withdrawing culture broth through a continuously operating pump. The pH of the culture was maintained at 7.0 by adding 2.0 M NaOH or 2.0 M HCl. The agitation speed of 500 rpm with 1.0 L min⁻¹ of air flow ensured the dissolved oxygen concentration above 60% of air saturation.

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 “ 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 Cultures” in Biochemical Engineering Journal for publication.

I have attended several national and international conferences and presented my research successfully. 50^th NIBB conferences was one of them that held at Aichi prefecture, Japan , during February 8-10, 2005.

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)