A ptn IdhA Double Mutant Escherichia coli SS373 and the Method of Producing Succinic Acid therefrom BACKGROUND OF THE INVENTION Field of the Invention This invention relates to a mutant Escherichia coli SS373 and the production of succinic acid by using the above strain. In detail, a novel E. coli SS373 (W3110 pta::Tnl0 IdhA::Km) with the deficiency in the acetate and lactate forming pathways was constructed by genetic engineering technique. An aerobically grown SS373 was then cultured by means of the anaerobic condition shift during the succinate producing stage, which resulted in the efficient production of succinic acid with a higher yield.

Description of the Prior Art Succinate is one of the basic metabolites and an intermediate in the TCA cycle of the biological system. In the petrochemical industry, succinate serves a precursor of 1, 4-butandiol, tetrahydrofuran, y-butyrolactone. It is also useful as an ingredient in the food and cosmetic industry. Succinic acid is commercially produced by a chemical process. Recently, the biological process has been of interest for an environmentally clean process. In addition, the biological process could produce succinate from low- cost renewable resources. For the reasons of as above, the biological succinate production has been intensely studied in the recent years. Among these studies, strict anaerobic Anaerobiospirillum succiniciproducens has been particularly well examined (US Patent 5573931, 5521075, 5504004). A.succiniciproducens, however, has a complex nutrient requirement and slow growth rate as well as difficulty in the production process associated with the strict anaerobe.

SUMMARY OF THE INVENTION To solve the problems of a strict anaerobe in the succinate production, a

facultative anaerobic E.coil was genetically engineered. By using the mutated E. coli, the succinate production with higher yield was achieved. Therefore, the objective of the invention herein is the construction of a mutant E.coli and enhanced production of succinate by using the mutant E.coli.

Brief Description of the Drawings Figure 1 represents the metabolic pathway of SS373 based on various carbon sources. Figure 2 indicates the succinate production profile by SS373.

Detailed Descrlptionof the Invention The invention herein is characterized by Escherichia coli SS373(W3110 pta::TnlO IdhA::Km). The method of the anaerobic production of succinate after aerobic growth of cells is also involved. The detailed descriptions are as follows: As reported in the Bergey's Manual, E.coli has the following characteristics: facultative anaerobe, rod shaped, Gram-positive, simple nutrient demand, fast growth rate (doubling time w 20 min.), temperature optimum of 37"C, and pH optimum of 7.0.

Especially, E.coli yields a mixture of acetate, lactate, formate, succinate, and ethanol from glucose in the anaerobic condition. The physiology and genetics of E.coli have been well studied, and the E.coli metabolism could be easily controlled and estimated.

In addition, the metabolic engineering could be readily applied by means of genetic engineering technique.

Principle of Succinate Mass-production: To produce succinate by using E. coli, an E. coil W3 110 was modified genetically. The modified E. coli was further optimized to lead to an enhanced production.

Because E.coli carries out a mixed acid-fermentation, the metabolic pathway of E.coli should be altered to efficiently produce succinate. By means of the genetic

block of the pathway involved in the other products, the succinate production would be improved. At first. the genes ofpta and IdhA ofE.coli, which encode the first enzyme of acetate and lactate pathway, were mutated.

The constructed strain was cultured in an aerobic condition with high growth rate. which in turn produced succinate in the anaerobic condition. The succinate therefrom is able to penetrate the cell membrane to accumulate in the medium. which in turn prevents a feedback control of cells. The accumulated succinate can be recovered with high purity by electrodialysis technique (Hongo, M., Appl. Environ. Microbiol., 52-2. 314-319 (1986)).

Construction of a Double Mutated E.coli: Construction of a double mutated E.coli was carried out by the method suggested by Silhavy.

Step 1. Preparation of Transformed P1 Phage: P1 lysates of a E.coli CP993 (pta::TnlO-lacZ1)( Shin, S. A. and C. K. Park, J.

Bacteriol., 177, 4696-4702 (1995)) and a Ecoli NZN117 (IdhA::Km)( Bunch, P. K. et al, Microbiology, 143, 187-195 (1997)) were prepared respectively.

Step 2. P1 Transduction of thepta::TnlO4acZl to W3110 An E. coli W3110 (E. coli genetic stock center collection number (CGSC) 4474) was used as a recipient strain. The insertion mutated gene (pta::TnlO-lacZ-l) of Ecoli CP993 was transferred to E.coli W3110 by P1 transduction. The mutant E. coli strains were selected on the tetracycline selection plate, which yielded an E.coli W3110 pta: :Tnl O-lacZ-1.

Step 3. P1 transduction of the ldhA::Km to W3110pta::Tn10-lacZ-1: To obtain a lactate-production deficient strain, P1 lysate of NZN 117 was

infected with E.coli W3110 pta::TnlO-lacZ-J. The selected strain on the kanamycin plate was an double mutated W3 10 pta::TnlO-lacZ-l ldhA::Km.

Principle of Succinate Production in SS373 from Various Carbon-sources: Though the pathways to succinate slightly differ from one another depending on the carbon source, the phosphorylation is a common process (Fig. 1). In the case of glucose. which is the most common carbon source, the main phosphate donor has been known to be phosphoenolpyruvate (PEP) when glucose is transported by phosphate transferase system (PTS). The PEP involved in the glucose uptake converts to pyruvate and the chance for the succinate production is relatively reduced because succinate is derived from oxaloacetate (OAA). Hence, the phosphate groups are delivered from ATP in the cases of galactose, xylose, and maltose, the PEP would be saved as compared with that of the case with glucose. The conservation of PEP, which serves as a phosphate donor in the PTS, would lead to the increase of succinate production as well as a decrease of by-product formation.

This invention will be described detail in the following examples but is not limited thereby.

Example 1: Construction of a Double Mutated E.coli for the Succinate Production Step 1. Preparation of Transformed P1 Phage: The P1 transduction was carried out by the Silhavy method. Each E.coli strain of pta::TnlO-lacZ-1 and IdhA::Km was pre-cultured in 3ml of TGC media (0.1% glucose, lacto-tryptone, l0mM CaCl2). The overnight grown cells were transferred to the 3ml of TGC media and cultured for Ihr at 35 C in a shaking incubator. When the absorbance (600nm) of cells was reached at 0.1, the P1 phage (30p1 in the concentration of 1010 pfu/ml) was infected and cultured for 2-3 hrs. After the cell lysis, chloroform (0.1my) was added. and then supernatant was prepared by centrifuge. The supernatant. termed P1 lysate. contained some P1 phage withpta::TnJO-lacZ-i and IdhA: :Km.

Step 2. P1 Transduction of the pta:: Tnl O-lacZl to W3110: The overnight grown E. coli W3 110 was prepared by centrifuge. After the

dispersion of cells with 0.5ml of divalent ion solution (lOmM MgSO4, 5mM CaCl2). the P1 lysate of pta::TnlO-lacZ1 (0.01-0.lml) was appended. The mixture was left to stand for 15 minutes at room temperature. The cells were collected by centrifuge and then washed twice by lml of 1M sodium citrate. After the activation in LB medium. the mutant cells were selected on the LB-agar plate containing tetracycline (13pg/ml).

Step 3. P1 Transduction of the ldhA::Km to W3110 pfa::TnlO-lacZ1: The P1 lysate of ldhA::Km from step 1 was infected to the strain obtained from <BR> <BR> <BR> <BR> <BR> <BR> step 2. After the same procedure of step 2, a double mutant of W31 10 pta: :Tnl O-lacZ- 1 IdhA ::Km was obtained on the LB-agar plate containing kanamycin (20,ug/ml).

The finally obtained E.coli W3110 pta::TnlO-lacZ-1 IdhA::Km was named E.coli SS373. The E.coli SS373 was deposited on the 28th of July 1997 in the Korea Collection of Type Culture(KCTC; 52, Ereun-dong, Yusong-ku, Taejeon 305-333, Republic of Korea), which is an international strain deposit institute by the Budapest Convention, and the deposit number was assigned as KCTC 8818P. For the purpose of PCT international application, a conversion of the original deposit under the Budapest Treaty was made on July 29, 1998, and a new deposit number was obtained. e.g., KCTC 0506BP.

The E.coli SS373 could be cultured on a glucose medium in an anaerobic condition because it could produce acetyl-CoA while E.coli NZN1 1 1(Clark D. P.

FEMS Microbiol. Rev., 63, 223-234 (1989)) could not.

Example 2: Succinate Production in the Glucose Medium: The Ecoli SS373 was cultured on a glucose-based medium. The components of medium were represented as Table 1.

Table 1 Component Glucose Na2HPO4-H2O NaH2PO4 Yeast Extract Na2CO32 Concentration(g/ l ) 15 7 3 5 3.18

Note: The pH was pre-set to 7.0 by adding a few drops of conc. H2SO4.

A single colony of SS373 was sub-cultured in a 15ml test tube at 370C for 12 hours. Cells were transferred to a 50ml medium in 250ml Erlenmeyer flask and cultured until absorbance reached 0.5 at 600nm. The actively grown cells from above were inoculated to a 2.5-liter jar-fermentor containing 1-liter medium and cultured at 370C pH 7.0 in aerobic condition (350 rpm, 1 vvm). When the absorbance(600nm) reached 4.0 aeration was stopped and mixed gas (5% CO2, 95% N2) was fluxed in. Glucose solution 500ml (60g/l) was appended with anaerobic shift. Thereafter, 1 lg/l of succinate was produced with 0.8g/l of pyruvate in 34 hours of culture. (Fig. 2) The concentrations of succinate and pyruvate were estimated by using a HPLC- UV system (Gilson, France) with carbohydrate analysis column (HPX-87H , Bio-Rad).

The glucose concentration was measured by the Glucose-Analyzer (2300STAT. Yellow Spring Instruments).

Example 3: Succinate Productions Based on Various Carbohydrates The E.coli SS373 and W3 110 were cultured in the media containing different carbon sources (Table 2). The carbon sources used were glucose, galactose. maltose. and xylose, respectively.

Table 2 Component *Carbon source Na2HPO4-H2O NaH2PO4 Yeast Extract NarC03 Concentration(g/ # ) 10 7 3 5 3.18 Note : The pH was set to 7.0 by adding a conc. H2SO4. *Carbon sources were glucose. galactose, maltose, and xylose, respectively.

A single colony of SS373 was sub-cultured in a 15ml test tube at 370C for 12 hours. Cells were transferred to a 10ml medium in 100ml Erlenmeyer flask. The biomass was set to an approximate absorbance of 1.0 at 600nm. The flask was flushed

with 5% C02 gas and sealed by using a silicon stopper to maintain anaerobic condition.

Cells were cultured for 8 hrs at 370C, and organic acids formation were investigated (Table 3).

In the cases of wild strains, e.g., W3110, the major organic acids were lactate and acetate, while succinate and pyruvate were the major factors in the SS373. In the So373 the proportions of succinate to pyruvate were varied depending on the carbon sources used. The glucose medium showed 1:2 of succinate to pyruvate with 1:0.8 for maltose and 1:0.3 for galactose and xylose. Nearly pure succinate was obtained in the concentration of 1.9 and 1.6 g/l from galactose and xylose, respectively. Therefore, the use of non-PTS carbohydrates was preferable in producing succinate with high purity and yield because PEP used in phosphorylation was conserved.

Table 3 Effect of Carbon Sources on the Succinate Production in E.coli SS373 Carbon Succinate Pyruvate Lactate Acetate Strain source (g/ Q ) (gl C) (g/ Q ) (g/ Q ) Glucose 0.6 0 1.0 1.0 Maltose 0.5 0 1.6 1.6 W3110 Galactose 2.1 0 0.8 2.3 Xylose 1.6 0.2 0.9 1.6 Glucose 2.7 5.3 0 0.6 Maltose 2.3 1.8 0 0.1 SS373 Galactose 1.9 0.6 0 0.3 Xylose 1.6 0.4 0 0.4 As noted, succinate in a novel E. coli SS373 could be produced with less effort to maintain strict anaerobic condition and without complex nutrient supply. In addition, E. coli SS373 showed fast growth rate due to the efficient succinate production.

Moreover. nearly pure succinate could be produced by using a carbon source with the result of conserving PEP.