Malic enzyme의 발현이 증폭된 재조합 대장균에의한 숙신산 생산 및 대사회로분석

홍순호, 이상엽

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과 및 생물공정연구센터

Metabolic flux analysis for succinic acid production by recombinant Escherichia coli with amplified malic enzyme activity

Soon Ho Hong and Sang Yup Lee

Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST 373-1 Kusong-dong, Yusong-gu, Taejon 305-701

Introduction

Succinic acid is an intermediate of cellular metabolism. Succinic acid and its derivatives are widely used as specialty chemicals with applications in polymers, foods, pharmaceuticals, and cosmetics. Succinic acid is currently manufactured by hydrogenation of maleic anhydride to succinic anhydride, followed by hydration to succinic acid. Production of succinic acid through fermentation represents an alternative synthesis route via the utilization of renewable feedstocks.

The Anaerobiospirillum succiniciproducens (A. succiniciproducens) fermentation employs a phosphoenolpyruvate carboxykinase (PEP carboxykinase) to direct phosphoenolpyruvate (PEP) toward succinic acid with concomitant generation of ATP. In Escherichia coli (E. coli) and most other organisms, on the other hand, the carboxykinase is gluconeogenic. Instead, a phosphoenolpyruvate carboxylase (PEP carboxylase) channels PEP toward succinic acid and dissipates the energy of PEP through hydrolysis to inorganic phosphate. A possible alternative route to direct to dicarboxylic acid from pyruvate instead of PEP, involving the reductive carboxylation of pyruvate to malic acid catalyzed by malic enzyme. Here we evaluate the possibility of constructing a nonnatural metabolic pathway to dicarboxylic acids through expression of the E. coli NAD+-dependent malic enzyme in a host which blocked in its normal fermentative metabolism of pyruvate.

Materials and methods

E. coli strain NZN111(F- □pfl::Cam ldhA::Kan) was used. This strain lacks pyruvate-formate lyase (PFL) and fermentative lactate dehydrogenase (LDH) due to insertional inactivation of the genes pfl and ldhA.

Fermentation studies were carried out using BioFlo 3000 bioreactor (5 L, New Brunswick Scientific, Edison, NJ, USA) containing 3 L of medium at 30℃. LB broth was used with the appropriate antibiotics and instead of MgCO3, 5 M NaOH was used to maintain pH. The dissolved oxygen (DO) level was maintained over 40% during aerobic cultivation, then IPTG was added to various final concentration and at various time. After induction, anaerobic condition was achieved by lowering the DO level with various decreasing rates. Under anaerobic condition, CO2-H2 (1:1) gas was purged.

Fermentation products were analyzed by high-performance liquid chromatography (HPLC) using Bio-Rad Aminex HPX-87H column and a Hitachi chromatography system equipped with refractive index detector.

Results and discussion

Fermentation

When the strain cultured at 30℃, pH 6.7 with 0.01 mM IPTG induction. acetic acid concentration was increased in aerobic condition and then decreased in anaerobic condition (Figure 1 and 2). However, after 60 hr, concentrations of ethanol, formic acid, and acetic acid were begin to increase gradually. At 50 hr, glucose concentration was 3 g/L and malic acid accumulation was initiated. This result was consistent with the fact that malic enzyme was inhibited by glucose. Therefore, glucose concentration was maintained below 5 g/L by adding small amount of glucose occasionally. Succinic acid concentration was steadily increased and final concentration was 9.4 g/L.

When fermentation was carried out at 30℃, pH 7.5 with 0.01 mM IPTG induction. Concentrations of formic acid and ethanol were increased. In the late stage of culture, concentration of malic acid was decreased while that of acetic acid was increased. In the cases of pH 7.5, succinic acid productivity was low and much byproducts were produced compare to cultures which carried out at pH 6.7.

Metabolic flux analysis

The result of fed-batch culture was analyzed by two metabolic flux analyses techniques. One is the conventional method and the other is a new method in which the flux to intracellular pyruvate pool was introduced. In conventional metabolic flux analysis, Four reaction rates (glucose, succinic acid, ext-Mal, and acetic acid) and maintenance energy were used to calculate volumetric formation rates of intracellular metabolites. The residual lactic acid, formic acid, and ethanol formation rates were used to check the validity of pseudo-steady state assumption by comparing calculated formation rates with the measured ones. Average intracellular metabolic flux distribution during the entire anaerobic condition was calculated and (result not shown).

As mentioned earlier, intracellular pyruvate accumulation is necessary to change the direction of reaction of malic enzyme. This means that the intracellular pyruvate concentration, which is initially low, needs to be increased to a certain level. Therefore, the pseudo-steady state assumption (no net formation of intermediate) was not satisfied. Consequently, intracellular pyruvate pool was introduced to mimic the pyruvate accumulation while satisfying the pseudo-steady state assumption. In this new method, five reaction rates (glucose, succinic acid, ext-Mal, acetic acid, and lactic acid) and maintenance energy were used to calculate volumetric formation rates of intracellular metabolites. Intracellular metabolic flux distributions were calculated and are shown in Fig. 3.

Figure 1. Time profiles of (A) the culture OD600 and glucose concentration, and (B) concentrations of succinic acid, malic acid, acetic acid, lactic acid, formic acid, and ethanol. Arrows indicate the time points of glucose addition.

Figure 2. Pictorial representation of the flux network with reaction numbers and flow rates in culture at 30℃ (mM/hr)

Acknowledgement

E. coli strain NZN111 was provided by Dr. David Clark. This work was supported by Ministry of Science and Technology(MOST).

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