1 Systems metabolic engineering of Escherichia coli for the production of

2 fumaric acid

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4 Chan Woo Song1, 3, Dong In Kim1, 2, Sol Choi1, 3, Jae Won Jang1, 3, Sang Yup Lee1, 2, 3*

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6 1Metabolic and Biomolecular Engineering National Research Laboratory, Department

7 of Chemical and Biomolecular Engineering (BK21 program), Center for Systems and

8 Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of

9 Science and Technology (KAIST), Daejeon 305-701, Republic of Korea.

10 2BioInformatics Research Center, KAIST, Daejeon 305-701, Republic of Korea.

11 3BioProcess Engineering Research Center, KAIST, Daejeon 305-701, Republic of

12 Korea.

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14 * Corresponding author: Department of Chemical and Biomolecular Engineering,

15 KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.

16 Fax: +82 42 350 3910. E-mail: leesy@kaist.ac.kr

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18 Abstract

19 Fumaric acid is a naturally occurring organic acid which is an key intermediate of the

20 tricarboxylic acid cycle. It can be used for diverse purposes such as food additives,

21 resins, plasticizers, and precursor for diverse chemicals. In this study, Escherichia coli

22 was metabolically engineered for the production of fumaric acid under aerobic

23 condition. To design optimal production pathway for fumaric acid, firstly the iclR gene

24 was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the

25 three known fumarase genes (fumA, fumB and fumC) were also deleted to enhance

26 fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric

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1 acid from 15 g/L of glucose in flask culture. This base strain was further engineered by

2 plasmid-based overexpression of the native ppc gene, encoding phosphoenolpyruvate

3 carboxylase (PPC), based on in-silico aided prediction strategy, which resulted in the

4 production of 4.09 g/L of fumaric acid. And then, the arcA and ptsG genes were

5 sequentially deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was

6 deleted to block the conversion of fumaric acid into L-aspartic acid. Since it is desirable

7 to avoid the use of inducer, the lacI gene was also deleted. The native promoter of the

8 galP gene was replaced with the strong trc promoter to increase glucose uptake rate and

9 fumaric acid productivity. Fed-batch culture of the final strain CWF812 allowed

10 production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of

11 0.389 g fumaric acid/g glucose and 0.448 g/L/h. This study demonstrates the possibility

12 for the efficient production of fumaric acid by metabolically engineered E. coli.

13 (Development of systems metabolic engineering platform technologies for biorefineries;

14 NRF-2012-C1AAA001-2012M1A2A2026556) funded by the Ministry of Education,

15 Science and Technology)

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