Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 99, Issue 20, Pages 8455-8464Publisher
SPRINGER
DOI: 10.1007/s00253-015-6816-6
Keywords
Fumaric acid; Escherichia coli; Flux optimization; Metabolic engineering
Categories
Funding
- Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea [NRF-2012-C1AAA001-2012M1A2A2026556]
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Fumaric acid is an important C4-dicarboxylic acid widely used in chemical, food, and pharmaceutical industries. Rational metabolic engineering together with flux optimization were performed for the development of an Escherichia coli strain capable of efficiently producing fumaric acid. The initial engineered strain, CWF4N overexpressing phosphoenolpyruvate carboxylase (PPC), produced 5.30 g/L of fumaric acid. Optimization of PPC flux by examining 24 types of synthetic PPC expression vectors further increased the titer up to 5.72 g/L with a yield of 0.432 g/g center dot glucose. Overexpression of the succinate dehydrogenase complex (sdhCDAB) led to an increase in carbon yield up to 0.493 g/g center dot glucose. Based on this mutant strain, citrate synthase (CS) was combinatorially overexpressed and balanced with PPC using 48 types of synthetic expression vectors. As a result, 6.24 g/L of fumaric acid was produced with a yield of 0.500 g/g center dot glucose. Fed-batch culture of this final strain allowed production of 25.5 g/L of fumaric acid with a yield of 0.366 g/g center dot glucose. Deletion of the aspA gene encoding aspartase and supplementation of aspartic acid further increased the fumaric acid titer to 35.1 g/L with a yield of 0.490 g/g center dot glucose.
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