Abstract
BackgroundFumarate is a multifunctional dicarboxylic acid in the tricarboxylic acid cycle, but microbial engineering for fumarate production is limited by the transmission efficiency of its biosynthetic pathway.ResultsHere, pathway engineering was used to construct the noncyclic glyoxylate pathway for fumarate production. To improve the transmission efficiency of intermediate metabolites, pathway optimization was conducted by fluctuating gene expression levels to identify potential bottlenecks and then remove them, resulting in a large increase in fumarate production from 8.7 to 16.2 g/L. To further enhance its transmission efficiency of targeted metabolites, transporter engineering was used by screening the C4-dicarboxylate transporters and then strengthening the capacity of fumarate export, leading to fumarate production up to 18.9 g/L. Finally, the engineered strain E. coli W3110△4-P(H)CAI(H)SC produced 22.4 g/L fumarate in a 5-L fed-batch bioreactor.ConclusionsIn this study, we offered rational metabolic engineering and flux optimization strategies for efficient production of fumarate. These strategies have great potential in developing efficient microbial cell factories for production of high-value added chemicals.
Highlights
Fumarate is a multifunctional dicarboxylic acid in the tricarboxylic acid cycle, but microbial engineering for fumarate production is limited by the transmission efficiency of its biosynthetic pathway
Coli W3110△4 were measured, and we found that E. coli W3110△4 was able to produce 6.8 g/L pyruvate, 10.5 g/L α-ketoglutarate, and 3.2 g/L fumarate (Fig. 2b)
To rewire the noncyclic glyoxylate pathway for fumarate production, pyruvate carboxylase (AfPYC) [7], citrate synthase (EcCS) [7], aconitase (EcACN) [7], isocitrate lyase (EcICL) [7], and succinate dehydrogenase (EcSDH) [22] were selected and overexpressed in E. coli W3110△4 (Fig. 2a). By overexpressing these five enzymes simultaneously, the specific activities of AfPYC, EcCS, EcACN, EcICL, and EcSDH were increased by 3.3, 5.5, 3.3, 4.3, and 0.5-fold compared with these of E. coli W3110△40, respectively (Fig. 2c)
Summary
Fumarate is a multifunctional dicarboxylic acid in the tricarboxylic acid cycle, but microbial engineering for fumarate production is limited by the transmission efficiency of its biosynthetic pathway. Two of the challenges in metabolically engineering the noncyclic glyoxylate cycle for fumarate production are how to identify and remove its potential bottlenecks and how to engineer and improve its transmission efficiency. Both challenges may benefit from the development of systems biology and synthetic biology. To identify and remove potential bottlenecks, several strategies have been developed, such as dynamic pathway analysis [9], X-omic technology [10], reverse metabolic engineering [11], in vitro metabolic engineering [12], and CRISPRi system [7]. To engineer and improve its transmission efficiency, many strategies have shown great potential, such as periplasmic engineering [13], mitochondrial engineering [14], DNA scaffold [15], protein scaffold [16], enzyme engineering [17], modular pathway engineering [18]
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