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. 2020 Jul 23:13:132.
doi: 10.1186/s13068-020-01771-3. eCollection 2020.

Engineering the transmission efficiency of the noncyclic glyoxylate pathway for fumarate production in Escherichia coli

Xiulai Chen  1   2   3 , Danlei Ma  1   2   3 , Jia Liu  1   2   3 , Qiuling Luo  1   4 , Liming Liu  1   2   3
Affiliations

Engineering the transmission efficiency of the noncyclic glyoxylate pathway for fumarate production in Escherichia coli

Xiulai Chen et al. Biotechnol Biofuels. .

Abstract

Background: 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.

Results: Here, 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.

Conclusions: In 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.

Keywords: Escherichia coli; Fumarate; Metabolic engineering; Pathway optimization; Transporter engineering.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Major metabolic pathways for the formation of fumarate in E. coli. PEP: phosphoenolpyruvate; ldhA: lactate dehydrogenase; pflB: pyruvate formate lyase; poxB: pyruvate oxidase; pta: phosphotransacetylase; ackA: acetate kinase A; PYC: pyruvate carboxylase; CS: citrate synthase; ACN: aconitase; ICL: isocitrate lyase, SDH: succinate dehydrogenase; fumABC: fumarase; frdBC: fumarate reductase; dcuBC: the C4-dicarboxylate transporter
Fig. 2
Fig. 2
Constructing the noncyclic glyoxylate pathway for fumarate production. a Schematic representation of fumarate biosynthesis through the noncyclic glyoxylate pathway. b Effect of gene expression on the production of organic acids. c The specific activities of AfPYC, EcCS, EcACN, EcICL, and EcSDH. d The expression level of genes AfPYC, EcCS, EcACN, EcICL, and EcSDH. Error bars represent standard deviation from three biological replicates
Fig. 3
Fig. 3
Optimizing the noncyclic glyoxylate pathway for fumarate production. a Effect of individual gene expression on fumarate production. b A series of AfPYC and EcICL expression cassettes were designed at different expression levels. c The concentrations of fumarate were achieved by different AfPYC and EcICL expression cassettes. Error bars represent standard deviation from three biological replicates
Fig. 4
Fig. 4
Improving fumarate production by transporter engineering. a Schematic representation of the C4-dicarboxylate transporters. b Effect of the C4-dicarboxylate transporters on the concentrations of intracellular fumarate, succinate, and oxaloacetate. c Effect of the C4-dicarboxylate transporters on fumarate production. Error bars represent standard deviation from three biological replicates
Fig. 5
Fig. 5
Production of fumarate by strain E. coli W3110△4-P(H)CAI(H)SC in a 5-L fed-batch bioreactor. Error bars represent standard deviation from three biological replicates
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