Evolution-assisted engineering boosts succinic acid production in E. coli

By evolving E. coli to tolerate acetate and then equipping it with exogenous carboxykinases and bicarbonate transporters, the team achieved titers exceeding 84 g/L SA.

Succinic acid is a key platform chemical in the bio-based economy, used in products ranging from bioplastics to pharmaceuticals. Traditional production methods rely on petrochemical feedstocks, such as maleic anhydride, but rising concerns about climate change and fossil fuel dependency have accelerated the search for renewable alternatives. Microbial fermentation has shown promise, yet wild-type strains often struggle with low yields and poor tolerance to industrial conditions. Meanwhile, biodiesel manufacturing generates large surpluses of glycerol, a highly reduced carbon substrate with excellent theoretical efficiency for SA production. However, unlocking this potential requires strains capable of efficiently metabolizing glycerol under anaerobic conditions—a challenge that existing microbes have not fully overcome.

A study (DOI:10.1016/j.bidere.2025.100022) published in BioDesign Research on 25 March 2025 by Hui Wu’s team, East China University of Science and Technology, not only demonstrates the potential for sustainable conversion of biodiesel byproducts into high-value chemicals but also paves the way for greener biomanufacturing of industrial precursors for plastics, pharmaceuticals, and food additives.

The researchers employed adaptive laboratory evolution (ALE) in combination with metabolic engineering to improve Escherichia coli’s capacity for succinic acid (SA) biosynthesis from glycerol. Starting with the NZN111 strain, which lacks ldhA and pflB and is limited in redox balance, they subjected it to successive cultivation in sodium acetate (NaAC) medium under aerobic conditions, progressively increasing the NaAC concentration from 5 g/L to 30 g/L. This evolutionary pressure selected for mutants with enhanced tolerance and metabolic efficiency. Among 49 evolved isolates, strain PG23 and its clone PG23-05 demonstrated the most robust growth and productivity. To further refine metabolic flux toward SA production, the team analyzed transcriptional profiles and found that ALE had upregulated key genes in the glyoxylate shunt (aceA, aceB), tricarboxylic acid (TCA) cycle (mdh, aceK), and gluconeogenesis (pckA, ppsA, maeB), thereby increasing precursor supply for SA synthesis. They next introduced heterologous phosphoenolpyruvate carboxykinase (pck) genes from Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, and Actinobacillus succinogenes, as well as bicarbonate transporter proteins (ecaA and bicA), to enhance CO₂ uptake and fixation. The corresponding results were significant: in anaerobic fermentation with 100 g/L glycerol, PG23-05 consumed more substrate and produced 71.16 g/L SA, an 18.99% improvement over NZN111, with higher yields and faster production rates. When engineered with exogenous pck and transporter genes, the strain achieved 84.27 g/L SA with a yield of 1.25 g per gram of glycerol—1.38 times the titer and 1.16 times the yield of the parental strain. Collectively, these findings show that ALE reshaped global regulation and stress tolerance, while metabolic engineering synergistically redirected carbon flux, culminating in a highly efficient microbial platform for converting glycerol into valuable industrial chemicals

In summary, the integration of ALE and targeted metabolic engineering enabled E. coli to efficiently convert glycerol, a low-value biodiesel byproduct, into large amounts of succinic acid, a high-value industrial precursor. This approach highlights a generalizable framework for valorizing other waste carbon streams through microbial cell factories. By transforming waste into resources, the study provides a powerful example of how synthetic biology can drive sustainable bio-manufacturing and reduce reliance on fossil fuels.

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References

DOI

10.1016/j.bidere.2025.100022

Original Source URL

https://doi.org/10.1016/j.bidere.2025.100022

Funding information

This work was supported by the National Key R&D Program of China (2022YFC2105400), The Science and Technology Commission of Shanghai Municipality (24HC2820800), Fundamental Research Funds for the Central Universities (DUT24RC(3)029). Partially supported by Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

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