Escherichia coli remains one of the most extensively studied and versatile chassis in synthetic biology, metabolic engineering, and industrial biotechnology. Its well-characterized genome, rapid growth, high-density cultivation kinetics, and extensive genetic toolbox have established it as a foundational host for fundamental research and translational engineering [1]. Beyond its traditional role in high-yield recombinant protein production, recent advances in metabolic pathway reconstruction, genome editing, and systems biology are transforming E. coli into a reprogrammable platform capable of utilizing sustainable alternative feedstocks, generating structurally complex biomolecules, enhancing cellular robustness, and advancing downstream bioprocessing, alongside novel diagnostic and therapeutic platforms [2,3].
Despite these remarkable advances, significant bottlenecks continue to hinder the broader translation of engineered E. coli systems across different applications. Cellular fitness is routinely challenged by the metabolic burden imposed by synthetic circuits, competing cofactor demands, and the accumulation of toxic intermediates. Maintaining physiological robustness, particularly membrane integrity and viability under shifting thermal, osmotic, and mechanical stresses, remains a major operational hurdle [4]. At the same time, producing complex heterologous proteins is frequently hindered by the bacterium’s lack of post-translational machinery and the tendency of target proteins to misfold or form insoluble aggregates [5]. Furthermore, transitioning from food-competing carbohydrates to alternative carbon streams or industrial side-streams often results in low biomass yields and slower growth [6]. Finally, downstream recovery and purification from complex fermentation broths remain a primary cost bottleneck, demanding more selective and cost-effective separation strategies.
This Special Issue brings together five original contributions that address several of these operational bottlenecks while also demonstrating the broader utility of engineered E. coli in biosensing and diagnostics.
To improve membrane robustness, Pu et al. [7] modulated membrane lipid profiles by heterologously co-expressing an acyl-ACP thioesterase (AtFatA) and a fatty acid desaturase (AtFab2) from Arabidopsis thaliana in E. coli. Their work demonstrated that fine-tuning the ratio between saturated and unsaturated fatty acids effectively reshapes membrane composition, providing a rational framework to optimize membrane fluidity and enhance environmental robustness under suboptimal bioprocess conditions.
Addressing the need for sustainable carbon sources, Wang et al. [8] engineered E. coli to synthesize poly-β-hydroxybutyrate directly from methanol, an attractive one-carbon feedstock that can be derived from captured CO2 or industrial emissions. By co-expressing a synthetic methanol oxidation cascade with the non-oxidative glycolysis pathway, the authors bypassed carbon loss via CO2, demonstrating a promising strategy to produce bioplastics without relying on food-derived sugars.
To overcome the limitations of bacterial expression systems in carrying out mammalian post-translational modifications, Cheng et al. [9] produced functional human-like collagen without relying on complex eukaryotic enzymes. By engineering a proline-auxotrophic E. coli strain and supplementing the growth medium with a balanced mix of proline and trans-4-hydroxy-L-proline, they enabled the direct incorporation of hydroxylated residues into the target protein, yielding stable collagen biomaterials.
Expanding the role of engineered bacteria beyond traditional biosynthesis, Nastouli et al. [10] used E. coli as a selective bio-purification tool to tackle downstream recovery bottlenecks. By deploying a lactate dehydrogenase-deficient strain inside a membrane bioreactor, the authors selectively removed residual sugars and nutrient impurities from fermentation broths without consuming the accumulated lactic acid. This work demonstrates the practical utility of engineered cells for biological polishing in circular downstream bioprocesses.
Finally, Sande et al. [11] leveraged bacterial surface engineering to design an electrochemical aptasensor targeting the ubiquitous surface protein A2 (UspA2) of Moraxella catarrhalis. By displaying the recombinant target on the outer membrane of E. coli during Cell-SELEX, the authors isolated high-affinity DNA aptamers and integrated them into a label-free sensor, demonstrating how microbial surface display can facilitate point-of-care diagnostics.
The research gathered in this collection reflects how versatile E. coli remains across modern biotechnology. Rather than serving solely as a standard production host, the organism is increasingly treated as a programmable platform capable of utilizing non-conventional feedstocks, producing complex materials, assisting downstream processing, and driving diagnostic technologies. The broader challenge ahead is translation: turning these specialized genetic and cellular strategies into robust, scalable systems that perform reliably outside carefully controlled laboratory settings. As synthetic biology tools expand, E. coli will continue to redefine its utility well beyond its traditional boundaries.
Funding
The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/04469/2025 research unit, https://doi.org/10.54499/UID/04469/2025 and LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020, https://doi.org/10.54499/LA/P/0029/2020.
Acknowledgments
We sincerely thank all contributing authors for their valuable scientific work, the reviewers for their constructive evaluations, and the Bioengineering editorial staff for their support in bringing this diverse collection together.
Conflicts of Interest
The authors declare no conflicts of interest.
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