Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Artículo científico |
| Language: | en |
| Published: |
Journal of biotechnology
2026
|
| Subjects: | |
| Online Access: | https://pubmed.ncbi.nlm.nih.gov/41202982/ |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
Table of Contents:
- Engineering of endogenous plasmids in probiotic Escherichia coli Nissle 1917 for autonomous accumulation of 5‑aminolevulinic acid. Yu, Dong Zhu, Lin Ding, Ya-Xue Mao, Li-Jing Xiong, Min Jin, Xiao-Xuan Ma, Yujie Wang, Feng-Qing Xiong, Liang-Bin Escherichia coli Aminolevulinic Acid Plasmids Metabolic Engineering Probiotics Escherichia coli Proteins Operon 5-aminolevulinic acid (ALA), a non‑proteinogenic δ‑amino acid, is a versatile compound with applications as a tumor-sensitizing agent in photodynamic therapy and a plant biostimulant that enhances stress tolerance and photosynthetic efficiency. This study aimed to leverage the endogenous cryptic plasmids pMUT1 and pMUT2 of the probiotic Escherichia coli Nissle 1917 (EcN) to construct a self-sufficient strain with autonomous ALA biosynthesis capability, eliminating dependencies on exogenous plasmids or chemical inducers. A synthetic operon was designed on a re-engineered cryptic plasmid to express core genes of the C5 pathway (gltX, hemA, and hemL). Systematic evaluation demonstrated that overexpression of gltX alone resulted in minimal ALA accumulation (6.8 mg/L). In contrast, coordinated co-expression of hemA and hemL significantly increased ALA titers to 854.5 mg/L, highlighting their synergistic role in channeling carbon flux through the C5 pathway in EcN. Subsequent optimization of the hemA‑hemL cassette further elevated production to 925.1 mg/L. Notably, this enhanced ALA synthesis perturbed the cellular NADP /NADPH balance. To address this, we integrated pos5, encoding a NADP⁺‑dependent transhydrogenase, into the endogenous plasmid, enabling in situ NADPH regeneration and boosting ALA titers to 1306.8 mg/L. Scale up to a 5-L bioreactor with fed-batch cultivation and controlled glycerol feeding achieved an ALA titer of 3372.5 ± 162.7 mg/L at 108 h. This endogenous plasmid-centric approach establishes an inducer-free, antibiotic-independent microbial cell factory, positioning EcN as a universal platform for ALA production with potential in biomedical and agricultural applications.