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Related Concept Videos

Bioreactor Controls-III01:22

Bioreactor Controls-III

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Strain improvement is a foundational strategy in industrial microbiology aimed at maximizing microbial productivity, particularly because natural isolates typically yield commercially valuable products in very low concentrations. Although optimizing the culture medium and environmental conditions can improve yields, these adjustments are inherently limited by the organism’s genetic potential. As a result, the focus shifts toward genetic modifications to enhance biosynthetic capacity. The...
54

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Related Experiment Video

Updated: Apr 8, 2026

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1
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Optimization of Plasmid Curing from Genetically Engineered Clostridium autoethanogenum.

Victoria Chinonyerem Udemezue1, Kurshedaktar Majibullah Shaikh1, Mariia Vorontsova1

  • 1Institute of Bioengineering, University of Tartu, 50411 Tartu, Estonia.

ACS Synthetic Biology
|December 2, 2025
PubMed
Summary

We optimized plasmid curing for acetogens, crucial for carbon capture and biomanufacturing. New methods significantly accelerate genetic engineering workflows for microbes like Clostridium autoethanogenum.

Keywords:
CRISPR/Cas9Clostridium autoethanogenumacetogengas fermentationplasmid curing

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Biotechnology

Background:

  • Greenhouse gas accumulation from fossil fuels drives climate change.
  • Microbial gas fermentation offers a sustainable route for CO2 capture and biomanufacturing.
  • Acetogens, like Clostridium autoethanogenum, are promising biocatalysts using CO2 and H2.

Purpose of the Study:

  • To optimize plasmid curing methods for genetically engineered acetogens.
  • To accelerate genetic engineering workflows for Clostridium autoethanogenum.
  • To develop efficient plasmid elimination techniques for microbial cell factories.

Main Methods:

  • Developed and tested CRISPR/Cas9-based and non-targeting control plasmids for curing.
  • Utilized electrocompetent cells (ECCs) and non-transformative electroporation.
  • Applied methods to buffer-washed glycerol stocks for plasmid elimination.

Main Results:

  • CRISPR/Cas9-based curing did not outperform non-targeting controls.
  • Electroporation methods achieved 14-100% curing efficiencies across five engineered C. autoethanogenum strains.
  • Non-transformative electroporation of glycerol stocks efficiently cured plasmids in C. autoethanogenum and E. coli (~97%).

Conclusions:

  • Optimized plasmid curing significantly accelerates genetic engineering in Clostridium autoethanogenum.
  • Developed efficient and time-saving plasmid curing methods applicable to other microbial systems.
  • Enhanced workflows facilitate the use of acetogens for sustainable biomanufacturing and carbon capture.