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bioRxiv · 10.1101/2025.10.19.683307

Sulbactam-mediated inhibition of TEM-1 β-lactamase increases specific yields of ampicillin-resistant plasmids

Pepeliaev, S.
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Abstract

Ampicillin resistance is the most widely used selection marker in molecular biology. Most ampicillin-resistant plasmids originate from highly active TEM-1 {beta}-lactamase cassette which causes very fast antibiotic decay and negatively affects long-term plasmid retention. The present work proposes a method of controlling the {beta}-lactamase activity by the addition of an irreversible {beta}-lactamase inhibitor. The positive effect of a {beta}-lactamase inhibitor, specifically sulbactam, on plasmid retention is confirmed by much higher specific plasmid yields: 4-8 times higher titres in shake-flask and almost 3 times higher in bioreactor scales were observed. Among all commercially available {beta}-lactamase inhibitors sulbactam provides the best combination of affordability and activity. The sulbactam addition is simple, fast and economic solution allowing to screen thousands of legacy pBR322 or pUC-derived plasmids harbouring TEM-1 {beta}-lactamase cassette without engineering their sequence.

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Pepeliaev, S.. 2025-10-21. Sulbactam-mediated inhibition of TEM-1 β-lactamase increases specific yields of ampicillin-resistant plasmids. https://doi.org/10.1101/2025.10.19.683307

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