A portable C-terminal rheostat calibrates RecE/Redα exonuclease activity for enhanced bacterial recombineering
- 1Shandong University
Abstract
Recombineering enables precise genome engineering across diverse bacteria, yet efficient tools for large DNA integration remain underdeveloped for Streptomyces and other biotechnologically relevant bacteria. Here we identify a panel of highly active RecET recombinase pairs from actinobacteriophages that mediate efficient homologous recombination in Streptomyces. The lead candidate, Che9cRecET, supports genomic integration of kilobase-scale DNA with only 50bp homology arms and enables accurate insertion of 79kb biosynthetic gene clusters, far outperforming conventional genome editing approaches. Structural and biochemical analyses reveal Che9cRecE as a distinct C3-symmetric toroidal homotrimeric 5'-3' exonuclease, whose extended C-terminal loop acts as a tunable rheostat calibrating exonuclease activity to an optimal window for recombination. We further show this portable C-terminal loop enhances recombineering efficiency across phylogenetically diverse exonucleases including Escherichia coli Redalpha/RecE and Pseudomonas A. Our work uncovers a conserved regulatory mechanism of the RecE/Redalpha exonuclease family and delivers a generalizable strategy for advancing bacterial genome editing.
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BibTeX
@misc{luan2026portable,
title = {{A portable C-terminal rheostat calibrates RecE/Redα exonuclease activity for enhanced bacterial recombineering}},
author = {J. Luan and C. Jiang and R. Guo and C. Wang and S. Wang and A. F. Stewart and J. Fu and Y. Zhang and H. Wang},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.29.762211},
url = {https://www.biorxiv.org/content/10.64898/2026.10.29.762211v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A portable C-terminal rheostat calibrates RecE/Redα exonuclease activity for enhanced bacterial recombineering
AU - J. Luan
AU - C. Jiang
AU - R. Guo
AU - C. Wang
AU - S. Wang
AU - A. F. Stewart
AU - J. Fu
AU - Y. Zhang
AU - H. Wang
PY - 2026
DA - 2026/10/29
PB - bioRxiv
DO - 10.64898/2026.10.29.762211
UR - https://www.biorxiv.org/content/10.64898/2026.10.29.762211v1
AB - Recombineering enables precise genome engineering across diverse bacteria, yet efficient tools for large DNA integration remain underdeveloped for Streptomyces and other biotechnologically relevant bacteria. Here we identify a panel of highly active RecET recombinase pairs from actinobacteriophages that mediate efficient homologous recombination in Streptomyces. The lead candidate, Che9cRecET, supports genomic integration of kilobase-scale DNA with only 50bp homology arms and enables accurate insertion of 79kb biosynthetic gene clusters, far outperforming conventional genome editing approaches. Structural and biochemical analyses reveal Che9cRecE as a distinct C3-symmetric toroidal homotrimeric 5'-3' exonuclease, whose extended C-terminal loop acts as a tunable rheostat calibrating exonuclease activity to an optimal window for recombination. We further show this portable C-terminal loop enhances recombineering efficiency across phylogenetically diverse exonucleases including Escherichia coli Redalpha/RecE and Pseudomonas A. Our work uncovers a conserved regulatory mechanism of the RecE/Redalpha exonuclease family and delivers a generalizable strategy for advancing bacterial genome editing.
ER -