Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon

Hadeel Waleed Abdulmalek, Ayten Yazgan-Karataş*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

Original languageEnglish
Pages (from-to)410-418
Number of pages9
JournalJournal of Microbiology and Biotechnology
Volume33
Issue number3
DOIs
Publication statusPublished - Mar 2023

Bibliographical note

Publisher Copyright:
© 2023 by the authors.

Funding

This work was supported by the Istanbul Technical University Scientific Research Foundation (Grant No. 42659). Bacteria shapes in the graphical abstract were picked from Starline-Freepick.com. We would like to acknowledge J. Altenbucher for providing the plasmid pJOE9958.1. We thank our PhD student Büşra Öztürk for providing technical assistance in the RT-qPCR experiments.

FundersFunder number
Istanbul Technical University Scientific Research Foundation42659

    Keywords

    • bacABCDEF
    • Bacillus subtilis
    • bacilysin
    • CRISPR/Cas9
    • ribosome binding site

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