The construction of a PAM-less base editing toolbox in Bacillus subtilis and its application in metabolic engineering

Yan Xia, Lichao Sun, Zeyu Liang, Yingjie Guo, Jing Li, Dan Tang, Yi Xin Huo*, Shuyuan Guo

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

The necessity of a specific protospacer adjacent motif (PAM) greatly limited the editing scope and the application range of Cas9-dependent base editors. Here, we showed that SpRY breaks the PAM recognition barrier in Bacillus subtilis with a preference toward NRN than NYN while the PAM specificity is slightly distinct from other organisms. We developed a highly efficient PAM-less base editing toolbox for B. subtilis based on a variant of Cas9 nickase, nSpRY, yielding PAM-less adenine and/or cytosine base editors (PLABE/PLCBE/PLACBE) for A-to-G and/or C-to-T conversions. The conversion efficiency was optimized to 100% at high-activity sites and the editing window was refined to 1–2 nucleotides at pre-selected sites. Multiplexed editing for double, triple, and quadruple genes was validated simply by one cycle of editing. As a proof of concept, PLCBE was utilized to introduce premature stop codons for yielding gene knockouts and PLABE was applied to change start codons from ATG to GTG for realizing translation control. A website was designed to accelerate the inactivation of any genes of interest in B. subtilis by incorporating PAM specificity information for identifying the high-efficiency sites. Facilitated by multiplexed PAM-less editing, we successfully obtained a diversified library of strain variants for chemical production with multi-level regulations in central and competing metabolism pathways. We recruited this library to modulate metabolic fluxes in B. subtilis, achieving up to a 26.3% increase in acetoin production with a final titer of 20.8 g/L in shake-flask fermentation. By enabling in situ base editing of nearly all As and Cs in the Bacillus genome, this toolbox shows great potential in applications of metabolic engineering and codon expansion.

Original languageEnglish
Article number143865
JournalChemical Engineering Journal
Volume469
DOIs
Publication statusPublished - 1 Aug 2023

Keywords

  • Bacillus subtilis
  • Base editor
  • Gene editing
  • Multiplex
  • PAM-less
  • SpRY

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