ABSTRACT
In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Streptomyces , Streptomyces/genetics , Streptomyces/metabolism , Gene Editing/methods , Acidaminococcus/genetics , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Multigene Family , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , Genome, BacterialABSTRACT
The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects the cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.
