Abstract
Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain.
Highlights
Bacillus subtilis, a well-characterized gram-positive bacterium, has been widely used for the production of heterologous proteins
Clustered regularly interspaced short palindromic repeat (CRISPR) systems, which are composed of CRISPR RNAs, trans-activating CRISPR RNAs and CRISPR-associated (Cas) proteins constitute an immune system in bacteria and archaea that efficiently cleaves foreign DNA entering the cell, including phages and plasmids[12]
This study describes the establishment and optimization of a CRISPR/Cas[9] system in B. subtilis American Type Culture Collection (ATCC) 6051a
Summary
A well-characterized gram-positive bacterium, has been widely used for the production of heterologous proteins. B. subtilis ATCC 6051a has some undomesticated properties that hamper the extracellular production of recombinant proteins It can produce large amounts of foam, highly resistant spores, multiple types of extracellular protease, and high level of amylase during fermentation, which related to srfC5, spoIIAC6, nprE7, aprE8 and amyE9, respectively. As an efficient genome editing technology, the type II CRISPR/Cas[9] system has been proved to be feasible in point mutation, single gene deletion/ insertion and large-size gene cluster deletion[19] Until recently, it has been widely applied in various organisms including, but not restricted to, Escherichia coli[14], Streptococcus pneumonia[14], Saccharomyces cerevisiae[20], Lactobacillus reuteri[21], Bombyx mori[22], Drosophila[23], mice[24] and humans cell lines[17]. The increase of β-CGTase secretion from Bacillus subtilis can largely reduce the cost of β-cyclodextrin production, which shows the high industrial value of BS5
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