Fragment Exchange Plasmid Tools for CRISPR/Cas9-Mediated Gene Integration and Protease Production in Bacillus subtilis

Abstract

Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains.IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.

Keywords: Bacillus subtilis; CRISPR; FX cloning; genome editing; microbial fermentation; protease; subtilisin.

FIG 1

Customization of FX-compatible vectors for genome editing in B. subtilis. (A) The pCC9X plasmid was constructed by introducing an FX counterselection cassette into the backbone of pJOE8999, which contains a pUC minimal origin of replication for E. coli, the temperature-sensitive replication origin pET194^ts for B. subtilis, and a kanamycin resistance gene working in both organisms. The cassette harbors a lethal ccdB gene (indicated in red) flanked by two opposite outward-oriented SapI sites (triangles) useful for directional cloning. (B) An FX-compatible editing cassette is readily cloned into pCC9X by SapI restriction and ligation to obtain the final stable SapI-free editing vector, pCC9X:sgRNA(spoIIAC). The editing cassette is flanked by two opposite inward-oriented and compatible SapI sites (triangles) and contains an inducible sgRNA and a short insert between two homology arms (HA1 and HA2) against spoIIAC. Gene lengths are not to scale.

FIG 2

Demonstration of the asporogenic phenotype in B. subtilis KO7S2 introduced by using the FX-compatible pCC9X vector. The CRISPR/Cas9-based pCC9X:sgRNA(spoIIAC) plasmid was used to knock out the spoIIAC sporulation gene. Sporulation was induced by metabolic stress, and vegetative cells were killed by heat treatment. Only the wild-type KO7 strain produces colonies from heat-resistant spores in all serial dilutions (top halves of plates in the top panel), whereas KO7S2 did not (top halves of plates in the bottom panel). Nontreated cells (bottom halves of plates in both panels) served as controls of cell viability.

FIG 3

Efficient FX cloning for CRISPR-based editing in the amyE locus of B. subtilis. (A) An sgRNA is FX cloned into pCC9X to induce Cas9 cleavage in the amyE locus. (B) Building the repair plasmid pAHX by replacing the chloramphenicol resistance (Cam^r) gene in pDG1662Δspc with a BamHI/SalI-flanked fragment containing a xylose-inducible promoter (P_xylA) and a ccdB-based counterselection cassette (red). The counterselection region is flanked with opposite outward-oriented SapI sites (triangles) to make it compatible with FX cloning. The homology arms (HA1 and HA2) (gray) in pAHX ensure homologous recombination of genes into the amyE locus of B. subtilis. (C) The repair plasmid pAHX serves as the destination for FX cloning of a gene of interest (here, aprE encoding subtilisin) from a delivery plasmid, pINITIAL. Gene lengths are not drawn to scale.

FIG 4

Batch fermentation with Bacillus KO7S2 amyE::aprE. (A) OD₆₀₀ of fermentations of the KO7S2 strain (control) and the aprE-containing KO72S amyE::aprE strain (two uninduced replicates, aprE, and two induced replicates, aprE+). The doubling times in hours (T_d) are inserted into the chart with the corresponding color codes. No significant differences in cell density were observed over time between strains, even after the induction of recombinant subtilisin expression. The dotted vertical line shows the transition from exponential phase to stationary and/or death phase. (B) Proteolytic activity measured as relative fluorescence counts (RFC) from supernatants of the fermentation cultures in panel A during exponential phase (<5.5 h). Color codes are the same as the ones for panel A. Although uninduced and aprE-negative strain controls resulted in substantial proteolytic activity in the extracellular medium toward the end of the exponential phase, the response was delayed more than 2 h compared to the induced strain, altogether suggesting the induced expression of AprE from KO72S amyE::aprE.

FIG 5

Overview of the compatible FX cloning platform for quick transitions from high-throughput cloning and the expression of new enzymes in E. coli and B. subtilis (enzyme discovery and optimization phases) to stable CRISPR/Cas-mediated integration of genes for the production of enzymes through B. subtilis fermentation (upscaling and manufacture).