Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

Post-translational secretion stress regulation in Bacillus subtilis is controlled by intra- and extracellular proteases.

The Gram-positive bacterium Bacillus subtilis is a prolific producer of industrial enzymes that are effectively harvested from the fermentation broth. However, the high capacity of B. subtilis for protein secretion has so far not been exploited to the full due to particular bottlenecks, including product degradation by extracellular proteases and counterproductive secretion stress responses. To unlock the Bacillus secretion pathway for difficult-to-produce proteins, various cellular interventions have been explored, including genome engineering. Our previous research has shown a superior performance of genome-reduced B. subtilis strains in the production of staphylococcal antigens compared to the parental strain 168. This was attributed, at least in part, to redirected secretion stress responses, including the presentation of elevated levels of the quality control proteases HtrA and HtrB that also catalyse protein folding. Here we show that this relates to the elimination of two homologous serine proteases, namely the cytosolic protease AprX and the extracellular protease AprE. This unprecedented posttranslational regulation of secretion stress effectors, like HtrA and HtrB, by the concerted action of cytosolic and extracellular proteases has important implications for the biotechnological application of microbial cell factories. In B. subtilis, this conclusion is underscored by extracellular degradation of the staphylococcal antigen IsaA by both AprX and AprE. Extracellular activity of the cytosolic protease AprX is remarkable since it shows that not only extracellular, but also intracellular proteases impact extracellular product levels. We therefore conclude that intracellular proteases represent new targets for improved recombinant protein production in microbial cell factories like B. subtilis.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Enhancing bacterial fitness and recombinant enzyme yield by engineering the quality control protease HtrA of Bacillus subtilis.

IMPORTANCE: In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.