Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

Improving gene set enrichment analysis (GSEA) by using regulation directionality.

To infer the biological meaning from transcriptome data, it is useful to focus on genes that are regulated by the same regulator, i.e., regulons. Unfortunately, current gene set enrichment analysis (GSEA) tools do not consider whether a gene is activated or repressed by a regulator. This distinction is crucial when analyzing regulons since a regulator can work as an activator of certain genes and as a repressor of other genes, yet both sets of genes belong to the same regulon. Therefore, simply averaging expression differences of the genes of such a regulon will not properly reflect the activity of the regulator. What makes it more complicated is the fact that many genes are regulated by different transcription factors, and current transcriptome analysis tools are unable to indicate which regulator is most likely responsible for the observed expression difference of a gene. To address these challenges, we developed the gene set enrichment analysis program GINtool. Additional features of GINtool are novel graphical representations to facilitate the visualization of gene set analyses of transcriptome data, the possibility to include functional categories as gene sets for analysis, and the option to analyze expression differences within operons, which is useful when analyzing prokaryotic transcriptome and also proteome data.IMPORTANCEMeasuring the activity of all genes in cells is a common way to elucidate the function and regulation of genes. These transcriptome analyses produce large amounts of data since genomes contain thousands of genes. The analysis of these large data sets is challenging. Therefore, we developed a new software tool called GINtool that can facilitate the analysis of transcriptome data by using prior knowledge of gene sets controlled by the same regulator, the so-called regulons. An important novelty of GINtool is that it can take into account the directionality of gene regulation in these analyses, i.e., whether a gene is activated or repressed, which is crucial to assess whether a regulon or functional category is affected. GINtool also includes new graphical methods to facilitate the visual inspection of regulation events in transcriptome data sets. These and additional analysis methods included in GINtool make it a powerful software tool to analyze transcriptome data.

Serial propagation in water-in-oil emulsions selects for Saccharomyces cerevisiae strains with a reduced cell size or an increased biomass yield on glucose.

In S. cerevisiae and many other micro-organisms an increase in metabolic efficiency (i.e. ATP yield on carbon) is accompanied by a decrease in growth rate. From a fundamental point of view, studying these yield-rate trade-offs provides insight in for example microbial evolution and cellular regulation. From a biotechnological point of view, increasing the ATP yield on carbon might increase the yield of anabolic products. We here aimed to select S. cerevisiae mutants with an increased biomass yield. Serial propagation of individual cells in water-in-oil emulsions previously enabled the selection of lactococci with increased biomass yields, and adapting this protocol for yeast allowed us to enrich an engineered Crabtree-negative S. cerevisiae strain with a high biomass yield on glucose. When we started the selection with an S. cerevisiae deletion collection, serial propagation in emulsion enriched hxk2Δ and reg1Δ strains with an increased biomass yield on glucose. Surprisingly, a tps1Δ strain was highly abundant in both emulsion- and suspension-propagated populations. In a separate experiment we propagated a chemically mutagenized S. cerevisiae population in emulsion, which resulted in mutants with a higher cell number yield on glucose, but no significantly changed biomass yield. Genome analyses indicate that genes involved in glucose repression and cell cycle processes play a role in the selected phenotypes. The repeated identification of mutations in genes involved in glucose-repression indicates that serial propagation in emulsion is a valuable tool to study metabolic efficiency in S. cerevisiae.