Open Access

For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.

For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.

Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.