Stephan Michalik

Bacillus subtilis is a Gram-positive bacterium that is well-known for its high secretory capacity. Although secretion of homologous proteins is extremely efficient, secretion of heterologous proteins imposes what is known as “secretion stress” on the cells. This is not desirable in an industrial setting, because the bacterial secretion stress responses can adversely affect product yields. So far, the main approach to reduce such counterproductive secretion stress responses has been to remove components of the CssR-CssS two-component regulatory system, including the CssR-CssS-regulated quality control proteases HtrA and/or HtrB. However, there are several examples indicating that a complete loss of these proteases does not correlate with improved protein secretion or a reduced secretion stress response. In this study, an alternative approach to limit the effects of secretion stress by modulating the protease activity of HtrA rather than completely removing this protease was investigated. Indeed, expression of the proteolytically inactive HtrA led to a generally improved fitness of the bacteria when producing the heterologous α-amylase AmyQ. Moreover, compared to control strains, the strain expressing the mutant HtrA provided higher AmyQ yields, which correlated with superior α-amylase enzymatic activity in shake flasks and fermenter-scale cultivations. Proteome analysis of the engineered strains revealed critical differences in stress responses and metabolism. Altogether, our present findings show how proteolytically inactive HtrA can be employed to achieve improved bacterial fitness and higher enzyme yields. 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.

In proteomics, fast, efficient, and highly reproducible sample preparation is of utmost importance, particularly in view of fast scanning mass spectrometers enabling analyses of large sample series. To address this need, we have developed the web application MassSpecPreppy that operates on the open science OT‐2 liquid handling robot from Opentrons. This platform can prepare up to 96 samples at once, performing tasks like BCA protein concentration determination, sample digestion with normalization, reduction/alkylation and peptide elution into vials or loading specified peptide amounts onto Evotips in an automated and flexible manner. The performance of the developed workflows using MassSpecPreppy was compared with standard manual sample preparation workflows. The BCA assay experiments revealed an average recovery of 101.3% (SD: ± 7.82%) for the MassSpecPreppy workflow, while the manual workflow had a recovery of 96.3% (SD: ± 9.73%). The species mix used in the evaluation experiments showed that 94.5% of protein groups for OT‐2 digestion and 95% for manual digestion passed the significance thresholds with comparable peptide level coefficient of variations. These results demonstrate that MassSpecPreppy is a versatile and scalable platform for automated sample preparation, producing injection‐ready samples for proteomics research.