MassIVE MSV000091841

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

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Description

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. Sample processing 16 OD600 units of bacterial culture were harvested from shake flasks or fermenters. Tubes were briefly immersed in liquid nitrogen to stop the metabolism of the cells. Cells were pelleted by centrifugation at 4000 rpm for 2 min at 4 degC and washed with 20 mM HEPES. Cell pellets were frozen in liquid nitrogen and stored at -80 degC until processing. After benzonase treatment, reduction and alkylation, and digestion with trypsin in an enzyme-to-sample mass ratio of 1:25 overnight at 37 degC, peptides were desalted on SP3 beads as previously described (Blankenburg et al. 2019). Lyophilized peptide samples were reconstituted in mass spectrometry Buffer A (2% acetonitrile in 0.1% acetic acid), For LC-MS/MS analyses tryptic peptide solutions were separated on an Ultimate 3000 nano-LC system (Thermo Fisher Scientific, MA, USA) and analysed on a Q Exactive HF mass spectrometer in data independent acquisition mode. Peptides were separated on a 25 cm Accucore column (75 um inner diameter, 2.6 um, 150 A, C18) at a flow rate of 300 nl/min in a binary buffer system of aqueous Buffer A (0.1% acetic acid, 2% acetonitrile in water) and organic Buffer B (0.1% acetic acid in 100% acetonitrile) by applying a linear gradient of buffer B from 5% up to 25% within 120 min. Data processing Identification and quantitation were performed via the DirectDIA approach in the Spectronaut software (Biognosys, Germany) using a predicted synthetic library fasta-file with database search against a Uniprot/Swissprot database restricted to entries from B. subtilis (version 20210705) with the addition of AmyQ (P00692), Chloramphenicol acetyltransferase (P00485), Kanamycin nucleotidyltransferase (P05057) and rRNA adenine N-6-methyltransferase (P13956). Analysis settings: tryptic peptides; variable modifications: oxidation at Met and protein N-terminal acetylation; missed cleavages: >= 2; iRT peptide-based correlation of retention time ratios. Quantitation: with Interference Correction; protein grouping by Protein Group ID; peptide grouping by Stripped Sequence; precursor Q-value < 0.001, without any imputation. [doi:10.25345/C5J679651] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: secretion stress ; alpha amylase ; HtrA ; Bacillus subtilis

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Principal Investigators:
(in alphabetical order) 		Prof. Uwe Voelker, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, germany
Submitting User: gesell
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