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Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

ABSTRACT: Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.

SUBMITTER: Boock JT

PROVIDER: S-EPMC4576832 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Boock Jason T JT King Brian C BC Taw May N MN Conrado Robert J RJ Siu Ka-Hei KH Stark Jessica C JC Walker Larry P LP Gibson Donna M DM DeLisa Matthew P MP

Journal of molecular biology 20150112 6 Pt B

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation ...[more]

PMID: 25591491

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Project 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.

Dataset Information

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Publications

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

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