Project description:To determine the affect of CRISPR interference-mediated silencing of glycosylation in Burkholderia diffusa harbouring a pglL (WL85_RS02125)-targeted guide, and a nontarget guide using Data Independent Acquisition
Project description:To determine whether CRISPR interference can be used to silence the expression of pglL in Burkholderia diffusa and therefore modulate glycosylation
Project description:To interrogate the glycoproteome following knockdown of pglL and subsequent knockdown of glycosylation in Burkholderia diffusa through Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) glycopeptide enrichment
Project description:To interrogate the glycoproteome following knockdown of pglL and subsequent knockdown of glycosylation in Burkholderia diffusa through Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) glycopeptide enrichment – loading control pre-enrichment
Project description:Ruminiclostridium thermocellum DSM 1313 strain adhE*(EA) expression was studied along with ∆hydG and ∆hydG∆ech mutants strains deposited under GSE54082. All strains have been described in a study entitled Elimination of hydrogenase post-translational modification blocks H2 production and increases ethanol yield in Clostridium thermocellum. Biswas, et .al. Biotechnology for Biofuels 2015 8:20 Ruminiclostridium (Clostridium) thermocellum is a leading candidate organism for implementing a consolidated bioprocessing (CBP) strategy for biofuel production due to its native ability to rapidly consume cellulose and its existing ethanol production pathway. C. thermocellum converts cellulose and cellobiose to lactate, formate, acetate, H2, ethanol, amino acids, and other products. Elimination of the pathways leading to products such as H2 could redirect carbon flux towards ethanol production. Rather than delete each hydrogenase individually, we targeted a hydrogenase maturase gene (hydG), which is involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes by assembling the active site. This functionally inactivated all three Fe-Fe hydrogenases simultaneously, as they were unable to make active enzymes. In the ∆hydG mutant, the [NiFe] hydrogenase-encoding ech was also deleted to obtain a mutant that functionally lacks all hydrogenase. The ethanol yield increased nearly 2-fold in ∆hydG∆ech compared to wild type, and H2 production was below the detection limit. Interestingly, ∆hydG and ∆hydG∆ech exhibited improved growth in the presence of acetate in the medium. Transcriptomic and proteomic analysis reveal that genes related to sulfate transport and metabolism were up-regulated in the presence of added acetate in ∆hydG, resulting in altered sulfur metabolism. Further genomic analysis of this strain revealed a mutation in the bi-functional alcohol/aldehyde dehydrogenase adhE gene, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities, whereas the wild type strain can only utilize NADH. This is the exact same adhE mutation found in ethanol-tolerant C. thermocellum strain E50C, but ∆hydG∆ech is not more ethanol tolerant than the wild type. Targeting protein post-translational modification is a promising new approach to target multiple enzymes simultaneously for metabolic engineering. This GEO study pertains to expression profiles generated for C. thermocellum DSM 1313 strain adhE*(EA)