Genome-scale identification and characterization of ethanol tolerance genes in Escherichia coli.

The identification of relevant gene targets for engineering a desired trait is a key step in combinatorial strain engineering. Here, we applied the multi-Scalar Analysis of Library Enrichments (SCALEs) approach to map ethanol tolerance onto 1,000,000 genomic-library clones in Escherichia coli. We assigned fitness scores to each of the ∼4,300 genes in E. coli, and through follow-up confirmatory studies identified 9 novel genetic targets (12 genes total) that increase E. coli ethanol tolerance (up to 6-fold improved growth). These genetic targets are involved in the processes related to cell membrane composition, translation, serine biosynthesis, and transcription regulation. Transcriptional profiling of the ethanol stress response in 5 of these ethanol-tolerant clones revealed a total of 700 genes with significantly altered expression (mapped to 615 significantly enriched gene ontology terms) across all five clones, with similar overall changes in global gene expression between two clone clusters. All ethanol-tolerant clones analyzed shared 6% of the overexpressed genes and showed enrichment for transcription regulation-related GO terms. iTRAQ-based proteomic analysis of ethanol-tolerant strains identified upregulation of proteins related to ROS mitigation, fatty acid biosynthesis, and vitamin biosynthesis as compared to the parent strain's ethanol response. The approach we outline here will be useful for engineering a variety of other traits and further improvements in alcohol tolerance.

Inverse metabolic engineering to improve Escherichia coli as an N-glycosylation host.

An inverse metabolic engineering strategy was used to select for Escherichia coli cells with an increased capability to N-glycosylate a specific target protein. We developed a screen for E. coli cells containing extra-chromosomal DNA fragments for improved ability to add precise sugar groups onto the AcrA protein using the glycosylation system from Campylobacter jejuni. Four different sized (1, 2, 4, and 8 kb) genomic DNA libraries were screened, and the sequences that conferred a yield advantage were determined. These advantageous genomic fragments were mapped onto the E. coli W3110 chromosome. Five candidate genes (identified across two or more libraries) were subsequently selected for forward engineering verification in E. coli CLM24 cells, utilizing a combination of internal standards for absolute quantitation and pseudo-selective reaction monitoring (pSRM) and Western blotting validation. An increase in glycosylated protein was quantified in cells overexpressing 4-α-glucantransferase and a phosphoenolpyruvate-dependent sugar phosphotransferase system, amounting to a 3.8-fold (engineered cells total = 5.3 mg L(-1) ) and 6.7-fold (engineered cells total = 9.4 mg L(-1) ) improvement compared to control cells, respectively. Furthermore, increased glycosylation efficiency was observed in cells overexpressing enzymes involved with glycosylation precursor synthesis, enzymes 1-deoxyxylulose-5-phosphate synthase (1.3-fold) and UDP-N-acetylglucosamine pyrophosphorylase (1.6-fold). To evaluate the wider implications of the engineering, we tested a modified Fc fragment of an IgG antibody as the target glycoprotein with two of our engineered cells, and achieved a ca. 75% improved glycosylation efficiency.

Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae.

Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (α = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing.

Proteomic analysis of the impact of static culturing on the expansion of rat bone marrow mesenchymal stem cells.

The clinical potential of mesenchymal stem cells (MSC) in tissue engineering and regenerative medicine is due to their self-renewal, proliferation and multi-lineage differentiation potential. Clinical use requires large cell numbers; which can, theoretically, be generated by ex vivo expansion of plastic adherent, MSC subpopulation, of bone marrow cells (BMC). Effects of serial culture on MSC phenotype were investigated using non-gel based quantitative proteomic methodology for static monolayer cultures of rat BMC. In total, 382 proteins were relatively quantified (≥ 2 peptides). Nine proteins were up-regulated and seven down-regulated at passage 4 relative to passage 2 (p ≤ 0.05). We propose that serial culture impacts on MSC expansion (observed decline in colony forming potential and colony size) is through a combination of osteogenic differentiation and ageing/senescence and propose six novel protein biomarkers as candidates for quality control purposes in bioprocessing.

The conserved Trp155 in human frataxin as a hotspot for oxidative stress related chemical modifications.

Frataxin is a mitochondrial protein that is defective in Friedreich's ataxia resulting in iron accumulation and an environment prone to Fenton reactions. We report that frataxin is susceptible to carbonylation and nitration modifications in residues from the beta-sheet surface (Tyr143, Tyr174, Tyr205 and Trp155). Frataxin functions are not significantly affected: frataxin-mediated protection against ROS is still observed, as well as iron-binding (5 Fe(3+)mol(-1), K(d) from 13-36 microM) necessary for the metallochaperone activity. However, the protein is up to 1.0 kcal mol(-1) destabilized, with conformational opening. Interestingly, the strictly conserved Trp155, which is mutated in patients, may be a functional hotspot in frataxin.