5 research outputs found

    A <i>p</i>‑Coumaroyl-CoA Biosensor for Dynamic Regulation of Naringenin Biosynthesis in Saccharomyces cerevisiae

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    In vivo biosensors that can convert metabolite concentrations into measurable output signals are valuable tools for high-throughput screening and dynamic pathway control in the field of metabolic engineering. Here, we present a novel biosensor in Saccharomyces cerevisiae that is responsive to p-coumaroyl-CoA, a central precursor of many flavonoids. The sensor is based on the transcriptional repressor CouR from Rhodopseudomonas palustris and was applied in combination with a previously developed malonyl-CoA biosensor for dual regulation of p-coumaroyl-CoA synthesis within the naringenin production pathway. Using this approach, we obtained a naringenin titer of 47.3 mg/L upon external precursor feeding, representing a 15-fold increase over the nonregulated system

    Condensin subunits co-purify with MAP::SMCL-1.

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    <p>(A-B) Proteins that co-purified with MAP::SMCL-1 but not untagged control adult extracts, identified by tandem affinity purification and MudPIT mass spectrometry. Numbers represent average NSAF values from two replicas. Co-purified proteins with the highest NSAF values are shown in (A), values for other condensin subunits are shown in (B), and all other proteins are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s011" target="_blank">S5 Table</a>. Condensin SMC subunits are highlighted.</p

    Presence of predicted orthologs of SMCL-1, DPY-27 (I<sup>DC</sup>), and SMC-4 (I & II) in various species.

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    <p>Phylogenetic tree built from all available <i>Caenorhabditis</i> species with sequenced and well-assembled genomes, other selected nematode species, and other selected model organisms. “<i>+”</i> symbol denotes the presence of SMCL-1-like protein based on similarity in a BLAST search and the additional criteria of short length, imperfect signature motif, and a Walker B motif lacking the catalytic glutamate (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#sec014" target="_blank">Methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s003" target="_blank">S3 Fig</a>). “1” denotes orthologs detected using a high-confidence Ensemble-COMPARA method and “2” denotes orthologs detected using BLAST-neighbor-joining tree methods.</p

    SMCL-1 expression and protein features.

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    <p>(A) Adult hermaphrodites from wild-type (WT) and a strain carrying the <i>map</i>::<i>smcl-1</i> transgene driven by endogenous <i>smcl-1</i> 5’ and 3’ elements. A section of the germline is shown, imaged by DIC to show structures and fluorescent microscopy to detect mVenus expression from the MAP tag. Arrowheads denote the first four oocytes. (B) A typical SMC protein folds back on itself at a hinge domain, bringing coil regions together and creating a “head domain” (yellow) from ATPase domains in the N- and C-termini. SMCL-1 lacks predicted coil and hinge domains, but has N- and C-terminal ATPase domains that may be capable of forming a head domain (purple). (C) SMC head domain and the ATPase cycle, showing binding of ATP (red circle), ATP-dependent engagement of heads from two SMC proteins, and disengagement upon ATP hydrolysis. (D) SMCL-1 amino acid sequence aligned to <i>C</i>. <i>elegans</i> condensin SMC proteins. Shown are regions surrounding three conserved motifs found in SMCs and related ATPases: the Walker A motif, ABC transporter signature motif, and Walker B motifs, and their consensus sequences. SMCL-1 shares a conserved Walker A motif, but differs from consensus signature motif and Walker B motif at residues shown in red. Asterisk denotes catalytic amino acid required for ATP hydrolysis. x = any amino acid and h = hydrophobic amino acid.</p

    SMCL-1 overexpression in the gut disrupts condensin I<sup>DC</sup> localization on the X chromosomes.

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    <p>(A) Heat shock regimen for data in (B-E). Bolt represents the single heat-shock pulse given to young adult hermaphrodites from the wild-type or inducible <i>hs</i>:<i>smcl-1(+)</i> transgenic strain. (B-E) Adult hermaphrodite gut tissue of the indicated strain and treatment was stained with DAPI to image DNA (green in merge) and immuno-stained with antibody against CAPG-1(B-C), DPY-27 (D) and DPY-28 (E), (red in merges). Antibody against SMCL-1 was also included in (B and C), showing overexpression upon heat shock (blue in merge). The foci of staining created by condensin I<sup>DC</sup> subunit association with the X chromosomes are lost when SMCL-1 is overexpressed. (C) A mosaic animal in which anti-SMCL-1 staining indicates that one cell lacks the transgene (left) and shows foci of anti-CAPG-1, while a neighboring cells has the <i>smcl-1</i> overexpression transgene (right) and CAPG-1 staining is weak and not localized to foci (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s006" target="_blank">S6C and S6D Fig</a>). HS = heat shock.</p