The quality score and the number of counts of <i>ginjo</i> sake by 15 sensory evaluation panelists.

<p>The quality score and the number of counts of <i>ginjo</i> sake by 15 sensory evaluation panelists.</p

Protein profiles of 13 sake samples showing that yeast cellular protein leakage is linked to low sake quality.

<p>A) Overall protein profiling by Coomassie brilliant blue (CBB) staining after SDS-PAGE. For each sample, 10 μL of denatured protein was applied to a lane, and 5 μL of marker was utilized. Protein bands indicated by double black triangles are <i>Saccharomyces cerevisiae</i> triosephosphate isomerase (TPI). B) Immunoblot analysis for <i>S</i>. <i>cerevisiae</i> TPI1 in sake. Pyruvic acid concentrations in sake (n = 2) determined by colorimetric assay is shown in the upper rectangle. C) <i>Upper panel</i>; Immunoblot analysis for glucoamylase (GlaB) derived from <i>Aspergillus oryzae</i> in sake. The <i>N</i>-glycosylated forms of GlaB were detected at 47 kDa and as a blurred band beyond 65 kDa. <i>Middle panel</i>; Immunoblot analysis for α-amylase. <i>Lower panel</i>; Immunoblot analysis for the acid protease, PepA. D) Protein profiling following PNGaseF treatment by CBB staining after SDS-PAGE. For each sample, 10 μL of denatured protein was applied to a lane, and 5 μL of marker was applied. The asterisk at 34 kDa shows PNGaseF. E) Immunoblot analysis for glucoamylase (GlaB) following PNGaseF treatment of sake. The blurred band and multiple bands at 55–70 kDa and 37–50 kDa correspond to GlaB.</p

The patterns of representative metabolites associated with sake quality.

<p>A) Compounds involved in the methionine cycle. 5′-deoxi-5′-methylthioadenosine is shown as relative value. <i>S</i>-Adenosylmethionine and methionine are shown as μmol/L. B) A simple summarization of the methionine cycle pathway. C) Compounds involved in glutathione or ophthalmic acid biosynthesis pathways. Cysteine and glutathione (GSSG; oxiglutathione) are shown as μmol/L. Gamma-glutamyl-2-aminobutyric acid, ophthalmic acid, and cysteine glutathione disulfide are shown as relative values. D) The metabolites that participate in part of the glutathione (GSH) and ophthalmic acid biosynthesis pathways. E) Other amino acid-related chemical compounds associated with lower sake quality. Saccharopine and cystine are shown as relative values. Homoserine is shown as μmol/L. Data are shown as means ± SEM (n = 2).</p

Principal component analysis (PCA) of Japanese sake.

<p>After selecting 202 compounds using Fisher ratio criteria, PCA was performed for 13 sake samples in duplicate. Same symbols indicate the same sake samples. Highly ranked sake (n = 7) are shown in red, sake with fatty acid odor (n = 3) are shown in green, and inharmonious bitter tasting sake (n = 3) are shown in blue.</p

Chemical compounds whose levels were positively or negatively correlated with either taste or flavor quality.

<p>Chemical compounds whose levels were positively or negatively correlated with either taste or flavor quality.</p

Chemical compounds contributing to fatty acid odor and inharmonious bitter taste.

<p>A) Representative chemical compounds involved in fatty acid odor generation are medium-chain fatty acids and medium-chain fatty acid analogues. B) Two representative polyamines (cadaverine and agmatine) involved in inharmonious bitter taste generation. All chemical compounds are shown as relative values. Data are shown as means ± SEM (n = 2).</p

Relative heatmap for the metabolites of 13 sake samples.

<p>Heatmap showing the metabolic profiles of 13 sake samples (7 of highly ranked sake, 3 of inharmonious bitter tasting sake, and 3 of sake with fatty acid odors) analyzed in duplicate. Maximum to minimum level is represented by a red-gray-green color scheme. The values of <i>m/z</i> of the unknown compounds XA0004, XA0012, XA0017 are 144.0292, 166.0154, and 186.1124, respectively, by the anion detection mode. The values of <i>m/z</i> of the unknown compounds XC0016, XC0017, XC0040, and XC0120 are 129.0646, 130.0977, 174.0861, and 298.0518, respectively, by the cation detection mode.</p

Deep Sequencing Analysis of Aptazyme Variants Based on a Pistol Ribozyme

Chemically regulated self-cleaving ribozymes, or aptazymes, are emerging as a promising class of genetic devices that allow dynamic control of gene expression in synthetic biology. However, further expansion of the limited repertoire of ribozymes and aptamers, and development of new strategies to couple the RNA elements to engineer functional aptazymes are highly desirable for synthetic biology applications. Here, we report aptazymes based on the recently identified self-cleaving pistol ribozyme class using a guanine aptamer as the molecular sensing element. Two aptazyme architectures were studied by constructing and assaying 17 728 mutants by deep sequencing. Although one of the architectures did not yield functional aptazymes, a novel aptazyme design in which the aptamer and the ribozyme were placed in tandem yielded a number of guanine-inhibited ribozymes. Detailed analysis of the extensive sequence-function data suggests a mechanism that involves a competition between two mutually exclusive RNA structures reminiscent of natural bacterial riboswitches