Functional improvement of Saccharomyces cerevisiae to reduce volatile acidity in wine

Abstract Control of volatile acidity (VA) is a major issue for wine quality. In this study, we investigated the production of VA by a deletion mutant of the fermentation stress response gene AAF1 in the budding yeast Saccharomyces cerevisiae. Fermentations were carried out in commercial Chardonnay grape must to mimic industrial wine-making conditions. We demonstrated that a wine yeast strain deleted for AAF1 reduced acetic acid levels in wine by up to 39.2% without increasing the acetaldehyde levels, revealing a potential for industrial application. Deletion of the cytosolic aldehyde dehydrogenase gene ALD6 also reduced acetic acid levels dramatically, but increased the acetaldehyde levels by 41.4%, which is not desired by the wine industry. By comparison, ALD4 and the AAF1 paralog RSF2 had no effects on acetic acid production in wine. Deletion of AAF1 was detrimental to the growth of ald6D and ald4Dald6D mutants, but had no effect on acetic acid production. Overexpression of AAF1 dramatically increased acetic acid levels in wine in an Ald6p-dependent manner, indicating that Aaf1p regulates acetic acid production mainly via Ald6p. Overexpression of AAF1 in an ald4Dald6D strain produced significantly more acetic acid in wine than the ald4Dald6D mutant, suggesting that Aaf1p may also regulate acetic acid synthesis independently of Ald4p and Ald6p

Metabolic engineering of malolactic wine yeast

Abstract Malolactic fermentation is essential for the deacidification of high acid grape must. We have constructed a genetically stable industrial strain of Saccharomyces cerevisiae by integrating a linear cassette containing the Schizosaccharomyces pombe malate permease gene (mae1) and the Oenococcus oeni malolactic gene (mleA) under control of the S. cerevisiae PGK1 promoter and terminator sequences into the URA3 locus of an industrial wine yeast. The malolactic yeast strain, ML01, fully decarboxylated 5.5 g/l of malate in Chardonnay grape must during the alcoholic fermentation. Analysis of the phenotype, genotype, transcriptome, and proteome revealed that the ML01 yeast is substantially equivalent to the parental industrial wine yeast. The ML01 yeast enjoys 'Generally Regarded As Safe' status from the FDA and is the first genetically enhanced yeast that has been commercialized. Its application will prevent the formation of noxious biogenic amines produced by lactic acid bacteria in wine.

The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs

Transcription and Metabolism Pathways of Anthocyanin in Purple Shamrock (Oxalis triangularis A.St.-Hil.)

Anthocyanins are water-soluble pigments that can impart various colors to plants. Purple shamrock (Oxalis triangularis) possesses unique ornamental value due to its purple leaves. In this study, three anthocyanins, including malvidin 3-O-(4-O-(6-O-malonyl-glucopyranoside)-rhamnopyranosyl)-5-O-(6-O-malonyl-glucopyranoside), delphinidin-3-O-rutinoside and malvidin-3,5-di-O-glucoside, were characterized with ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) in purple shamrock. To investigate the molecular mechanism of anthocyanin biosynthesis in green shamrock (Oxalis corymbosa) and purple shamrock, RNA-seq and qRT-PCR were performed, and the results showed that most of the anthocyanin biosynthetic and regulatory genes were up-regulated in purple shamrock. Then, dark treatment and low temperature treatment experiments in purple shamrock showed that both light and low temperature can induce the biosynthesis of anthocyanins

Genome-Wide Identification, Classification and Expression Analysis of m6A Gene Family in Solanum lycopersicum

Advanced knowledge of messenger RNA (mRNA) N6-methyladenosine (m6A) and DNA N6-methyldeoxyadenosine (6 mA) redefine our understanding of these epigenetic modifications. Both m6A and 6mA carry important information for gene regulation, and the corresponding catalytic enzymes sometimes belong to the same gene family and need to be distinguished. However, a comprehensive analysis of the m6A gene family in tomato remains obscure. Here, 24 putative m6A genes and their family genes in tomato were identified and renamed according to BLASTP and phylogenetic analysis. Chromosomal location, synteny, phylogenetic, and structural analyses were performed, unravelling distinct evolutionary relationships between the MT-A70, ALKBH, and YTH protein families, respectively. Most of the 24 genes had extensive tissue expression, and 9 genes could be clustered in a similar expression trend. Besides, SlYTH1 and SlYTH3A showed a different expression pattern in leaf and fruit development. Additionally, qPCR data revealed the expression variation under multiple abiotic stresses, and LC-MS/MS determination exhibited that the cold stress decreased the level of N6 2′-O dimethyladenosine (m6Am). Notably, the orthologs of newly identified single-strand DNA (ssDNA) 6mA writer–eraser–reader also existed in the tomato genome. Our study provides comprehensive information on m6A components and their family proteins in tomato and will facilitate further functional analysis of the tomato N6-methyladenosine modification genes

The Fermentation Stress Response Protein Aaf1p/Yml081Wp Regulates Acetate Production in <em>Saccharomyces cerevisiae</em>

<div><p>The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of <em>ALD4 and ALD6</em>, which encode a cytosolic acetaldehyde dehydrogenase (ACDH) and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of <em>ALD6</em>, YML081W had no effect on acetic acid levels, suggesting that this transcription factor’s effects are mediated primarily through this gene. <em>lacZ</em> reporter assays revealed that Yml081wp stimulates <em>ALD6</em> transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in <em>Saccharomyces cerevisiae.</em> We propose <em>AAF1</em> as a gene name for the YML081W ORF.</p> </div

Yml081Wp regulates acetaldehyde dehydrogenase activity.

<p>M2 yeast cells were grown to mid-log phase, then harvested and lysed. ACDH specific activity was assayed as described in the Materials and Methods section. The numbers represent (nmol NAD(P)H formed/min) per mg protein. Cells without Yml081Wp contained lower ACDH activity, while cells overexpressing YML081W contained higher ACDH activity. These differences were statistically significant (p<0.05).</p

Yml081Wp regulates <i>ALD</i> gene expression and protein levels.

<p>(<b>A</b>) Acetate biosynthesis pathway, adapted from Saint-Prix <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051551#pone.0051551-SaintPrix1" target="_blank">[6]</a>. (<b>B</b>) <i>ALD</i> gene expression was measured in mid-log phase wild-type and YML081W-null cells by quantitative PCR. Removal of YML081W resulted in a significant reduction in <i>ALD4</i> and <i>ALD6</i> mRNA levels. (<b>C</b>) <i>ALD</i> gene expression was measured in mid-log phase yeast cells overexpressing YML081W under the control of the <i>PGK1</i> promoter, and compared to wild-type cells. YML081W-overexpressing cells produced significantly higher levels of ALD4 and ALD6 mRNA compared to wild-type cells. (<b>D</b>) <i>ALD4</i> and <i>ALD6</i> genes were tagged with the FLAG epitope at the C-terminus in wild-type, YML081W-null and YML081Woverexpressing strains. The cells were grown to mid-log phase, then harvested and lysed. The lysates were immunoblotted with an α-FLAG antibody to detect the levels of Ald4p-FLAG and Ald6p-FLAG protein. Cells lacking YML081W produced lower levels of FLAG-tagged Ald4p and Ald6p, compared to wild-type cells. Cells overexpressing YML081W produced significantly higher levels of Ald4p-FLAG protein compared to wild-type cells. ALD6p-FLAG levels did not appear to change significantly. Membrane staining shown below the immunoblots indicates the equivalance of total protein loading between lanes.</p