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

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request

IgG and fibrinogen driven nanoparticle aggregation

A thorough understanding of how proteins induce nanoparticle (NP) aggregation is crucial when designing in vitro and in vivo assays and interpreting experimental results. This knowledge is also crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extraction of immunoglobulin G (IgG) from cow serum results in lower polystyrene NPs aggregation. Moreover, addition of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concentration; i.e., low protein concentrations induced large aggregates, whereas high protein concentrations induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, respectively. Here, we propose a mechanism behind the protein concentration dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins.We thank Professor Sara Linse for scientific discussions and advice and Professor Patrik Brundin for enabling access to the light microscope. The project received financial support from Nanometer structure consortium at Lund University (nmC@LU), Lars Hierta Foundation, and the research school FLAK of Lund University

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge

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

Functional analyses of NSF1 in wine yeast using interconnected correlation clustering and molecular analyses.

Analyzing time-course expression data captured in microarray datasets is a complex undertaking as the vast and complex data space is represented by a relatively low number of samples as compared to thousands of available genes. Here, we developed the Interdependent Correlation Clustering (ICC) method to analyze relationships that exist among genes conditioned on the expression of a specific target gene in microarray data. Based on Correlation Clustering, the ICC method analyzes a large set of correlation values related to gene expression profiles extracted from given microarray datasets. ICC can be applied to any microarray dataset and any target gene. We applied this method to microarray data generated from wine fermentations and selected NSF1, which encodes a C2H2 zinc finger-type transcription factor, as the target gene. The validity of the method was verified by accurate identifications of the previously known functional roles of NSF1. In addition, we identified and verified potential new functions for this gene; specifically, NSF1 is a negative regulator for the expression of sulfur metabolism genes, the nuclear localization of Nsf1 protein (Nsf1p) is controlled in a sulfur-dependent manner, and the transcription of NSF1 is regulated by Met4p, an important transcriptional activator of sulfur metabolism genes. The inter-disciplinary approach adopted here highlighted the accuracy and relevancy of the ICC method in mining for novel gene functions using complex microarray datasets with a limited number of samples

Yml081Wp regulates acetic acid levels.

<p>(<b>A</b>) Wild-type, YML081W-null and YML081W-overexpressing M2 yeast cells were used in a wine fermentation with sterile Chardonnay grape juice. Once fermentation was complete, the wine was assayed for acetic acid by HPLC. YML081W-null cells produced significantly less acetic acid, while YML081W-overexpressing cells produced significantly more acetic acid. In this figure, and all subsequent figures, * indicates p<0.05 for a two-tailed Student t-test, compared to wild-type. (<b>B</b>) These same strains were grown in triplicate in YPD media. At 4 hour intervals, media samples were removed, and assayed for acetic acid as above. YML081-null cells (▪, dotted line) produced significantly less acetic acid in the media than wild-type cells (•, solid line), while YML081W-overexpressing cells (⧫, dashed line) produced significantly more acetic acid.</p