Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome.

The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.

Current Research on Flavor Compounds in Fermented Food Products.

Recent advancements in the field of food science have spurred a surge of research focused on unraveling the intricate world of flavor compounds in fermented food products [...

Parallel laboratory evolution and rational debugging reveal genomic plasticity to synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of chromosome XIV as part of the Synthetic Yeast Genome project.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H.

The Influence of Addition on the Aroma Profile of a Kombucha Tea Fermentation.

Traditional kombucha is a functional tea-based drink that has gained attention as a low or non-alcoholic beverage. The fermentation is conducted by a community of different microorganisms, collectively called SCOBY (Symbiotic Culture of Bacteria and Yeast) and typically consists of different acetic acid bacteria and fermenting yeast, and in some cases lactic acid bacteria that would convert the sugars into organic acids-mostly acetic acid. In this study, the effect of including a starter culture in a kombucha fermentation was investigated.

Exploring future applications of the apiculate yeast .

As a metaphor, lemons get a bad rap; however the proverb '' is often used in a motivational context. The same could be said of in winemaking. Despite its predominance in vineyards and grape must, this lemon-shaped yeast is underappreciated in terms of its contribution to the overall sensory profile of fine wine.

Engineering a fermenting yeast able to produce the fragrant β-ionone apocarotenoid for enhanced aroma properties in wine.

Wine is composed of multitudinous flavour components and volatile organic compounds that provide this beverage with its attractive properties of taste and aroma. The perceived quality of a wine can be attributed to the absolute and relative concentrations of favourable aroma compounds; hence, increasing the detectable levels of an attractive aroma, such as β-ionone with its violet and berry notes, can improve the organoleptic qualities of given wine styles. We here describe the generation of a new grape-must fermenting strain of Saccharomyces cerevisiae that is capable of releasing β-ionone through the heterologous expression of both the enzyme carotenoid cleavage dioxygenase 1 (CCD1) and its substrate, β-carotene.

Synthesis of Aroma Compounds as a Function of Different Nitrogen Sources in Fermentations Using Non- Wine Yeasts.

Non- yeasts are prevalent at the onset of grape must fermentations and can have a significant influence on the final wine product. In contrast to , the biosynthetic pathways leading to aroma compound formation in these non-conventional yeasts, in particular those that are derived from amino acid metabolism, remains largely unexplored. Within a synthetic must environment, we investigated the amino acid utilization of four species (, , , ) and .

The film-forming Pichia spp. in a winemaker's toolbox: A simple isolation procedure and their performance in a mixed-culture fermentation of Vitis vinifera L. cv. Gewürztraminer must.

Certain yeast species belonging to the Pichia genus are known to form a distinctive film on grape must and wine. In a mixed-culture type fermentation, Pichia spp. (P.

Construction and Analysis of a Yeast for the Simultaneous Release and Esterification of the Varietal Thiol 3-Sulfanylhexan-1-ol.

Polyfunctional thiols like 3-sulfanylhexan-1-ol (3SH) and its ester 3-sulfanylhexyl acetate (3SHA) are important aroma determinants in wine with exceptionally low odor thresholds. 3SH is largely found in grape must bound to glutathione and cysteine and requires enzymatic action to be perceived sensorially. The wine yeast is ineffective in releasing volatile thiols from their precursor configuration.

Development of Genetic Modification Tools for .

Apiculate yeasts belonging to the genus are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking.

Functional Characterization of the -Acetylmuramyl-l-Alanine Amidase, Ami1, from .

Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells.

Effect of Isomixing on Grape Must Fermentations of -Overexpressing Wine Yeast Strains.

Speeding up grape must fermentation would be of great economic benefit. We subjected Saccharomyces cerevisiae VIN13 and two recombinant VIN13-strains expressing alleles under two different promoters (either or ) to four styles of grape must fermentations; we then assessed the effect of constantly stirring a must fermentation (isomixing). The four different fermentation setups were as follows: in an ANKOM Rf Gas productions system; in a stirred tall tube cylinder; constituting a conventional fermentation in a wine bottle equipped with an airlock and static; and in a tall tube cylinder (without stirring).

Blending wine yeast phenotypes with the aid of CRISPR DNA editing technologies.

In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e.

The Whiff of Wine Yeast Innovation: Strategies for Enhancing Aroma Production by Yeast during Wine Fermentation.

Despite being used chiefly for fermenting the sugars of grape must to alcohol, wine yeasts (most prominently ) play a pivotal role in the final aroma profiles of wines. Strain selection, intentionally incorporating non- yeast in so-called mixed-culture fermentations, and genetic modifications of have all been shown to greatly enhance the chemical composition and sensory profile of wines. In this Review, we highlight how wine researchers employ fermenting yeasts to expand on the aroma profiles of the wines they study.

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors.

Mycobacterial nucleoid associated proteins: An added dimension in gene regulation.

Nucleoid associated proteins (NAPs) are known organisers of chromosomal structure and regulators of transcriptional expression. The number of proposed NAPs in mycobacteria are significantly lower than the number identified in other organisms. An interesting feature of mycobacterial NAPs is their low sequence similarity with those in other species, a property that has hindered their identification.

Characterization of a mycobacterial cellulase and its impact on biofilm- and drug-induced cellulose production.

It was recently shown that Mycobacterium tuberculosis produces cellulose which forms an integral part of its extracellular polymeric substances within a biofilm set-up. Using Mycobacterium smegmatis as a proxy model organism, we demonstrate that M. smegmatis biofilms treated with purified MSMEG_6752 releases the main cellulose degradation-product (cellobiose), detected by using ionic chromatography, suggesting that MSMEG_6752 encodes a cellulase.

Binding of NADP triggers an open-to-closed transition in a mycobacterial FabG β-ketoacyl-ACP reductase.

The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the genome uncovered several putative FabG-like proteins.

Current perspectives on the families of glycoside hydrolases of Mycobacterium tuberculosis: their importance and prospects for assigning function to unknowns.

Glycoside hydrolases (GHs) are enzymes that catalyze the hydrolysis of glycosidic bonds in glycoconjugates, oligo- and polysaccharides. A classification of these enzymes based on conserved sequence and structure motifs supported by the Carbohydrate Active Enzyme (CAZy) database has proven useful in the systematic groupings of similar enzymes into families. The human pathogen Mycobacterium tuberculosis employs 30 GHs to perform a variety of different functions, which can be divided into four broad categories: α-glucan metabolism, peptidoglycan remodeling, β-glycan hydrolysis and α-demannosylation.

Sequence and structure-based prediction of fructosyltransferase activity for functional subclassification of fungal GH32 enzymes.

Sucrolytic enzymes catalyse sucrose hydrolysis or the synthesis of fructooligosaccharides (FOSs), a prebiotic in human and animal nutrition. FOS synthesis capacity differs between sucrolytic enzymes. Amino-acid-sequence-based classification of FOS synthesizing enzymes would greatly facilitate the in silico identification of novel catalysts, as large amounts of sequence data lie untapped.

Comparative secretome analysis of Trichoderma asperellum S4F8 and Trichoderma reesei Rut C30 during solid-state fermentation on sugarcane bagasse.

Background: The lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF).

Results: Comparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities.

Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae.

Background: β-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries.

Results: We identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family.

Heterologous co-production of Thermobifida fusca Cel9A with other cellulases in Saccharomyces cerevisiae.

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.