Whole genome sequencing of Canadian Saccharomyces cerevisiae strains isolated from spontaneous wine fermentations reveals a new Pacific West Coast Wine clade.

Vineyards in wine regions around the world are reservoirs of yeast with oenological potential. Saccharomyces cerevisiae ferments grape sugars to ethanol and generates flavor and aroma compounds in wine. Wineries place a high-value on identifying yeast native to their region to develop a region-specific wine program. Commercial wine strains are genetically very similar due to a population bottleneck and in-breeding compared to the diversity of S. cerevisiae from the wild and other industrial processes. We have isolated and microsatellite-typed hundreds of S. cerevisiae strains from spontaneous fermentations of grapes from the Okanagan Valley wine region in British Columbia, Canada. We chose 75 S. cerevisiae strains, based on our microsatellite clustering data, for whole genome sequencing using Illumina paired-end reads. Phylogenetic analysis shows that British Columbian S. cerevisiae strains cluster into 4 clades: Wine/European, Transpacific Oak, Beer 1/Mixed Origin, and a new clade that we have designated as Pacific West Coast Wine. The Pacific West Coast Wine clade has high nucleotide diversity and shares genomic characteristics with wild North American oak strains but also has gene flow from Wine/European and Ecuadorian clades. We analyzed gene copy number variations to find evidence of domestication and found that strains in the Wine/European and Pacific West Coast Wine clades have gene copy number variation reflective of adaptations to the wine-making environment. The "wine circle/Region B", a cluster of 5 genes acquired by horizontal gene transfer into the genome of commercial wine strains is also present in the majority of the British Columbian strains in the Wine/European clade but in a minority of the Pacific West Coast Wine clade strains. Previous studies have shown that S. cerevisiae strains isolated from Mediterranean Oak trees may be the living ancestors of European wine yeast strains. This study is the first to isolate S. cerevisiae strains with genetic similarity to nonvineyard North American Oak strains from spontaneous wine fermentations.

CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli.

Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics in high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here, we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct Escherichia coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications.