S14: Higher throughput continuous culture for strain selection in yeast

Directed evolution is a tool used to derive microbes with desired properties, such as improved growth in nutrient limited or toxic environments. The productivity of this approach relies on the ability to target specific selection conditions. Chemostat continuous cultures enable evolution in a constant environment over hundreds of generations of growth, thus improving reproducibility of results and the ability to relate identified mutations which arise to the imposed selection. However, the large size and high cost of commercial fermentors makes it difficult to attain high throughput with this approach.

We have developed a multiplexed array of miniature chemostats with similar design and outcomes to larger volume chemostats at a fraction of the cost. This platform allows us to perform higher-throughput evolution and competition experiments. In the past year we have performed hundreds of evolution experiments, using haploid and diploid yeast strains, across three different nutrient environments (glucose, phosphate, and sulfate-limitation). Analysis with array CGH and deep sequencing show amplifications of relevant nutrient transporter genes specific to each selection environment. We are also applying deep sequencing approaches to quantitate the frequency of SNVs, indels, and more complex rearrangements in these populations with the goal of identifying the range of adaptive strategies adopted during these replicate evolution experiments. Additionally we have evolved knockouts of commonly amplified nutrient transporters such as SUL1 and PHO84 to identify alternative strategies for adaptation. Our multiplexed screening approach facilitates unprecedented bandwidth for experimental prototyping and hypothesis testing for directed evolution applications and fitness determination.