Team:UIUC Illinois/Description
Background:
We are exploring symbiotic co-culture of Lactococcus lactis, a lactic acid bacteria (LAB), and Saccharomyces cerevisiae, brewer’s yeast, as a means to naturally produce lactic acid. This precursor is valuable for the synthesis of poly-lactic acid, a widely used biodegradable plastic. In many food and beverage industries, LAB is a common contaminant of yeast. This suggests that yeast and LAB form a complex microbiome where both species act in symbiosis. Studying the symbiotic relationship between yeast and LAB could increase carbon flux to the production of lactic acid.
Monoculture Growth and Media Optimization:
Monoculture growth of both brewer’s yeast and LAB have been well studied and documented. Yeast grows best in yeast extract peptone dextrose, also known as YPD, supplemented in two percent glucose (w/v). LAB, on the other hand, has been shown to grow in a variety of media but best in M17 broth supplemented in various concentrations of lactose or glucose, ranging from zero to two percent w/v. Co-culture dynamics have not been thoroughly studied, as a result, our team had to experiment with various media that would allow optimal growth of both brewer’s yeast and LAB individually. A mixture 1X YPD media and 1X M17 broth supplemented with two percent glucose weight by volume (YPDM17 w/v 2% glucose) were determined to be optimal for both yeast and M17.
Co-culture Growth and Dynamics:
We obtained bacterial and yeast fluorescence reporter strains, GFP and RFP respectively, and performed a systematic analysis of co-culture dynamics, including optimization of media characteristics and ratios of initial cell numbers. Individual growth was measured by determining OD600 and fluorescence readings of the co-cultures. Co-cultures were grown in tandem with monoculture biological replicates that were used to compare co-culture growth to monoculture growth. Starting optimal densities of 0.04 LAB and 0.2 yeast were determined to lead to best co-culture growth when grown in YPDM17 w/v 2% glucose media.
CRIPSR:
While conducting co-culture growth we determined that our yeast reporter strain began to only work in the presence of G418 antibiotic. G418 resistance could not be incorporated into our LAB and yeast growth strains genome so a new strain of yeast had to be created. Using Cas9/CRISPR technology we were able to successfully integrated both RFP and GFP into yeast genomes allowing our co-culture experiments to continue.
Future Direction:
Using the information we have collected from a summer of research and experimentation we can begin to work on metabolic engineering of yeast and LAB. Using metabolic engineering we would be able to create high throughput products like polylactic acid using glucose flux.