BIOSYNTHESIZING EVOLUTION FOR THE ENHANCE PRODUCTION OF CAROTENOIDS

1. BIOSYNTHESIZING EVOLUTION FOR THE ENHANCE PRODUCTION OF CAROTENOIDS Saniyyah Thomas Gilliam Collegiate Academy Dallas ISD Dr. Katy Kao Assistant Professor Department of Chemical Engineering Texas A&M University

2. B.S. University of California • Ph.D. University of California • Her research team focuses primarily on: • using genomics (discipline of genetics) and biological systems to study adaptations in microbial populations • evolving microorganisms such as E. coli and yeasts for enhanced tolerance to toxicity of specific bio-products Dr. Katy Kao

3. Research Question??Can we efficiently and economically evolve strains of yeasts with enhanced production of carotenoids?If so, can we identify the parameters (or causes) involved in the enhanced production of these carotenoids?

4. Organic pigments – hydrocarbons with no oxygen • Natural occurring in the chloroplasts of plants, algae, photosynthetic bacteria, and some fungi. • Have potential positive effects on human health • Antioxidants • Precursors for vitamin A Carotenoids • Total market value of $1.2 billion in 2010 • Supplements • Fortified foods • Cosmetics

5. CHEMICAL vs. BIOLOGICALSYNTHESIS Due to the structural complexity of carotenoids, traditional chemical synthesis of these compounds proved to be challenging and expensive. Since only few carotenoids are produced via chemical synthesis on an industrial level, bio-genetic engineering of microbials became the solution

6. A change that happens in successive generations throughout time. • These changes happen as mutations and are random. • A change has to represent a favorable characteristic or an adaptation. Evolution?

7. VERT System • Visualizing Evolution in Real - Time Is an adaptive evolution method that helps to facilitate the occurrence & expansion of desired mutants.3-colored VERT system, to generate baker’s yeast with enhanced production of carotenoids and subsequently characterize the molecular mechanisms involved in enhanced productivity. This will be accomplished via the following 2 specific aims: Specific aim 1: To engineer fluorescent strains of yeast capable of producing carotenoids.

8. Generation of Fluorescent Strains + Uracil 3 colored VERT system GFP, YYP, RFP (Selection of desired adaptation)

9. Courtesy of Dr. Kao & Group Using the colors to track the growth of the desired strains of yeast

10. Generation of Carotenoid Producing Fluorescent Counter Selection of Desired Mutations

11. CROSS CHECKING

12. Specific aim 2: To evolve the carotenoid producing strains using a 3-colored VERT system in the presence of hydrogen peroxide (The Chosen Selective Pressure) Finding the “cause” of enhanced productionIs it the increase of carotenoid production and/or cttl production??Eliminate cttl/ replace with antifungalPlate in dish w/& w/o antifugalWant strains that grow in antifungal dishLater run PCR

13. Determining the Optimal H2O2 [ ] Amount Place in TECAN microplate reader