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

Dr. Katy Kao 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

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?

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

CHEMICAL vs. BIOLOGICAL SYNTHESIS 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

Evolution? 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.

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. VERT System Visualizing Evolution in Real - Time

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

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

Counter Selection of Desired Mutations Generation of Carotenoid Producing Fluorescent

CROSS CHECKING

Finding the “cause” of enhanced production Is it the increase of carotenoid production and/or cttl production?? Eliminate cttl/ replace with antifungal Plate in dish w/& w/o antifugal Want strains that grow in antifungal dish Later run PCR Finding the “cause” of enhanced production Is it the increase of carotenoid production and/or cttl production?? Eliminate cttl/ replace with antifungal Plate in dish w/& w/o antifugal Want strains that grow in antifungal dish Later run PCR 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)

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