BIOSYNTHESIZING BIOSYNTHESIZING EVOLUTION FOR THE EVOLUTION FOR THE

ENHANCE PRODUCTION ENHANCE PRODUCTION OF CAROTENOIDSOF CAROTENOIDS

Saniyyah ThomasGilliam Collegiate Academy

Dallas ISD

Dr. Katy KaoAssistant Professor

Department of Chemical Engineering

Texas A&M University

Dr. Katy KaoDr. Katy Kao

B.S. University of CaliforniaPh.D. University of CaliforniaHer 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??Research Question??

Can we efficiently and Can we efficiently and economically evolve strains of economically evolve strains of yeasts with enhanced production yeasts with enhanced production of carotenoids?of carotenoids?

If so, can we identify the If so, can we identify the parameters (or causes) involved parameters (or causes) involved in the enhanced production of in the enhanced production of these carotenoids?these carotenoids?

CarotenoidsCarotenoids

Total market value of $1.2 billion in 2010

o Supplementso Fortified foodso Cosmetics

Have potential positive effects on human health

o Antioxidantso 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. BIOLOGICALCHEMICAL vs. BIOLOGICALSYNTHESIS 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?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 Is an adaptive evolution method that helps to facilitate the occurrence & expansion of desired mutants.the occurrence & expansion of desired mutants.

3-colored VERT system, to generate baker’s yeast with enhanced 3-colored VERT system, to generate baker’s yeast with enhanced production of carotenoids and subsequently characterize the production of carotenoids and subsequently characterize the molecular mechanisms involved in enhanced productivity. This molecular mechanisms involved in enhanced productivity. This will be accomplished via the following 2 specific aims: will be accomplished via the following 2 specific aims:

Specific aim 1: To engineer fluorescent strains of Specific aim 1: To engineer fluorescent strains of yeast capable of producing carotenoids. yeast capable of producing carotenoids.

VERT SystemVisualizing Evolution in Real - Time

3 colored VERT system GFP, YYP, 3 colored VERT system GFP, YYP, RFP RFP (Selection of desired adaptation)(Selection of desired adaptation)

Generation of Fluorescent Strains + Uracil

Using the colors to track the Using the colors to track the growth of the desired strains of growth of the desired strains of yeastyeast

Courtesy of Dr. Kao & Group

Counter Selection of Desired Counter Selection of Desired MutationsMutations

Generation of Carotenoid Producing Fluorescent

CROSS CHECKINGCROSS CHECKING

• Finding the “cause” of enhanced productionFinding the “cause” of enhanced production

Is it the increase of carotenoid production Is it the increase of carotenoid production and/or cttl production??and/or cttl production??

Eliminate cttl/ replace with antifungalEliminate cttl/ replace with antifungal

Plate in dish w/& w/o antifugalPlate in dish w/& w/o antifugal

Want strains that grow in antifungal dishWant strains that grow in antifungal dish

Later run PCRLater 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 readerPlace in TECAN microplate reader

Determining the Optimal H2O2 [ ] Amount