BIOSYNTHESIS AND ENHANCED PRODUCTION OF CAROTENOIDS J. Miguel Ramirez Hanna High School Brownsville 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  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

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.  Charles Darwin – “Father of Evolution”

BAKERS YEAST  Saccharomyces cerevisiae  Eukaryotic unicellular microorganisms classified in the kingdom Fungi  Reproduce asexually by a division process known as budding  Main use is in fermentation in which they convert carbohydrates into carbon dioxide (baking) and alcohols (brewing)  Along with E. coli, used in molecular and cell biology research as a model bacterium.

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

MEMBERS OF CAROTENOID FAMILY  β -carotene - Gives carrots and other vegetables color  lycopene - Gives tomatoes red color  Astaxanthin - Found mainly in sea animals like salmon, shrimp, krill, and crustaceans

FIRST OBJECTIVE  Develop a mutated strain of yeast (S. cerevisiae) with enhanced production of carotenoids.  Traditional chemical synthesis of carotenoids is difficult because of its unique structure and cost.  This will be accomplished by applying a selective pressure to the cells which will cause generation of mutants with enhanced properties.  Hydrogen peroxide (H 2 O 2 ) was chosen as the selective pressure.  Carotenoids have a natural antioxidant property  Organisms that are able to grow in an oxidized environment will have enhanced carotenoid production

STREAKING BACTERIA ON PETRI DISH

Fluorescent yeasts grown GFPRFP YFP

FLUORESCENT STRAINS UNDER UV LIGHT GFP RFP YFP

Yeast populations that grew in 5-FOA are mutants missing URA3 gene

Counter-Selection: Uracil GFP RFP YFP

PIPETTING CELLS ON MICROPLATE

0mM 2mM TECAN MICROPLATE READER  GFP, YFP, and RFP yeast cells are plated at different concentrations of H 2 O 2  A growth kinetic curve is given for each of the 96 wells plotting the number of individuals vs. time.  Allows us to pick a good concentration of our selective pressure to start with.

0.00 mM H 2 O TECAN® Microplate Reader Growth Kinetics Curves

VISUALIZING EVOLUTION IN REAL-TIME  Identify when adaptive events occur  Facilitate the isolation of adaptive mutants VERT is based in the generation of fluorescent strains by integrating different fluorescent proteins into microbial cells Red Discosoma Coral Aequoreavictoria Property of Dr. Kao Group Second Objective: Identify parameters involved in enhanced production of carotenoids

AN IDEAL SCENARIO! 17 AABABABCABC Time Individuals Adapted from Desai and Fisher, 2007 Property of Dr. Kao Group

CLONAL INTERFERENCE! A Time Individuals B ACAC ADAD ACEACE Adapted from Desai and Fisher, 2007 Parameters that contribute to clonal interference: -Size of the population -Rate of mutation -Fitness of the engineered strain -Time to grow -Medium used – volume -Concentration, etc. Isolated Mutant ACH Property of Dr. Kao Group

CLASSROOM APPLICATIONS  Fluorescent Activated Cell Sorting (FACS) machine  Counts heterogeneous and homogeneous mixtures of cells  One at a time  Extremely fast and efficient  Distinguishes cells by their light scattering and fluorescence emitted.  Compares to particular wavelengths of visible light  Sorts and plots them using the electromagnetic spectrum.

GFP YFP RFP FL2-H FL1-H 510/21 nM FL2-H 585/42 nM FL1-H 650 nM FL3-H F2-H RFP FLUORESCENT ACTIVATED CELL SORTER Property of Dr. Kao Group

ACKNOWLEDGEMENTS  TAMU E³ Program  National Science Foundation  Nuclear Power Institute  Dr. Katy Kao  Luis H. Reyes (Ph.D. student)  Other lab members: Priscila Almario Mian Huang Yuqi Guo Sisi Thomas (partner) 16 ACKNOWLEDGMENTS