INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrougb, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Z eeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9307735 Engineering strategies to optimize plasmid stability and protein production in recombinant Saccharomyces cerevisiae fermentation Cheng, Chinyuan, Ph.D. The Ohio State University, 1992 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 Engineering Strategies to Optimize Plasmid Stability and Protein Production in Recombinant Saccharomyces cerevisiae Fermentation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Chinyuan Cheng, B.S., M.S. The Ohio State University 1992 Dissertation Committee: Approved by Dr. S. T. Yang Dr. J. J. Charlmers Dr. J. F. Davis Adviser Department of Chemical Engineerings To My Parents ACKNOWLEDGMENT I wish to express my appreciation to Dr. S. T. Yang for his support and insight throughout the research. I would also like to thank the other members of my advisory committee, Dr. Jeffrey J. Chalmers, Dr. Jim F. Davis and Dr. Jacques L. Zakin for their useful suggestions and comments. I also would like to acknowledge some friends who have been most helpful in various part of this study. Design engineer Mike Kukla and machinist Roy R. Renshaw helped me to set up the control system. Mr. Gary L. Kleman and Mr. Don E. Ordaz provided me technical assistance in using the computer-controlled glucose-stat. I sincerely thank for my friends who helped me get through my study, especially Dr. Jacques L. Zakin for his constant support and encouragement, C. H. Shu and J. W. Yen for their friendship and care, Mr. Raghavan V. Venkat for proofreading my dissertation. Finally, I appreciate my parents for their priceless love and support to make my education possible. VITA October 29, 1960 ..................................... Bom in Tainan, Taiwan, Republic of China June, 1983 B.S. Chemical Engineering Tunghai University Taichung, Taiwan, R.O.C. 1983-1985 ................................................Military Service Chinese Navy 1986-1988 ................................................M.S. Chemical Engineering The Ohio State University Columbus, OH 1988-Present ............................................Graduate Research and Teaching Associate Department of Chemical Engineering, The Ohio State University Columbus, OH FIELDS OF STUDY Major Field : Chemical Engineering Minor Field : Biochemical Engineering TABLE OF CONTENTS ACKNOWLEDGMENTS----------------------------------------------------------- ii VITA--------------------------------------------------- iii LIST OF TABLES-------------------------------------------------------------------- ix LIST OF FIGURES.---------------------------------------------------- xi NOMENCLATURE------------------------------------------------------------------ xvi ABSTRACT-------------------------------------------------------------------------- xviii CHAPTER PAGE I. INTRODUCTION .............................................................. I II. LITERATURE REVIEW---------------------------------------------- 6 Introduction .........................................................—.................... 6 High Cell Density Fermentation ................................................ 8 Factors Affecting Cell Density ...................................... 11 Control Strategy for High Cell Density Fermentation— 18 Plasmid Stability ........................................................................ 18 Genetic Function of Cells and Recombinant Plasmid — 19 Types of Plasmid Instability -------------------------------- 21 Intrinsic Factors Affecting Plasmid Stability ------------- 22 Environmental Factors Affecting Plasmid Stability 23 (1) Effect of Growth Nutrients .............................. 23 (2) Effect of Temperature ----------------------------- 24 (3) Effect of Growth (Dilution) Rate ---------------- 26 (4) Effect of Oxygen Tension ------------------------ 26 Determination of Plasmid-Carrying Cells ----------------- 27 Strategies for Overcoming Plasmid Loss........................ 31 (1) Genetic Method ---------------------------------- 31 (a) Use of Genes Modulating ------------ 31 iv (b) Incorporation into Chromosome 31 (c) Natural Immunity ------------------------ 32 (d) Double Auxotrophic Mutant. ---------- 33 (e) Secretion ---------------------------------- 34 (2) Operation Strategies ----------------------------- 35 (a) Selective Pressure------------------------ 35 (b) Cell Immobilization --------------------- 40 (c) Separation of Growth and Production Phases 41 (d) Recycling Plasmid-Carrying Cells — 44 (e) Cyclic Oscillation in Fermentation Conditions 45 Cloning Vectors for Saccharomyces cerevisiae ------------------ 49 Plasmid with a Partition Sequence ------------------------- 50 Plasmid Based on the Yeast 2 Micron Circle ------------- 50 The Characteristics of Yeast Used in This Study 51 (1) Construction of Plasmid pSXR 125 ----------- 55 (2) The Characteristics of Auxotrophic Yeast— 57 References ------------------------------------------------------------- 59 III. EFFECT OF TEMPERATURE ON BATCH FERMENTATION OF A RECOMBINANT YEAST STRAIN CONTAINING A TEMPERATURE- SENSITIVE PROMOTER.............................................................. 72 Abstract ---------------------------------------------------------------- 72 Introduction ................................................................................ 73 Materials and Methods .............................................................. 75 Yeast and Plasmid -------------------------------------------- 75 Growth Media ------------------------ 75 Fermentation -------------------------------------------------- 76 Analytical Methods ........................................................ 76 Results and Discussion .............................................................. 81 Plasmid Stability in Flask ------------------ ---------------- 81 Fermentation Kinetics .................................................... 82 (1) Specific Growth Rate .................-.................. 96 (2)Cell Yield..................................-.................... 100 (3) Product Yield ................................................. 104 (4) Specific Production- ...................................... 107 (5) Plasmid Stability --------------------------------- 109 Mathematical Model ................ 114 (1) Parameter Estimation ----------------- 116 Conclusion ------------------------------------------------------------- 118 References ------------------------------------------------------------- 122 V IV. DYNAMIC RESPONSES OF GENE EXPRESSION TO TEMPERATURE SWITCH DURING FERMENTATION------------------------------ 124 Abstract ---------------------------------------------------------------- 124 Introduction ------------------------------------------------------------ 125 Materials and Methods ----------------------------------------------- 127 Yeast and Plasmid -------------------------------------------- 127 Growth Media ------------------------------------------------- 127 Fermentation -------------------------------------------------- 127 Analytical Methods ------------------------------------------ 128 Results and Discussion ----------------------------------------------- 129 Batch Fermentation ----------------------------------------- 129 Repeated Batch Fermentation ------------------------------- 132 (1) Turn-Off Action --------------- 132 (2) Turn-On Action ---------------------------------- 133 Continuous Fermentation ------------------------------------ 140 (1) Turn-Off Action ---------------------------------- 140 (2) Turn-On Action ---------------------------------- 140 (3) Turn-Off Then Turn-On ----------------------- 140 Mathematical Model ----------------------------------------- 145 (1) Parameter Estimation— ................................. 149 Conclusions and Recommendation -------------------------------- 153 References ------------------------------------------------------------- 157 V. EFFECT OF OSCILLATING GLUCOSE CONCENTRATION ON PLASMID STABILITY AND RECOMBINANT PROTEIN PRODUCTION IN REPEATED BATCH