METABOLIC ENGINEERING FOR ENHANCED PROPIONIC ACID FERMENTATION BY PROPIONIBACTERIUM ACIDIPROPIONICI DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Supaporn Suwannakham, B.Eng. ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Shang-Tian Yang, Adviser Professor Jeffrey J. Chalmers __________________________________ Professor Hua Wang Adviser Graduate Program in Chemical Engineering ABSTRACT Propionic acid is widely used in food and dairy industries. As a result of its antimicrobial activity, propionic acid and its salts are widely used as preservatives in foods and grains. Currently, the market of propionic acid is mainly supplied by production via petrochemical routes. Fermentation by propionibacteria produces mainly propionic and acetic acids from sugars; however, the fermentation suffers from low propionic acid production due to by-product formation and strong propionic acid inhibition on cell growth and the fermentation. The high demand of propionic acid for use as a natural preservative in foods and grains has stimulated developments of new fermentation processes to achieve improved propionic acid production from low-cost biomass and food processing wastes. In this research, novel approaches, at process engineering, metabolic engineering, and genetic engineering levels, were developed for enhanced propionic acid production by Propionibacterium acidipropionici. Fed-batch fermentation of glucose by P. acidipropionici immobilized in a fibrous- bed bioreactor (FBB) with a high cell density (>45 g/L) produced a high final propionic acid concentration of 72 g/L and a high propionate yield of up to 0.65 g/g. A mutant with improved propionate tolerance was obtained by adaptation in the FBB, which resulted in significant physiological and morphological changes. The mutant cells were less sensitive ii to propionate inhibition and had a higher saturated fatty acid content in the cell membrane and a slimmer shape with an increased specific surface area. Metabolic stoichiometric analysis was applied to quantitatively describe the global cellular mechanism in propionic acid fermentation. By feeding carbon sources with different oxidation states, different fermentation end-product compositions were obtained, indicating different controlling mechanisms involving various acid-forming enzymes with significant changes in their activities and overall protein expression pattern. In general, the metabolic pathway shifted toward more propionate formation with a more-reduced substrate. Gene inactivation via gene disruption and integrational mutagenesis was used to knock out the acetate kinase (ack) gene with the goal of eliminating acetate formation and further enhancing propionic acid production by P. acidipropionici. Mutants were obtained by transforming the cells with a partial ack gene fragment, which was introduced either as a linear DNA fragment with a tetracycline resistance cassette within the partial ack gene or in a non-replicative integrational plasmid containing the tetracycline resistance cassette. The ack inactivation in the mutants showed a profound impact on cell growth rate. Compared to the wild type, the ack-deleted mutants achieved ~10% increase in propionate yield and ~10% decrease in acetate yield. iii The FBB, the knowledge of the underlying mechanism in controlling propionic acid fermentation, and the mutants obtained in this research should allow us to develop an economical bioprocess for the production of propionic acid from sugars. iv Dedicated to my mother v ACKNOWLEDGMENTS My great appreciation goes to my adviser, Dr. Shang-Tian Yang, for intellectual and financial support as well as for his inspiring advice, encouragement, enthusiasm, and flexibility throughout my study. I have gained a lot from his insights and I have greatly enjoyed my staying here as his student. I would like to acknowledge with sincere gratitude to the members of my dissertation committee, Dr. Jeffrey J. Chalmers and Dr. Hua Wang. I am grateful for their helpful advices on a variety of topics. I wish to thank Dr. Yan Huang for her help in the work reported in Chapter 5. I am greatly indebted to Dr. Ying Zhu for teaching me the fundamental laboratory skills in conducting the fermentation experiments and the basic molecular biology skills in performing the genetic engineering experiments. I also appreciate Mr. Carl Scott, Mr. Leigh Evrard, and Mr. Paul Green for their technical assistance. My colleagues in my research group, especially Dr. Nuttha Thongchul and Ms. Suwattana Pruksasri, offered many kinds of support and help over the last four years. I benefited a lot from discussions with them and sharing the knowledge on my research. vi I would like to thank Dr. Daniel R. Zeigler (Bacillus Genetic Stock Center, The Ohio State University, Columbus, OH) for supplying pBEST309 and pDG1515, Dr. Desh Pal Verma (Department of Molecular Genetics, The Ohio State University, Columbus, OH) for supplying electroporation device, and Dr. Mitsuo Yamashita (Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan) for his suggestions on genetic engineering experiments. Financial supports from the U.S. Department of Agriculture (CSREES 99-35504- 7800) and the Consortium for Plant Biotechnology Research, Inc. to various phases of this work are also acknowledged. I appreciate Mrs. Panitee Panjakup for her kindly help and encouragement on pursuing my Ph.D. study. I wish to thank best friends of mine, Mr. Chanin Hunsakunathai, Mr. Vichian Suksoir, and Ms. Ratchat Chantawongvuti, for their warm support and understanding during the last four years. Special thank goes to my family for their love and warm support. With their love and support, I could be through ups and downs during my study. Finally, my heartfelt gratitude goes to my mother, Ms. Suwannee, my grandmother, Mrs. Sunee, and my aunt, Ms. Sumalee Peerapongsathorn, for their love and inspiration. My graduation could only be achievable with their warmest support and understanding. vii VITA August 5, 1978…………………………...............................Born – Bangkok, Thailand March, 1999……………………………...............................B.Eng. Chemical Engineering Chulalongkorn University Bangkok, Thailand September, 2000 – March 2005…………………………….Graduate Research Associate Chemical Engineering The Ohio State University PUBLICATION Suwannakham S, Yang S-T. 2005. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol Bioeng, in press. FIELD OF STUDY Major Field: Chemical Engineering Specialty: Biochemical Engineering viii TABLE OF CONTENTS Page Abstract……………………………………………………………………...............……ii Dedication………………………………………………………………………........……v Acknowledgments………………………………………………………………….....….vi Vita…………………………………………………………………………………..…viii List of Tables……………………………………………………………...................….xiv List of Figures……………………………………………………………………….….xvi Chapters: 1. Introduction……………………………………………………………………..……..1 2. Literature Review……………………………………………………………….….….9 2.1 Propionic Acid Fermentation….......................................................................… …..9 2.1.1 Propionic Acid………………………………………………………..…. ….9 2.1.2 Microorganisms……………………………………………………….……12 2.1.3 Metabolic Pathway…………………………………………………………14 2.1.4 Fermentation Processes……………………………………………….……17 2.2 Cell Immobilization and Fibrous-Bed Bioreactor…………………………….….30 2.2.1 Cell Immobilization…………………………………………………...…..30 2.2.2 Immobilized-Cell Bioreactor……………………………………………. …31 2.2.3 Fibrous-Bed Bioreactor……………………………………………….……32 2.3 Immobilized-Cell Fermentation……………………………………………...……33 2.4 Metabolic Engineering……………………………………………………….……36 2.4.1 Metabolic Flux Analysis……………………………………………………36 2.4.2 Applications of Other Metabolic Engineering Techniques…………...……39 2.5 Genetic Engineering of Propionibacteria…………………………………….……40 2.5.1 Acetic Acid Formation in Propionic Acid Fermentation……………..……40 2.5.2 Acetic Acid Formation Pathway Genes and Enzymes………………..……42 2.5.3 Genetics and Molecular Biology of Propionibacteria………………...……43 2.6 References…………………………………………………………………………50 ix 3. Enhanced Propionic Acid Fermentation by Propionibacterium acidipropionici Mutant Obtained by Adaptation in a Fibrous-Bed Bioreactor…………….........……64 Summary………………………………………………………………………....……64 3.1 Introduction…………………………………………………………………..……66 3.2 Materials and Methods……………………………………………………….……68 3.2.1 Culture and Media…………………………………………………….……68 3.2.2 Free-Cell Fermentation…………………………………………….....……68 3.2.3 Immobilized-Cell Fermentation……………………………………………69 3.2.4 Effect of Propionic Acid on Cell Growth………………………….....……70 3.2.5 Enzyme Assays…………………………………………………….....……70 3.2.6 Membrane-Bound ATPase Assay…………………………………….……71 3.2.7 Cell Membrane Fatty Acid Analysis………………………………….……72 3.2.8 Cell Viability Assay.………………………………………………….……72 3.2.9 Scanning Electron Microscopy……………………………………….…. …72 3.2.10 Analytical Methods…………………………………………………...……73 3.3 Results and Discussion……………………………………………………….……73 3.3.1 Fermentation Kinetics………………………………………………...……73 3.3.2 Propionic Acid Inhibition……………………………………………..……76 3.3.3 Acid-Forming Enzyme Activities…………………………………….…. …78 3.3.4 Membrane-Bound ATPase…………………………………………………81 3.3.5 Membrane Fatty Acid Composition…………………………………..……83 3.3.6 Morphological Change in Mutant…………………………………….……83 3.3.7 Effects of Cell Immobilization in FBB……………………………….……84