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Metabolic Engineering for Fumaric and Malic Acids Production

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Metabolic Engineering for Fumaric and Malic Acids Production Dissertation Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Baohua Zhang, M.S. Ohio State Biochemistry Program The Ohio State University 2012 Dissertation Committee: Professor Shang-Tian Yang, Advisor Professor Jeffrey Chalmers Professor Hua Wang Copyright by Baohua Zhang 2012 ABSTRACT Fumaric acid is a natural organic acid widely found in nature. With a chemical structure of two acid carbonyl groups and a trans-double bond, fumaric acid has extensive applications in the polymer industry, such as in the manufacture of polyesters, resins, plasticizers and miscellaneous applications including lubricating oils, inks, lacquers, styrenebutadiene rubber, etc. It is also used as acidulant in foods and beverages because of its nontoxic feature. Currently, fumaric acid is mainly produced via petrochemical processes with benzene or n-butane as the feedstock. However, with the increasing crude oil prices and concerns about the pollution problems caused by chemical synthesis, a sustainable, bio-based manufacturing process for fumaric acid production has attracted more interests in recent years. Rhizopus oryzae is a filamentous fungus that has been extensively studied for fumaric acid production. It produces fumaric acid from various carbon sources under aerobic conditions. Malic acid and ethanol are produced as byproducts, and the latter is accumulated mainly when oxygen is limited. Like most organic acid fermentations, the production of fumaric acid is limited by low productivity, yield and final concentration influenced by many factors including the microbial strain used and its morphology, medium composition, and neutralizing agents. Many efforts have been done to improve fumaric acid production through optimization of the fermentation process. The goal of ii this project was to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae. This is the first attempt to apply genetic engineering strategies to change the metabolic flux towards fumaric acid biosynthesis. The biosynthesis of fumaric acid in R. oryzae takes place in the cytosol and three enzymes: pyruvate carboxylase (PYC), malate dehydrogenase and fumarase are involved in the reaction. PYC is situated at the branch point of pyruvate metabolism, and its role in affecting fumaric acid and various metabolites accumulation was thus studied in this work. An expression plasmid containing native R. oryzae pyc gene, encoding pyruvate carboxylase, was transformed into the uracil auxotroph R. oryzae M16. Two transformants were obtained: pyc3 and pyc5, and were verified by Southern hybridization. Compared to the wild type, the PYC activity in the pyc tranformants increased 56%-83%. However, the pyc transformants grew poorly and had a low fumaric acid yield of less than 0.05 g/g glucose due to the formation of large cell pellets that limited oxygen supply and resulted in the accumulation of ethanol with a high yield of 0.13-0.36 g/g glucose. An exogenous phosphoenolpyruvate carboxylase (PEPC) was introduced in R. oryzae with the aim to increase CO2 fixation and the carbon flux toward Oxaloacetate. The pepc gene encoding PEPC was expressed in R. oryzae under the endogenous pgk1 promoter and pdcA terminator. The obtained pepc transformants exhibited significant PEPC activity of 3-6 mU/mg that was absent in the wild type. Compared to the fermentation kinetics of the wild type, the fumaric acid production by the pepc transformant increased 26% (0.78 g/g glucose vs. 0.62 g/g for the wild type). iii Fumarase catalyzing the final step of fumaric acid biosynthesis in R. oryzae was overexpressed to investigate its effects on cell growth and fumaric acid production. Three fumR fragments with different lengths of 5’ and 3’ untranslated regions (UTR) were used to express the fumR gene in R. oryzae. All transformants showed significantly increased fumarase activity during both the seed culture and fermentation stages. However, fumarase overpression yielded more malic acid, instead of fumaric acid in the fermentation. It was attributed to the catalytic prevalence on the direction of fumaric acid to malic acid by the overexpressed fumarase. The results suggested that the overepxressed fumarase by itself was not responsible for the overproduction of fumaric acid in R. oryzae. Fumaric acid is an intermediate in the succinic acid biosynthesis pathway in E. coli fermentation under anaerobic conditions. The fumarate reductase, encoded by frd, was disrupted in E. coli KJ060, a high succinic acid producer, to study its effect on the metabolic flux distribution. The frd gene was removed from E. coli chromosome through homologous recombination. Under anaerobic conditions, the frd disrupted mutant of E. coli KJ060M produced little succinic acid. However, it did not produce much fumaric acid either. Under aerobic conditions, the mutant produced malic acid as the main product, with a high yield of 0.72 g/g glucose, higher than that (0.65 g/g) produced by the parental strain. A high malic acid concentration of 48.4 g/L was produced in fed-batch fermentation in a 5-liter fermenter with a productivity of 2.38 g/L·h. The mutant thus has the potential for use in industrial production of malic acid, which is widely used as acidulants in foods and beverages. iv Dedicated to my parents and husband v ACKNOWLEDGEMENTS Firstly, I would like to express my special thanks and gratitude to my advisor, Dr. Shang-Tian Yang, who gave me guidance, encouragement and support throughout my graduate study. I learned a lot from his expertise in science and deep insights in my research. I would also like to thank Dr. Jeffrey Chalmers and Dr. Hua Wang for their time being on my committee and their valuable advices on my research project. I want to give my sincere thanks to all the previous and current laboratory members in our research group, especially Dr. Mingrui Yu, Mr. Kun Zhang, and Mr. Zhongqiang Wang for their helpful suggestions and support. In addition, I would like to specially thank Dr. Christopher D. Skory from USDA for providing experimental strains, plasmids as well as valuable technical advice and assistance. I would also like to thank Dr. Lonnie O. Ingram from University of Florida for providing E. coli strains. Financial supports from the United Soybean Board and the Consortium for Plant Biotechnology Research, Inc. (CPBR) to this research are also acknowledged. Finally, I wish to thank my parents for all their emotional support and my husband for helping me get through the difficult times. My entire family and all my friends are acknowledged in the depth of my heart. vi VITA 1999 - 2003…………………………………...B.S. Biological Science, Nankai University 2003 - 2006…………………………………..........M.S. Microbiology, Nankai University 2006 - 2007……………………..Graduate Fellowship, Ohio State Biochemistry Program The Ohio State University 2007 – 2012………………………………………………….Graduate Research Associate, Chemical & Biomolecular Engineering The Ohio State University PUBLICATIONS 1. Zhang, B., Skory, C.D., Yang, S.T. Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose. Metabolic Engineering. 2012. In Press. 2. Zhang, B., Yang, S.T. Metabolic engineering of Rhizopus oryzae: Effects of overexpressing fumR gene on cell growth and fumaric acid biosynthesis from glucose. Process Biochemistry. 2012. Accepted for publication. 3. Yang, S.T., Zhang, K., Zhang, B., Huang, H. Biobased Chemicals - Fumaric Acid. In: Moo-Young M (ed.) Comprehensive Biotechnology, 2nd edition. 2011. pp.163-177. 4. Wang, Z., Feng, S., Huang, Y., Qiao, M., Zhang, B., Xu, H. Prokaryotic expression, purification, and polyclonal antibody production of a hydrophobin from Grifola frondosa. Acta Biochim Biophys Sin (Shanghai). 2010, 42:388-95. 5. Yu, L., Zhang, B., Szilvay, G.R., Sun, R., Jänis, J., Wang, Z., Feng, S, Xu, H., Linder, M.B., Qiao, M. Protein HGFI from the edible mushroom Grifola frondosa is a novel 8 kDa class I hydrophobin that forms rodlets in compressed monolayers. Microbiology. 2008, 154:1677-1685. 6. Yu, L., Shao, B., Zhang, B. Isolation and purification of Trichoderma reesei hydrophobin HFBI. Food and Fermentation Industries. 2005, 31:129-132. FIELDS OF STUDY Major Field: Biochemistry Specialty: Biochemical Engineering vii Table of Contents Abstract ............................................................................................................................... ii Dedication………………………………………………………………………………....v Acknowledgements ............................................................................................................ vi Vita .................................................................................................................................... vii List of Figures ................................................................................................................... xv List of Tables ................................................................................................................. xviii 1. Introduction ..................................................................................................................... 1 1.1 Objectives .................................................................................................................. 3 1.2 Scope of study ..........................................................................................................
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