Recombinant Protein Production in Bacillus Species, Isolation and Methionine Production in Methionine-producing Microorganisms

Linoleic acid isomerase (LAI) is the enzyme that converts linoleic acid to conjugated linoleic acids (CLAs) which appear to be capable of lowering cancer risk and enhancing immunity. There is an interest in developing commercial processes for the production of single isomers of CLA by biotransformation of LA using microbial cells and enzymes. However, the evaluation of these strains suggest that growth and LA isomerase production levels by these anaerobes are insufficient to support economic commercial production of single CLA isomers. A better alternative is to clone the isomerase gene and to generate new production strains using recombinant technology. Bifidobacterium LAI genes were cloned into pET24a(+) as an expression vector and transferred into E. coli BL21 (DE3) as the expression host while Propionibacterium acnes LAI were cloned into Bacillus species as the expression hosts. Unfortunately, the expressed Bifodobacterium LAI formed inclusion bodies and did not exhibit detectable enzymatic activities. Propeptide Staphylococcus hyicus lipase fused with P. acnes LAI expressed in B. megaterium YYBm1 was secreted into the surrounding medium. Based on MALDI-TOF MS results showed that this propeptide S. hyicus lipase still attached with the secreted LAI and might inhibit activity. However, other propeptides (B. subtilis nprE, B. subtilis amyE, B. megaterium nprM) did not protect LAI from proteolytic degradation. Methionine is the first limiting amino acid in poultry feed and must be supplemented to the poultry feed mixture. Many studies attempted to isolate methionine producing microoragnisms from environments and genetically modified by using chemical mutagens. However, genetically-modified organisms are considered unacceptable for use in organic food production. Therefore, wild type strains with methionine-producing ability are necessary for the organic poultry industry. In this study, numerous bacteria were isolated from natural environments that appeared to produce high methionine; however, all of them were identified as pathogens. In addition, bacteria excreted methionine into the surrounding medium and it was concluded that this could be impractical for large scale recovery of methionine. Conversely, isolated yeast strain K1 yielded high methionine content, compared to other yeasts. Based on large subunit rRNA sequences, these isolated strains were identified as Pichia kudriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is recognized as a ′′generally recognized as safe′′ (GRAS) organism. Yeast strain K1 may be suitable as a source of methionine for dietary supplements in organic poultry feed. This dissertation is approved for recommendation to the Graduate Council. Dissertation Director: _______________________________________ Dr. Steven C. Ricke Dissertation Committee: _______________________________________ Dr. Michael F. Slavik _______________________________________ Dr. Young Min Kwon _______________________________________ Dr. Yuchun Du DISSERTATION DUPLICATION RELEASE I hereby authorize the University of Arkansas Libraries to duplicate this dissertation when needed for research and/or scholarship. Agreed __________________________________________ Suwat Saengkerdsub Refused __________________________________________ Suwat Saengkerdsub ACKNOWLEDGEMENTS I would like to thank my graduate advisor, Dr. Steven C. Ricke, for his guidance throughout my research projects for this dissertation. I would further like to thank my committee Dr. Michael F. Slavik, Dr. Young Min Kwon, Dr. Yuchun Du for his support on my research. This work was supported by an Arkansas Biosciences Institute Grant and the Methionine Task Force, Coleman Natural Foods, Petaluma, CA. I thank Dr. Simon Stammen, Dr. Rebekka Biedendieck, and Professor Dieter Jahn of Institute of Microbiology, Technische Universitat Braunschweig, Germany for providing plasmids and Bacillus megaterium YYBm1. I appreciate Robert Preston Story Jr. at the Center for Food Safety, Department of Food Science, the University of Arkansas for supporting Bidifobacterium longum ATCC15707, Bacillus subtilis, Bacillus licheniformis NRRL B-14212. I would like to thank Professor Ines Pinto, the Department of Biological Sciences, the University of Arkansas for providing yeasts from her collection and Rohana Liyanage, the Department of Chemistry and Biochemistry, the University of Arkansas for technical assistance. Finally, thanks are extended to my mom, my wife, sisters, brother, and Elizabeth Chadbourn McKee, a librarian at the University of Arkansas, for their encouragement. TABLE OF CONTENTS CHAPTER I: INTRODUCTION 1 References 3 CHAPTER II: LITERATURE REVIEW 5 Bacterial expression systems for heterologous protein production 5 Introduction 5 Escherichia coli as host 5 T7 RNA polymerase system 6 Bacilli as host 7 Brevibacillus choshinensis 10 Bacillus megaterium 10 The spac promoter 13 The xylose-inducible promoter 14 Prepeptide or signal peptide 17 Secretion systems of Bacillus 18 Propeptide 23 Linoleic acid isomerase 25 Methionine in the poultry industry 28 Methionine production by microorganisms 31 Methionine detection 33 Microbial sources for organic poultry industry 36 References 37 APPENDIX 45 CHAPTER III:LINOLEIC ACID ISOMERASE EXPRESSION IN E. coli BL21 (DE3) AND Bacillus SPECIES 56 Abstract 56 Introduction 57 Materials and methods 58 Results and discussion 61 Acknowledgements 64 References 64 CHAPTER IV: CONTRIBUTION OF THE PREAND PRO-REGIONS OF Staphylococcus hyicus LIPASE, Bacillus subtilis NEUTRAL PROTEASE, AND Bacillus megaterium THE MAJOR EXTRACELLULAR PROTEASE TO SECRETION OF HETEROLOGOUS PROTEINS BY Bacillus megaterium AND Bacillus licheniformis 78 Abstract 79 Introduction 80 Materials and methods 80 Results 83 Discussion 87