Lactobacillus Reuteri

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Lactobacillus Reuteri VITAMIN B12 SYNTHESIS IN Lactobacillus reuteri Filipe Santos Promotor: Prof. Dr. W. M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Co-promotor: Prof. Dr. J. Hugenholtz Hoogleraar Industrial Molecular Microbiology Universiteit van Amsterdam Samenstelling Promotiecomissie: Prof. Dr. John R. Roth University of California, Davis, USA Prof. Dr. Douwe van Sinderen University College Cork, Ireland Dr. Paul G. Bruinenberg DSM Food Specialties, Delft Prof. Dr. René H. Wijffels Wageningen Universiteit This research has been conducted at the VLAG Graduate School VITAMIN B12 SYNTHESIS IN Lactobacillus reuteri Filipe Santos Electronic version Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M. J. Kropff, in het openbaar te verdedigen op maandag 15 September 2008 des namiddags te vier uur in de aula Vitamin B12 Synthesis in Lactobacillus reuteri Filipe Santos Ph.D. thesis Wageningen University, Wageningen, The Netherlands (2008) 274 pages, with summary in Dutch and Portuguese ISBN 978 90 8504 990 6. (Electronic version) For my parents, Table of Contents of Electronic Version Abstract 9 Chapter 1 General introduction 11 Chapter 2 Pseudovitamin B12 is the corrinoid produced by Lactobacillus 43 reuteri CRL1098 under anaerobic conditions Chapter 3 The complete coenzyme B12 biosynthesis gene cluster of 57 Lactobacillus reuteri Chapter 4 The impact of glycerol on the metabolism of Lactobacillus 83 reuteri studied by functional genomics and genome-scale modeling Chapter 5 PocR regulates glycerol reduction, assembly of metabolosomes 179 and vitamin B12 biosynthesis in Lactobacillus reuteri JCM1112 as in otherwise unrelated enteric bacteria Chapter 6 Effect of amino acid availability on vitamin B12 production in 197 Lactobacillus reuteri Chapter 7 Functional enrichment of fermented foods by increased folate 229 production in the B12 producer Lactobacillus reuteri JCM1112 Chapter 8 Summary, discussion and concluding remarks 241 Supplements 261 Samenvatting (Dutch) 263 Súmario (Portuguese) 265 Acknowledgments 266 About the Author 269 List of Publications 271 Training and Supervision Plan (VLAG) 273 8 ABSTRACT Abstract Vitamin B12 is a fascinating molecule that acts as a co-factor in processes crucial to many living things. It is an essential constituent of our diet and an industrially important co-factor used in biocatalysis processes, such as the production of 1,3-propanediol. De novo synthesis of vitamin B12 is limited to a few representatives of bacteria and archaea. Lactobacillus reuteri has been reported to produce vitamin B12, but before this study, our knowledge about this process was very limited. The work presented here aimed at characterizing all aspects related to vitamin B12 production in L. reuteri using a multidisciplinary approach, and exploring its potential applications. The native corrinoid produced by L. reuteri under anaerobiosis was analyzed by HPLC, MS and NMR and found to contain adenine, instead of 5,6-dimethlbenzimidazole, in the Co-ligand. Commonly known as pseudovitamin B12, this compound could play a role in assessing the capability of vitamin B12-dependent enzymes to utilize alternative cofactors, and in understanding the impact of analogues in vitamin B12 metabolism. The anaerobic biosynthetic pathway converting glutamate into vitamin B12 was found to be encoded in one single stretch of the chromosome, neighbored by clusters of genes encoding glycerol reduction, metabolosome assembly and cobalt transport. The control of expression of these clusters appears to be part of the same regulon, in which a single regulatory protein plays a pivotal role. Genome-scale analysis tools such as cDNA microarrays and metabolic network modeling, exposed unsuspected liaisons between glycerol utilization, vitamin B12 synthesis and amino acid metabolism. This provided new leads that resulted in a 20-fold increase in vitamin B12 production without the usage of genetic manipulation. The possibility of combining the production of folate and vitamin B12 in the same L. reuteri cell was also illustrated and applied to the fermentation of substrates of plant origin. The findings reported here, besides their scientific relevance, can be applied to the improvement/development of fermentation process with increased vitamin B12 production. Ultimately, the work presented here might also be used to ensure an adequate vitamin B12 intake in humans. Keywords: vitamin B12, Lactobacillus reuteri, lactic acid bacteria, genome-scale model, omics, biosynthesis. 9 10 CHAPTER 1 GENERAL INTRODUCTION Abstract Vitamin B12 is a captivating molecule that is associated with several fundamental processes that sustain life. For humans, it is an essential nutrient in our diet and a co-factor of great relevance in biotechnological transformations. In this Chapter, a brief description of the illustrious history of B12 research will be presented, followed by some general aspects relative to its chemistry and biochemistry. We will review our current understanding of how this complex molecule is synthesized in Nature and its importance in human nutrition, describing a particular species of lactic acid bacteria, Lactobacillus reuteri, which unlike others in this group can synthesize B12. A brief account of the most modern research approaches is provided. The Chapter ends with a short overview of the thesis. CHAPTER 1 12 GENERAL INTRODUCTION Brief historical background of vitamin B12 research In resemblance to so many other discoveries, vitamin B12 was first stumbled upon while studying a condition caused by its absence. Minot and Murphy reported in 1926 that by submitting their patients to a diet including whole liver they were able to cure pernicious anemia (67). They explained their remarkable finding by postulating the presence of an “extrinsic factor” in liver, whose identity was going to remain elusive for more than 20 years to come. For their efforts related to liver therapy in cases of anemia, Minot and Murphy (along with Whipple) were attributed the first Nobel Prize related to vitamin B12 research in 1934. The race to identify the mysterious “extrinsic factor” culminated in the isolation of a red crystalline compound from liver that was able to cure pernicious anemia. Two principal pharmaceutical companies separated by the Atlantic Ocean achieved this independently within the same year. The discovery was carried by a group at Glaxo in the UK headed by Smith and a group at Merck in the USA lead by Folkers, and at this stage the “extrinsic factor” was designated vitamin B12 (78). Within the same year, vitamin B12 was also found to be present in milk powder, beef extract and culture broth of several bacteria (77), and was reported to be a growth factor for Lactobacillus lactis that lead to the development of a vitamin B12 bioassay (90, 91). Soon, the presence of cobalt was recognized (79), along with the identification of analogues that retained biological activity (29, 36), namely pseudo-B12 (72). The isolation of vitamin B12 along with the realization that this molecule was structurally much more complex than anything that had previously been solved, triggered research on the determination of its exact crystal structure. Hodgkin pioneered X-ray crystallography of complex molecules and in 1955 she revealed the intricately complex three-dimensional structure of vitamin B12 (cyanocobalamin) with a very high level of accuracy (48, 49). Shortly after Barker had crystallized the first biologically active coenzyme forms of (pseudo)-B12 (7), Lenhert and Hodgkin repeated the feat, and solved the structure of adenosylcobalamin in 1961 (55). For her efforts, Hodgkin was awarded in 1964 the second Nobel Prize related to vitamin B12 research in its relatively short history. In 1962, an alternative -axial ligand was found to be present in biologically active forms of vitamin B12. This methyl radical was demonstrated to be involved in methionine synthesis in bacteria (47). Another important event in the history of vitamin B12 was its chemical synthesis. This test of persistence that dragged throughout the ‘60s and ‘70s was headed by Eschenmoser and by yet another Nobel Prize laureate, Woodward. It involved the participation of over one hundred scientists and culminated in a highly complicated chemical synthesis process involving around 70 steps (35). It became apparent that the 13 CHAPTER 1 industrial production of vitamin B12 was not going to rely on chemical synthesis, since it was obviously far too technically challenging and economically unviable. At this stage, much attention turned to how this remarkable molecule was synthesized in Nature. It was only in 1985 that the first gene sequence associated with B12 biosynthesis was identified (115). With the arise of molecular genetics in combination with well- established biochemistry and chemistry expertise, it took less than ten years for the groups of Blanche at Rhône-Poulenc Rorer, Battersby at Cambridge UK and Scott in Texas to elucidate most of the aerobic biosynthesis pathway (8). The recognition of a biosynthetic route towards B12 that did not depend on molecular oxygen by the group of Scott constituted a turning point in B12 history. It lead to the intense study of B12 biosynthesis in Propionibacterium shermanii by Scott himself and co-workers, and additionally to the study of the more genetically accessible anaerobic B12 producer, Salmonella typhimurium, in the groups of Roth, Warren, amongst others (115). Even though much progress
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