UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems Permalink https://escholarship.org/uc/item/9n89c8zv Author Walker, Mark Chalfant Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems by Mark Chalfant Walker A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Michelle C. Y. Chang, Chair Professor Judith P. Klinman Professor Susan Marqusee Professor Mathew B. Francis Spring 2013 Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems © 2013 by Mark Chalfant Walker Abstract Expanding the scope of organofluorine biochemistry through the study of natural and engineered systems by Mark Chalfant Walker Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Michelle C. Y. Chang, Chair Fluorination has become a very useful tool in the design and optimization of bioactive small molecules ranging from pesticides to pharmaceuticals. Its small size allows a sterically conservative substitution for a hydrogen or hydroxyl, thus maintaining the overall size and shape of a molecule. However, the extreme electronegativity of fluorine can dramatically alter other properties of the molecule. As a result, the development of new methods for fluorine incorporation is currently a major focus in synthetic chemistry. It is our goal to use a complementary biosynthetic approach to use enzymes for the regio-selective incorporation of fluorine into complex natural product scaffolds through the fluoroacetate building block. Towards this goal, we initiated a study of the only known genetic host of a carbon-fluorine bond forming enzyme, the fluoroacetate- and fluorothreonine-producing bacterium Streptomyces cattleya. Sequencing and analysis of the genome identified several paralogs of enzymes predicted to be on the fluoroacetate and fluorothreonine biosynthetic pathway and whose function were probed by in vitro biochemistry and genetic knock-out studies. We sought to further explore how S. cattleya manages fluoroacetate, which is a potential biosynthetic building block but is also a potent toxin that operates by shutting down the tricarboxylic acid (TCA) cycle. Coordinated transcriptional changes of genes involved in central metabolism and organofluorine metabolism suggest transcriptional control may serve as the major mechanism for management of fluoroacetate toxicity. This hypothesis is further supported by biochemical analysis of S. cattleya enzymes involved in fluoroacetate toxicity, which showed that they were no more selective against fluorine than orthologs from non-producing bacteria. To explore the possibility of incorporating fluorinated building blocks into more complex natural products, we turned to type I polyketide synthases as their modular assembly line nature would make them ideal candidates for the engineering of site-selective incorporation of fluorinated subunits. We first developed methods to enzymatically synthesize fluoromalonyl- CoA, which is a fluorinated congener of the malonyl-CoA extender unit. With this building block in hand, we were able to observe incorporation of fluorine into a triketide polyketide. We then showed that it is possible to site-selectively incorporate fluorine into tetraketide products. These results suggest that the production of complex fluorinated natural products is possible and may allow us to explore the medicinal chemistry of these compounds using site-selective fluorination. 1 Table of Contents Table of Contents i List of Figures, Schemes, and Tables iii List of Abbreviations vi Acknowledgments vii Chapter 1: Introduction 1.1 Fluorine in pharmaceuticals 2 1.2 Halogens in biology 5 1.3 Polyketide synthases 8 1.4 Thesis motivation and organization 10 1.5 References 11 Chapter 2: Assembly and analysis of the Streptomyces cattleya draft genome 2.1 Introduction 17 2.2 Materials and methods 18 2.3 Results and discussion 24 2.4 Conclusions 36 2.5 References 37 Chapter 3: Fluoroacetate resistance in Streptomyces cattleya 3.1 Introduction 41 3.2 Materials and methods 42 3.3 Results and discussion 47 3.4 Conclusions 66 3.5 References 67 i Chapter 4: Engineering fluorinated polyketide biosynthesis 4.1 Introduction 71 4.2 Materials and methods 72 4.3 Results and discussion 83 4.4 Conclusions 97 4.5 References 97 Appendices Appendix 1: Plasmids and oligonucleotides 102 ii List of Figures, Schemes, and Tables Chapter 1 Figure 1.1 Potential impacts of fluorination on small molecules 2 Figure 1.2 Blockage of P450 degradation by fluorination 2 Figure 1.3 Tuning of pKa by fluorination 3 Figure 1.4 Fluorine in the design of a mechanism based inhibitor 3 Figure 1.5 Fluorination of the complex natural product erythronolide A 4 Figure 1.6 Naturally-occurring organochlorines and organobromines 4 Figure 1.7 Active sites of halogenases 5 Figure 1.8 Naturally-occurring organofluorines 5 Figure 1.9 Lethal synthesis of 4-hydroxy-transaconitate from fluoroacetate 6 Figure 1.10 Proposed biosynthetic pathway of fluoroacetate and fluorothreonine 7 in Streptomyces cattleya Figure 1.11 Pharmacologically important natural products produced by Type I 8 polyketide synthases Figure 1.12 A schematic diagram of the well studied DEBS polyketide synthase 9 Figure 1.13 Sequence alignment of malonyl-CoA and methylmalonyl-CoA 9 selective acyltransferase domains Figure 1.14 Proposed mechanism of acyl transfer in AT domains 10 Chapter 2 Scheme 2.1 The proposed biosynthetic pathway for fluoroacetate and 17 fluorothreonine in S. cattleya Figure 2.1 Electropherograms of paired-end libraries for Illumina sequencing 24 Table 2.1 Analysis of steps in draft genome assembly 24 Figure 2.2 Comparison of steps in draft genome assembly to the S. cattleya 25 closed genome Figure 2.3 Comparison of draft genomes produced with different assemblers 26 Table 2.2 Identity of gaps in draft genomes 27 Table 2.3 Comparison of fluoroacetate pathway paralogs in S. cattleya and 28 other sequenced streptomycetes iii Figure 2.4 Genomic contexts of methylthioribose-1-phosphate isomerases 29 in S. cattleya Figure 2.5 Genomic contexts of MRI2 orthologs in Nocardia farnicia 30 Figure 2.6 Expanded view of genomic context of MRI2 in S. cattleya 31 Figure 2.7 Characterization of MRI2 reaction product by LC/MS 32 Figure 2.8 Characterization of MRI knockout strains by 19F NMR 33 Figure 2.9 Growth and fluoride uptake by MRI knockout strains 33 Table 2.4 Prediction of PKS AT domain specificity in S. cattleya 34 Table 2.5 Prediction of NRPS A domain specificity in S. cattleya 35 Chapter 3 Scheme 3.1 Lethal synthesis of 4-hydroxy-transaconitate from fluoroacetate 41 Figure 3.1 Electropherograms of single-end library for Illumina sequencing 47 Figure 3.2 Comparison of RNA-seq and custom microarray results 48 Figure 3.3 Analysis of fluoride response by S. cattleya 49 Figure 3.4 Time- and fluoride-dependent response of predicted pathway 50 genes and their paralogs Figure 3.5 Transcription patterns of selected TCA cycle genes with respect 51 to fluoride and time Figure 3.6 Transcription patterns of all predicted TCA cycle genes with 52 respect to fluoride and time Table 3.1 Gene IDs and annotations for all predicted TCA cycle genes 53 Figure 3.7 COG categories of differentially-expressed genes 54 Figure 3.8 Transcriptional response of the crcB locus to the presence of 55 fluoride Figure 3.9 Sequence alignment of selectivity filter regions of selected eriC 56 orthologs Figure 3.10 Characterization of ∆flK strain 57 Figure 3.11 Heterologous expression and purification of acetate assimilation 58 enzymes Table 3.2 Kinetic parameters of acetate assimilation enzymes 59 Figure 3.12 Phylogenetic tree of selected citrate synthases 60 Figure 3.13 Heterologous expression of citrate synthases 61 Table 3.3 Kinetic parameters of citrate synthases 61 iv Figure 3.14 Dose response curves for citrate synthases 62 Figure 3.15 Production of citrate and fluorocitrate by citrate synthases 63 Figure 3.16 Sequence alignment of selected citrate synthases 63 Figure 3.17 Activation of citrate synthases by potassium chloride 64 Figure 3.18 Activation of streptomyces citrate synthases by AMP 65 Figure 3.19 Inhibition of the S. cattleya aconitase by fluorocitrate 65 Chapter 4 Figure 4.1 Synthetic biology of fluorine 71 Figure 4.2 Enzymes used in extender unit biosynthesis 84 Figure 4.3 Enzymatic synthesis of extender units from acetate and 85 fluoroacetate Figure 4.4 Kinetic parameters for malonate activation 86 Figure 4.5 Heterologous expression of NphT7 86 Figure 4.6 Chain extension and keto reduction with a fluorinated extender 87 Figure 4.7 Structural alignment of NphT7 and a DEBS ketosynthase domain 88 Figure 4.8 1H-19F HMBC NMR analysis of enzymatically produced 2-fluoro- 89 3-hydroxy-butyryl-CoA Figure 4.9 Enzymes used in the production of model polyketides 90 Figure 4.10 Production of a fluorinated polyketide using DEBSMod6+TE 91 Figure 4.11 GC-MS and 19F NMR analysis of F-TKL 92 Scheme 4.1 Hydrolysis and regeneration reactions for F-TKL synthesis 93 Figure 4.12 Test for covalent inhibition of DEBSMod6+TE by fluoromalonyl-CoA 93 Table