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Biosynthesis of the Antibiotic Mupirocin by Pseudomonas Fluorescens Ncimb 10586

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Biosynthesis of the Antibiotic Mupirocin by Pseudomonas Fluorescens Ncimb 10586 BIOSYNTHESIS OF THE ANTIBIOTIC MUPIROCIN BY PSEUDOMONAS FLUORESCENS NCIMB 10586 by RACHEL GURNEY A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences The University of Birmingham September 2012 ABSTRACT The mupirocin biosynthetic pathway belongs to the trans-AT group in which acyltransferase (AT) activity is provided by a separate polypeptide (MmpC) rather than in cis as found in the typical type I polyketide synthases. AT docking domains have been documented in trans-AT PKS clusters for ten years yet little functional evidence is available. The cluster shows many interesting features that must be understood to create novel products. Specificity studies demonstrated that AT2 performs the typical AT function of loading malonyl-CoA to ACPs throughout the cluster. Mutagenesis studies demonstrated the importance of AT active site residues for protein structural integrity, acquisition and transfer of malonate and propose an alternate role for AT1 as a proofreading enzyme responsible for hydrolysing truncated intermediates from the pathway. Consequently an edit, reload, reduce model for MmpC is proposed. Mutagenesis of docking domains led to a halt in mupirocin production and suggested that docking domains are required for structural integrity of the Mmps or for guiding the ACPs into the correct position for interactions with their respective partners. Studies involving a mutated ACP3 protein confirmed the importance of Trp55, as demonstrated by structural changes and the inability of the protein to accept malonate from AT2. ACKNOWLEDGEMENTS I would like to thank my supervisor, Chris Thomas, for giving me the opportunity to study for my PhD and for his unwavering support, guidance and knowledge over the last four years. Thank you to my co-supervisor, Jo Hothersall, for her advice, patience and friendship. Thank you to the BBSRC for funding my research, to our collaborators at Bristol for their invaluable input and contributions, and to all the behind the scenes technicians and cleaners that make media and keep us in a constant supply of glass wear while at the same time providing cheerful banter. Thank you to everyone in S101, past and present. In particular Yui, for saving my sanity and becoming a great friend; Yusra for ‘very good’ and for always paying me back when she borrowed from the bank of Rach; Tony for persistently inviting me for coffee despite the fact I don’t like it; Elton, for always being there and for knowing absolutely everything; Claire for being a cheerful presence during my last nine months; Daisuke for showing me the joys of SDS-PAGE; Jenny, for starting what I was able to continue; Harry for his amazing effort and golden touch when it came to lab work. Thanks to The Geeks for our lunchtime laughs – I really wouldn’t have made it without you. We should write that book. My parents have been a constant support throughout all of my years in education. Thank you - it would not have been possible without you, I hope I’ve done you proud! Finally I would like to thank my husband Nigel for continually supporting me. I couldn’t have done it without you, and I promise to get a job. TABLE OF CONTENTS 1 Introduction 1 1.1 Antibiotics 2 1.1.1 Major targets of antibiotics 4 1.1.2 Antibiotic resistance 8 1.1.3 Implications of antibiotic resistance 12 1.1.4 Methicillin-resistant Staphylococcus aureus 19 1.2 Polyketides 22 1.2.1 Fatty acid biosynthesis 24 1.2.2 Type I polyketides 26 1.2.2.1 Erythromycin biosynthesis pathway 26 1.2.3 Type II polyketides 30 1.2.4 Type III polyketides 32 1.3 Mupirocin - pseudomonic acid 33 1.3.1 Mupirocin biosynthetic cluster 36 1.3.2 Mupirocin biosynthesis 38 1.3.2.1 Monic acid biosynthesis 40 1.3.2.2 Tailoring the monic acid backbone 42 1.3.2.3 9-hydroxynonanoic acid biosynthesis 45 1.3.3 Regulation of mupirocin production 46 1.3.4 Special features of the mupirocin cluster 48 1.3.4.1 Dependence on in trans ATs 48 1.3.4.2. No integrated ER domains 48 1.3.4.3 Methyl group incorporation 49 1.3.4.4 Duplicated acyl carrier proteins 50 1.3.5 Clinical significance 51 1.3.6 Resistance to mupirocin 54 1.4 Non-ribosomal peptide synthases 59 1.4.1 The thiomarinols 60 1.5 Statement of objectives 64 2 Materials and methods 67 2.1 Bacterial strains, culture conditions and plasmids 68 2.2 Polymerase chain reaction 79 2.2.1 BIO-X-ACT long DNA polymerase kit 87 2.2.2 Invitrogen Taq polymerase 89 2.2.3 QuikChange® site-directed mutagenesis PCR 90 2.2.4 Velocity DNA polymerase 92 2.3 DNA manipulation 93 2.3.1 Plasmid extraction 93 2.3.1.1 Birnboim and Doly alkaline SDS method 93 2.3.1.2 Bioneer Accuprep® plasmid extraction kit 94 2.3.2 Restriction digests 94 2.3.3 Agarose gel electrophoresis 95 2.3.4 Extraction of DNA from an agarose gel 95 2.2.5 A-tailing blunt-ended PCR products 97 2.3.5 DNA ligations 97 2.3.6 DNA transformation 98 2.3.7 DNA sequencing 98 2.3.8 Sequence analysis 99 2.3.9 Conjugation and suicide vector excision 99 2.4 Bioassay for mupirocin production 100 2.5 High performance liquid chromatography 101 2.6 Liquid chromatography mass spectrometry 101 2.7 Protein expression and purification 103 2.7.1 Cell lysis 103 2.7.2 Nickel affinity chromatography 104 2.7.2.1 Purifying proteins under denaturing conditions 104 2.7.2.2 Refolding denatured protein 105 2.7.3 SDS-PAGE 108 2.7.4 Determination of protein concentration 109 2.8 Acyltransferase assays 109 2.9 Autoradiography 109 2.9.1. Analysis of phosphorimager screen 110 2.10 Circular dichroism 111 2.11 Protein crosslinking with glutaraldehyde 111 3 Expression and purification of mupirocin biosynthesis proteins 112 3.1 Introduction 113 3.2 Results 118 3.2.1 Expression and purification of acyltransferase proteins 120 3.2.1.1 Expression and purification of AT1 120 3.2.1.1.1 Alternative purification strategy for AT1 121 3.2.1.1.2 Expression of AT1 in E. coli Lemo21(DE3) 124 3.2.1.1.3 Expression of AT1 in Terrific Broth 126 3.2.1.1.4 Purification of AT1 with styrene maleic acid 127 3.2.1.1.5 Purifying AT1 under denaturing conditions 129 3.2.1.1.6 Refolding purified denatured AT1 130 3.2.1.1.7 Expression of AT1 with the GST tag 132 3.2.1.2 Expression and purification of AT2 133 3.2.1.3 Expression of thiomarinol acyltransferases 134 3.2.2 Expression and purification of ACPs 135 3.3 Discussion 138 4 Characterisation of AT1 and AT2 141 4.1 Introduction 142 4.2 Results 153 4.2.1 Phylogenetic analysis 153 4.2.2 Secondary structure prediction 156 4.2.4 Crosslinking of AT2 158 4.2.5 Substrate specificity assay of AT1 and AT2 160 4.2.6 Acquisition and transfer of malonate by AT2 162 4.2.6.1 Assessing the appropriate conditions for AT assays 163 4.2.6.2 AT2 malonylation of type I and type II ACPs 167 4.3 Discussion 170 5 Site directed mutational analysis of AT2 174 5.1 Introduction 175 5.2 Results 181 5.2.1 Sequence analysis 181 5.2.2 Construction of AT2 point mutants 186 5.2.3 Expression and purification of AT2 point mutants 189 5.2.4 Substrate specificity of mutant AT2 proteins 190 5.2.5 Circular dichroism of mutant AT2 proteins 193 5.2.6 Acquisition and transfer of malonate by mutant AT2 proteins 197 5.3 Discussion 200 6 Acyltransferase active site mutagenesis 206 6.1 Introduction 207 6.2 Results 210 6.2.1 Construction of point mutations in MmpC AT domains 210 6.2.2 Plate bioassay of antibiotic activity in MmpC point mutants 212 6.2.3 HPLC analysis of mupirocin production in MmpC point mutants 215 6.2.4 LCMS analysis of compounds produced by MmpC point mutants 215 6.3 Discussion 217 7 Genetic analysis of docking domains 224 7.1 Introduction 225 7.2 Results 228 7.2.1 Sequence analysis 228 7.2.2 Construction of P. fluorescens NCIMB 10586 deletion mutants 235 7.2.5 Bioassay and HPLC analysis of docking domain mutants 236 7.2.6 LCMS of docking domain mutants 238 7.3 Discussion 239 8 Mutagenesis of β-branching ACP3 244 8.1 Introduction 245 8.2 Results 249 8.2.1 Construction of ACP3 W55L point mutant 249 8.2.2 ACP3 mutant protein expression and purification 250 8.2.3 Circular dichroism of ACP3 WT and mutant proteins 251 8.2.4 Malonylation assay of ACP3 WT and mutant proteins 251 8.3 Discussion 253 9 General discussion and future work 256 9.1 Mupirocin proteins can be difficult to work with 259 9.2 AT2 exclusively prefers malonyl-CoA as a substrate and transfers it to mupirocin type I and type II ACPs 260 9.3 AT1 demonstrates potential hydrolase activity 263 9.4 Docking domains are essential for mupirocin biosynthesis 265 9.5 The role of Trp55 is essential for malonylation of ACP3 266 9.6 Future work on the third domain of MmpC 266 9.10 Model for MmpC function 267 Appendix Characteristics of trans-AT PKS systems 269 References 280 LIST OF FIGURES Chapter 1 1.1.
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