Expanding the potential of mutasynthetic approaches for pseudomonic acids by Yusra Alsammarraie A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Microbiology and infection School of Biosciences College of Life and Environmental Sciences The University of Birmingham August 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Natural products, particularly polyketides are among the most important sources of antimicrobial compounds. 20% of the top selling drugs are polyketide based. In recent years genetic engineering has played a critical role in modifying biosynthetic pathways of different polyketide compounds as a way to create novel structures with improved clinical properties. Further investigation and understanding of these giant multi-enzyme complexes is necessary to achieve efficient synthetic engineering. In many PKS systems including the mupirocin biosynthesis pathway, the thioesterase (TE) is normally considered as the end of the assembly line. However, expressing the CoA-ligase tmlU from the thiomarinol pathway in the mupirocin producer strain (Pseudomonas fluorescens NCIMB10586) revealed that TmlU could only release truncated pseudomonic acid when a TE domain was present. This finding led to the hypothesis that perhaps the TE domain could act as a tether for TmlU, in order for the latter to be able to capture the growing chain and perhaps load it onto the post TE pathway. This study also presents the first evidence of MmpB being involved in producing the 9- hydroxynonanoic acid in the mupirocin biosynthesis pathway. Table of Contents 1 Introduction ...................................................................................................................................... 2 1.1 Antibiotics ................................................................................................................................ 2 1.1.1 Antibiotic Targets ............................................................................................................. 3 1.1.2 Antibiotic resistance ......................................................................................................... 7 1.1.3 Implication of antibiotic resistance ................................................................................ 10 1.2 Polyketides ............................................................................................................................. 17 1.2.1 Fatty acids ...................................................................................................................... 19 1.2.2 Type I polyketide synthases ........................................................................................... 20 1.2.2.1 Erythromycin biosynthesis pathway .................................................................... 21 1.2.3 Type II polyketide synthases .......................................................................................... 23 1.2.4 Type III polyketide synthases......................................................................................... 25 1.3 Mupirocin ............................................................................................................................... 26 1.3.1 The mupirocin biosynthetic cluster ................................................................................ 28 1.3.2 Mupirocin biosynthesis .................................................................................................. 31 1.3.2.1 Biosynthesis of the monic acid backbone ............................................................ 33 1.3.2.2 Tailoring the monic acid backbone ...................................................................... 34 1.3.2.3 9-hydroxynonanoic acid biosynthesis .................................................................. 37 1.3.3 Regulation of Mupirocin production .............................................................................. 38 1.3.4 Clinical significance ....................................................................................................... 40 1.3.5 Resistance to mupirocin ................................................................................................. 42 1.3.6 Non-ribosomal peptide synthases ................................................................................... 43 1.3.7 Thiomarinols .................................................................................................................. 44 1.4 Aminocoumarin ...................................................................................................................... 49 1.5 Statement of objectives .......................................................................................................... 51 2 MATERIALS AND METHODS ................................................................................................... 55 2.1 Bacterial strains, culture conditions and plasmids ................................................................. 55 2.2 Polymerase chain reaction (PCR) .......................................................................................... 60 2.2.1 Velocity DNA polymerase kit ........................................................................................ 65 2.2.2 Invitrogen Taq polymerase ............................................................................................. 66 2.2.3 QuickChange site directed mutagenesis ......................................................................... 67 2.3 DNA manipulation ................................................................................................................. 68 2.3.1 Plasmid extraction .......................................................................................................... 68 2.3.1.1 ISOLATEII plasmid mini kit from Bioline .......................................................... 68 2.3.1.2 Bioneer Accuprep® plasmid mini extraction kit .................................................. 69 2.3.2 Restriction digest ............................................................................................................ 69 2.3.3 Agarose gel electrophoresis............................................................................................ 70 2.3.4 DNA extraction from TAE (Tris-acetate-EDTA) agarose gel ....................................... 70 2.3.5 A-tailing blunt-ended PCR products .............................................................................. 71 2.3.6 DNA ligation .................................................................................................................. 72 2.3.7 Splicing by overlap extension (gene SOEing) ............................................................... 72 2.3.8 DNA transformation ....................................................................................................... 74 2.3.9 DNA sequencing ............................................................................................................ 74 2.3.10 Sequencing analysis ....................................................................................................... 74 2.3.11 Conjugation and suicide vector excision ........................................................................ 75 2.4 Bioassay for Mupirocin production ........................................................................................ 76 2.5 Bacterial two hybrid system ................................................................................................... 76 2.6 High performance liquid chromatography ............................................................................. 78 2.6.1 Mupirocin detection ....................................................................................................... 78 2.6.2 Thiomarinol detection .................................................................................................... 78 2.7 β-galactosidase assay.............................................................................................................. 79 3 The effect of the amide synthetases NovL, CloL, CouL and SimL on production of pseudomonic acids ................................................................................................................................................ 81 3.1 Introduction ............................................................................................................................ 81 3.2 Results .................................................................................................................................... 85 3.2.1 Constructing pSIML, pCLOL, pNOVL and pCOUL ..................................................... 85 3.2.2 HPLC analysis of pseudomonic acids produced by P. fluorescens WT NCIMB10586, NCIMB10586 (pSIML), NCIMB10586 (pNOVL), NCIMB10586 (pCLOL), NCIMB10586 (pCOUL) 86 3.2.3 Effect of the amide synthetases on thiomarinol production and attempts to complement a ΔtmlU mutant .........................................................................................................