Biosynthesis of polyketide antibiotic mupirocin by Pseudomonas fluorescens
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
Abstract
Summary Biological systems build complex molecules for many different purposes - building blocks for cells, and supracellular structures, the catalytic and energy storage systems that drive living cells, the messenger molecules that allow communication and information storage within and between organisms and finally the molecules that allow defence or aggression against other organisms. Mankind has learnt to exploit many of these natural compounds, not least those that act as antibiotics. One class of compounds, the polyketides, are of great importance because they include many molecules with a great diversity of structures which cover a whole range of useful activities - not just antibacterials, but also antifungals, anticancer and anticholesterol agents, to name just a few. Typically, the molecular 'backbones' of these compounds are made by joining simple building blocks on an assembly line, each building block being added by a separate 'module' that also processes the new segment to one of a number (normally three) of modifications (known as the Type I PKS pathway). The resulting molecular chain can be of different lengths and combinations of modifications and can then be decorated with different side chains to produce a unique product ('tailoring'). However, an increasing number of atypical pathways are being uncovered that appear to use additional mechanisms not yet defined. These provide ways of producing new structures in a controlled way. One such pathway, found in Pseudomonas fluorescens, synthesises the clinically important antibiotic mupirocin. It is most active against Gram positive bacteria and is particularly used against MRSA (Methicillin Resistant Staphylococcus aureus), one of the most dangerous 'superbugs'. Biosynthesis of mupirocin involves an atypical Type I PKS along with a large number of 'tailoring' enzymes some of which we have discovered act in tandem with the PKS in building the backbone of mupirocin. This is in contrast to typical type I PKS modules which within themselves contain all the information needed to build the backbone. This project integrates microbial molecular genetics, biochemistry and chemistry to study the biosynthetic machinery both in living cells and with purified enzymes to understand the role of the different PKS and 'tailoring' components in building the final active product. It will explore the reactions carried out by different parts of the pathway and their flexibility to produce new compounds. These will be made available for screening for new biological activities that may be of use as prophylactic or therapeutic agents.
Technical Summary
Mupirocin is composed of a polyketide-derived acid, monic acid (MA) esterified by 9-hydroxy-nonanoic acid (9-HN). MA is formed via an 'AT-less' modular Type 1 PKS, with many features common to this increasing generic group. To understand how the tetrahydropyran ring and other features of MA are formed we will extend ongoing analysis of mutants defective in the pathway to study intermediates involved and create double mutants to establish the order in which particular gene products work. We will purify MmpD encoding the first four Type I PKS modules to explore how trans-acting tailoring genes mupC and mupD carry out reductions on module 3/4 intermediates. We will test MupC and MupF on synthetic substrates to establish their activity/specificity. We will determine whether MmpD processes altered substrates if module 1 is inactivated and alternative substrates are fed. We will assemble the predicted complement of genes in an heterologous host to reproduce the backbone of MA and determine if the tetrahydropyran ring is produced when predicted tailoring genes are added to the cloned PKS genes to complete biosynthesis of MA. We will test the need for the nascent MA to be esterified with 9-HN, and whether cassettes with mupW+T and mupOUV+macpE can function in biotransformations to convert model substrates to functionalised tetrahydropyrans. We will establish if 3-hydroxypropionate is the starter for 9-HN, how it is made and the possible role of mupQ, mupS and macpD. Then with isolated MmpB, the most likely candidate for the FAS/ PKS involved in 9-HN synthesis, supplemented with appropriate accessory genes/enzymes we will determine whether biosynthesis can be performed in vivo and in vitro. We will clone/sequence the genes from Alteromonas rava responsible for the biosynthesis of the thiomarinols - secondary metabolites closely related in structure to mupirocin. Genes unique to thiomarinols will be tested for ability to introduce thiomarinol features into mupirocin. Joint with BB/E022367/1 Co-funded by EPSRC.
Publications
Murphy AC
(2011)
Engineered thiomarinol antibiotics active against MRSA are generated by mutagenesis and mutasynthesis of Pseudoalteromonas SANK73390.
in Angewandte Chemie (International ed. in English)
Hothersall J
(2011)
Manipulation of quorum sensing regulation in Pseudomonas fluorescens NCIMB 10586 to increase mupirocin production.
in Applied microbiology and biotechnology
Gurney R
(2011)
Mupirocin: biosynthesis, special features and applications of an antibiotic from a gram-negative bacterium.
in Applied microbiology and biotechnology
Shields JA
(2010)
Phosphopantetheinylation and specificity of acyl carrier proteins in the mupirocin biosynthetic cluster.
in Chembiochem : a European journal of chemical biology
Gao SS
(2014)
Biosynthesis of mupirocin by Pseudomonas fluorescens NCIMB 10586 involves parallel pathways.
in Journal of the American Chemical Society
Medema MH
(2015)
Minimum Information about a Biosynthetic Gene cluster.
in Nature chemical biology
Thomas CM
(2010)
Resistance to and synthesis of the antibiotic mupirocin.
in Nature reviews. Microbiology
Fukuda D
(2011)
A natural plasmid uniquely encodes two biosynthetic pathways creating a potent anti-MRSA antibiotic.
in PloS one
Scott R
(2011)
Mupirocin F: structure elucidation, synthesis and rearrangements
in Tetrahedron
Trimba R
(2012)
Conjoined nerve roots of the lumbar spine.
in The spine journal : official journal of the North American Spine Society
Description | We sequenced the genome of the bacterium producing thiomarinol and identified all of the genes for thiomarinol biosynthesis, demonstrating that there are independent pathways to the antibiotics marinolic acid (an analogue of mupirocin) and a pyrrothine analogue of holomycin, plus a gene that joins these together. We demonstrated that thiomarinol can overcome the mupirocin resistance in Methicillin Resistant Staphylococcus aureus (MRSA) and that mutants of the thiomarinol producer can add holomycin to mupirocin, allowing it also to overcome this resistance. This opens the way to using mutasynthesis to generate new, more potent hybrid molecules that could be used against other resistant bacteria. We made significant progress in defining the steps of the mupirocin biosynthetic pathway to underpin manipulation and exploitation. We identified the gene responsible for the epoxide ring and showed that over-production of mupU eliminates pseudomonic acid B production, allowing fine tuning of composition of the final antibiotic mixture. |
Exploitation Route | We hope this research will lead to the development of new and useful bioactive compounds. We have been collaborating with GSK since 2012 and this work is on-going. We hope it will lead to a patent application in the near future. |
Sectors | Healthcare,Manufacturing, including Industrial Biotechology |
Description | We are exploiting the knowledge gained in collaborative projects with GSK |
First Year Of Impact | 2016 |
Sector | Manufacturing, including Industrial Biotechology |
Impact Types | Economic |
Description | Novel hybrid anti-MRSA antibiotics from manipulation of the mupirocin and thiomarinol biosynthetic pathways |
Amount | £650,000 (GBP) |
Funding ID | BB/I014373/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2011 |
End | 12/2014 |
Description | Synthetic biology to improve antibiotic production |
Amount | £200,000 (GBP) |
Funding ID | BB/L004453/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2013 |
End | 12/2014 |
Description | Birmingham-Bristol |
Organisation | University of Bristol |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Bacterial genetic analysis of genes involved in biosynthesis of mupirocin and thiomarinol |
Collaborator Contribution | Chemical analysis of intermediates in biosynthesis of mupirocin and thiomarinol from wild type and mutant bacteria |
Impact | All publication listed from our joint grants are outputs |
Description | Daiichi-Sankyo |
Organisation | Daiichi Sankyo Company |
Country | Japan |
Sector | Private |
PI Contribution | Research on the biosynthesis of the antibiotic thiomarinol |
Collaborator Contribution | Provided the bacterial strains that make thiomarinol; covered the salary and living costs for a Daiichi-Sankyo employee, Daisuke Fukuda, to work in Birmingham for two years; provided consumable costs at £12K pa for two years. |
Impact | Publications as already included in Research Fish |
Start Year | 2008 |
Description | Expert opinion |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | TV, radio and newspaper interviews Regular contact with Science Media Centre as an expert; Involvement with BBC Country File; Invitation to present a TV progamme. |
Year(s) Of Engagement Activity | 2009,2010,2011,2012,2013,2014,2015 |
Description | Schools lectures |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | With our current work on plasmid displacement and now that the patent has been filed so we can be more open about the details, I have changed my standard presentation to cover: the current AMR crisis; the fact that many resistance genes are carried and spread by plasmids; that our gut are a reservoir of resistance; that infections arising from antibiotic resistant bacteria will be difficult to treat; that plasmid displacment provides one possible way to address the current crisis; our technology - how it works and what it could be used for. Increased interest in microbiological research as a career |
Year(s) Of Engagement Activity | Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2019,2020 |
Description | Shenley Court School |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | pupils from a local Comprehensive School came into the department to do some experiment that were not possible at their school The pupils gained understanding but also seemed to mature as a result of the experience of doing a responsible piece of work. |
Year(s) Of Engagement Activity | Pre-2006,2006,2007,2008 |
Description | Society for General Microbiology |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Expert involvement with the Society for General Microbiology in preparing informative material/pamphlets on antibiotics Email questions from those who had seen the information on the SGM web site |
Year(s) Of Engagement Activity | 2009,2010,2011 |