Biosynthesis of polyketide antibiotic mupirocin by Pseudomonas fluorescens

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences


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.


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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 are exploring possible collaborations with manufacturers of mupirocin to identify collaboration to increase production or modify production.
Sectors Healthcare,Manufacturing/ including Industrial Biotechology

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 of Great Britain & Northern Ireland (UK)
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 of Great Britain & Northern Ireland (UK)
Start 07/2013 
End 12/2014
Description Birmingham-Bristol 
Organisation University of Bristol
Country United Kingdom of Great Britain & Northern Ireland (UK) 
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
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 Discussion about how antibiotics work, how resistance arises and why we need to use antibiotics carefully.

Increased interest in microbiological research as a career
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
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