Developing the Mupirocin QS system of P fluorescens into an efficient and economical way to control industrial production of high value products
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
Abstract
Industrial production of high value products by bacteria is a vital part of the modern biotechnology and pharmaceutical industries. Competition to produce such products as cheaply as possible means that the costs of the specialist chemicals added to fermenters to switch on production need to be controlled. The mupIRX gene system in Pseudomonas fluorescens regulates genes encoding the Mupirocin biosynthetic pathway, allows switch on of gene expression by an internal signal that accumulates as the bacteria grow and has already been manipulated to yield benefits for antibiotic production. We will define the elements of this control system to allow us to construct systems to put the production of other high value products under the control of this system. The innovation is in exploiting the native control system in an established and proven industrial host strain.
Technical Summary
The mupIRX gene system in Pseudomonas fluorescens regulates genes encoding the Mupirocin biosynthetic pathway, allows auto induction of gene expression and has already been manipulated to yield benefits for antibiotic production. We will define biochemical properties of the protein products and their DNA binding sites to allow more precise re-engineering of gene expression under the control of the positive regulator MupR and fine tuning of the N-acyl homoserine lactone quorum signal by expression of its synthase MupI and its modulation by MupX. We will test different configurations including a cascade controlled by mupR to induce expression of single genes encoding high value proteins and gene clusters encoding other biosynthetic pathways. We will optimise regulation via the quorum sensing (QS) circuitry and amplification of copy number of plasmids encoding key genes. The innovation is in exploiting the native QS system in an established and proven industrial host strain.
Planned Impact
As described in proposal submitted to IUK
People |
ORCID iD |
| Christopher Thomas (Principal Investigator) |
Publications
Haines AS
(2022)
High quality genome annotation and expression visualisation of a mupirocin-producing bacterium.
in PloS one
| Description | We have identified what we think is the binding site for the activator that switches on the genes that make the antibiotic mupirocin. We have confirmed the existence of gene expression signals in the region of at least some of these sites by analysing the RNA made from these regions. This gives us confidence that these are places where control of gene expression occurs. We have analysed the DNA sequences of these regions in more detail so that we can relate the DNA sequence to the strength of the expression signal. We have also shown that we can make big deletions of the gene cluster concerned so that we have the basis for engineering the bacteria to have the properties that we want. We have confirmed that foreign genes put under the control of this system are expressed well in a way that is consistent with what we know about the control mechanism. We have also developed a new vector to carry this expression system based on an endogenous plasmid. |
| Exploitation Route | The project is still underway and the objective is to generate a new, more efficient way to control the expression of industrially important genes whose products need to be produced by fermentation. |
| Sectors | Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | The findings have already been applied to the expression of some industrially useful model genes. |
| First Year Of Impact | 2015 |
| Sector | Manufacturing, including Industrial Biotechology |
| Impact Types | Economic |
| Title | New Vector for Suicide Mutagenesis |
| Description | A new plasmid vector was developed for suicide mutagenesis in Pseudomonas fluorescens by deleting the beta-lactamase gene (conferring ampicillin resistance) from pAKE604 to leave only the gene for amino-glycoside phosphotransferase that confers kanamycin resistance. Since P. fluorescens is naturally resistant to Ampicillin this allows ampicillin selection to kill the donor bacteria after conjugative transfer, thus speeding up the whole process which previously depended on selection with minimal medium, a much slower process. |
| Type Of Material | Improvements to research infrastructure |
| Year Produced | 2018 |
| Provided To Others? | Yes |
| Impact | The process of making mutations in P. fluorescens and related species has been accelerated so less time is devoted to mutation creation. |
| Title | Genome sequence of Pseudomonas NCIMB10586 |
| Description | Complete genome sequence of the mupirocin-producing strain NCIMB10586 |
| Type Of Material | Database/Collection of data |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | The genome sequence was compiled and annotated to a high stardard which will be described in a paper which is about to be submitted (March 2021). It has allowed reassignment of this strain to the species Pseudomonas synxantha, in the fluorescens group. This has underpinned analysis of RNASeq data over a timed growth cycle and the development of a novel way of displaying the RNAseq data. This will also be added Researchfish when the paper is published. |
| URL | https://www.ncbi.nlm.nih.gov/nuccore/PRJEB27737 |
| Description | GSK |
| Organisation | GlaxoSmithKline (GSK) |
| Country | Global |
| Sector | Private |
| PI Contribution | Academic research |
| Collaborator Contribution | Industrial Research; Loan of HPLC equipment |
| Impact | Research outputs covered by confidentiality agreement |
| Start Year | 2011 |
| 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 |