Principal Supervisor: Dr Manuela Tosin, Department of Chemistry
Co-supervisors: Dr Christophe Corre and Dr Alex Jones, School of Life Sciences
PhD project title: Investigation of secondary metabolite formation in Frischella perrara
University of Registration: University of Warwick
Gut microbiota play an important role in health and disease of humans and animals: their symbiotic behaviour is mediated by the production of secondary metabolites (mainly of polyketide-nonribosomal peptide origin) which are mostly beneficial to the gut but which can also prove harmful.1 The Western honey bee (Apis mellifera) harbors characteristic gut microbes which are currently of prominent interest: on one side the bee gut constitutes a relatively simple model for investigating gut community dynamics and host-microbe interactions;2 besides, bees play critical functions for agriculture and natural ecosystems as plant pollinators, hence it is important to understand factors that can affect their lifestyle and life-cycle.
Frischella perrara is a gammaproteobacterium commonly present in the gut of Apis mellifera.3 As such, it likely provides symbiotic functions that confer advantages to its host. However, the recent genome sequencing of F. Perrara has revealed the presence of the clb genomic island responsible for the biosynthesis of colibactin-related metabolites.4 Colibactin is a polyketide-nonribosomal peptide genotoxin of undetermined structure produced by E.coli B2 bacteria present in human and animal guts:5 these include E.coli Nissle 1917, which is utilised in several probiotic preparations. Other bacteria encoding for clb comprise Citrobacter, Ksebiella and Enterobacter species which can be also gut commensal; the widespread occurrence of the clb genomic island is due to horizontal gene transfer amongst Enterobacteriaceae.6
While the production of colibactin by E.coli in the gut has been unequivocally linked to the colorectal cancer in humans and other animals,5 the production of colibactin-related metabolites by F.perrara is thought to be a possible cause of scab formation in the gut of its honeybee host.7 A significant challenge in understanding the role of colibactin-related metabolites and others in their hosts is constituted by the lack of knowledge of their exact chemical structures as well as of the mechanisms leading to their assembly.
In the Tosin group we have recently developed and established the use of chemical probes for the in vivo capture of biosynthetic intermediates involved in polyketide8 and nonribosomal peptide formation;9 these tools have provided unique insights into natural product assembly in a variety of microorganisms, hence we envisage that they could be herein utilised to shed light on secondary metabolite formation in the gut of honeybees as well as of other animals relevant to the UK food industry and economy.
The aim of this PhD project is to elucidate the nature of colibactin-related metabolites and others produced by F. perrara. This will involve:
- the synthesis of functionalised chemical probes targeting polyketide synthase (PKS)- and nonribosomale peptide synthase (NRPS)- catalysed steps involved in the assembly of colibactin-related metabolites;
- the use of chemical probes in bacteria for the capture of biosynthetic intermediates and of the final metabolites;
- the engineering of microbial strains and the reconstitution of biosynthetic pathways in alternative hosts to favour the accumulation of such intermediates/metabolites;
- the structural elucidation of intermediates/metabolites by advanced MS and NMR techniques;
- the preliminary evaluation of intermediate/metabolite flux and bioactivity in the context of animal gut symbiosis.
W.L. Lee et al., Nat Chem Biol. 2014, 10, 416-24;
W. K. Kwong et al., Gut Microbes 2015, 6, 214-20;
P. Engel et al., Int. J. Syst. Evol. Microbiol. 2013, 63, 3646-51;
P. Engel et al., Appl. Environ. Microbiol. 2015, 81, 1502-12;
E. P. Balskus, Nat. Prod. Rep.2015,32, 1534-40;
J. Putze et al., Infect Immun.2009, 77, 4696-703;
P. Engel et al., mBio 2015, 6, e00193-15.
J. S. Parascandolo et al., Angew. Chem. Int. Ed.2016, 55, 3463-7; Y. T. C. Ho et al., Chem. Commun. 2017, 53, 7088-7091.
BBSRC Strategic Research Priority: Food Security
Techniques that will be undertaken during the project:
Organic chemistry: synthesis, purification and characterisation of chemical probes;Microbiology: growing and handling of F. perrara, E. coli and other relevant strains;Molecular biology: gene cloning and genetic disruptions in E.coli and other relevant strains using a PCR targeting methodology;
Analytical chemistry: identification, isolation and characterisation of natural products and biosynthetic intermediates by LC-MS, HPLC, HR-MS, GC, NMR.
Contact: Dr Manuela Tosin, University of Warwick