Creating new-to-nature antimicrobial peptides via ribosomal synthesis and posttranslational modification

Creating new-to-nature antimicrobial peptides via ribosomal synthesis

Studentprojects supervised by Fleur Ruijne

Introduction

Nature has been a source of inspiration and for the design and synthesis of many complex antimicrobials we are using in modern medicine to combat antibiotic resistance. Of particular interest to our lab are the Ribosomally synthesized and posttranslationally modified peptides (RiPPs). As the name RiPPs implies, these complex peptides are ribosomally produced and subsequently heavily modified by specific enzymes to create new chemical moieties; for instance a lanthionine ring in the case of the antibiotic nisin. Over the past few years, many of these posttranslational modification enzymes have been discovered, introducing many different types of modifications. These are unexplored enzymes from sponge symbionts, other marine environments and also the gut microbiome.

Aim

The aim of my PhD-project is to orchestrate several of these enzymes to work on the same substrate peptide in vivo to create complex new-to-nature antimicrobial peptides. This requires the design of a hybrid peptide that can be recognized by multiple modification enzymes in vivo. Ideally, we will design a plug-and-play system that can be combined with mutagenesis of the gene encoded peptide for facile development of novel antimicrobials.

Available student projects

Since many things are unknown about these enzymes and combining them in one heterologous host expression system, there are many challenging student projects available for either bachelor or master students and for different parts of the project. Potential projects could for example be about post-translational modification enzyme characterization, antimicrobial peptide design and production, creating and optimizing a heterologous expression system, natural product characterization produced by cyanobacteria, etcetera. You can also contact me if you have some nice project ideas of your own (related to antimicrobial peptide design)!

Techniques you can learn

Each project has its own specific lab skills that you will learn, but most projects will include these biochemistry techniques:

  • Cloning/molecular genetics techniques: PCR, restriction digestion and other novel cloning methods (for example Gibson Assembly, Golden Gate Assembly, USER cloning), transformations of different bacteria.
  • Bacterial culturing of mainly E. coli and potentially some cyanobacteria
  • Peptide and protein purification with in vitro activity assays
  • HPLC
  • Mass spectrometry: MALDI-TOF, LC-MS
  • Antimicrobial assays

How to contact me

If you are interested in doing a project with me in the Molecular Genetics lab, please contact Oscar and me with your interests, whether you are a bachelor or master student and when and how long you would be available to do the project:

Fleur Ruijne
E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Prof. Dr. Oscar Kuipers
E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Related articles

Montalbán-López, M. et al. New developments in RiPP discovery, enzymology and engineering. Nat. Prod. Rep. (2020) doi:10.1039/D0NP00027B  

Montalbán-Lopez M, van Heel AJ, Kuipers OP. (2017) Employing the promiscuity of lantibiotic biosynthetic machineries to produce novel antimicrobials. FEMS Microbiology Reviews, 41(1): 5–18. 

Hudson, G. A. & Mitchell, D. A. RiPP antibiotics: biosynthesis and engineering potential. Curr. Opin. Microbiol. 45, 61–69 (2018)  

Repka LM, Chekan JR, Nair SK, van der Donk WA (2017). Mechanistic understanding of lanthipeptide biosynthetic enzymes. Chem. Rev., 117(8): 5457-5520.
Burkhart, B. J., Kakkar, N., Hudson, G. A., Van Der Donk, W. A. & Mitchell, D. A. Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products. ACS Cent. Sci. 3, 629–638 (2017).  

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