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Overview

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Project

Introduction

Over the summer we worked with nonribosomal peptide synthetases (NRPSs). We developed a B. subtilis strain capable of oligo-mediated genome engineering and used this strain to alter a NRPS. We also investigated methods of screening for novel products with desired activities.

Background

Nonribosomal peptide synthases (NRPSs) are large multimodular enzymes that synthesize nonribosomal peptides, which are short bioactive peptides with a broad range of functions, including antibiotics, immunosuppressants and anticancer drugs.

MAGE subtilis

Multiplex automated genome engineering (MAGE) utilises cyclical recombination with short oligonucleotides in order to achieve a high allelic replacement efficiency and can be used to quickly generate cell populations with varying phenotypes. We introduced oligo-mediated genome engineering into Bacillus subtilis and used it to alter a nonribosomal peptide synthetase.

Tyrocidine

Tyrocidine is a mixture of non-ribosomal peptides. It can only be used topically due to its toxicity. We sought to express the tyrocidine synthase cluster in B. subtilis to make novel derivatives with oligo-mediated recombineering.

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Lab-on-a-disc

Lab-on-a-disc is a potential screening method for our MAGE NRPS products. After screening for antimicrobial activity, prospective MAGE edited NRPS products could be tested for different beneficial properties. One example is a quick screening method for cytotoxicity.

Intein

When one method fails the Synthesizer team has to innovate! We explored an alternative approach for generating short, cyclized peptides with similar length to tyrocidine by using self-splicing proteins. These short, self-splicing proteins that have no function in the proteins they are a part of, besides catalyzing their own excision after translation, are called inteins. The splicing creates a peptide bond between the two adjacent amino acids next to the inteins and we hypothesized that it could be used to make our cyclic peptides.

NRP Detection

Separation and identification of our non-ribosomal peptide synthetase (NRPS) products was determined by ultra-high performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOFMS). NRPS variants were identified by changes in mass-to-charge ratio and column retention time.

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