Genomic screening for novel peptide antibiotics in biofilm cyanobacteria by in-silico analysis and PCR

Abstract

Cyanobacteria are a group of phototrophic prokaryotes commonly known as blue-green algae. They grow embedded as biofilms in a thick matrix of extracellular polymeric substances (EPS) and can produce a highly diverse range of secondary metabolites, which are interesting in terms of their antimicrobial activity. Among these components, polyketide and polypeptide molecules are dominating. Antimicrobial polypeptide molecules are usually post-translational-modified or synthesised by non-ribosomal peptide synthetase (NRPS). Standard screening for antibiotics by inhibition tests is very time consuming and expression of antimicrobic activity highly depend on cultivation conditions. Therefore, they can vary between different cultivations. On a genomic level existing, but in this cultivation not synthesized, antibiotics are completely neglected. Due to the increasing amount of available genomic sequence data, screening for novel antibiotics can also be done in-silico. Highly homologous sequences to known antibiotic gen clusters can be determined in cyanobacterial genomes and eventually be detected in-vivo through PCR analysis. Compared to inhibition tests, a major advantage of PCR is the little amount of biomass needed. As the growth of cyanobacteria is slow, e.g. Trichocoleus sociatus (0.44 d^-1) compared to bacteria like Escherichia coli (2.08 h^-1), this leads to significant shorter cultivation and screening time. In addition, qPCR can be used to determine gene expression quantity of the considered genes. PCR with degenerated primers for specific gen cluster like NRPS, polyketide synthetases, lanthipeptides etc. can also be used to screen non-sequenced cyanobacteria for the possible origin of an unidentified antibiotic.

The following work is part of the iProcess project, whose overall scientific goal is to develop the process engineering fundamentals for using fungi and cyanobacteria as production organisms for pharmaceutically active substances. As part of the iProcess project, a semi-continuous process for the production of antibiotics from cyanobacteria biofilms in aerosol reactors shall be developed. Aim of the following work is the in-silico search for new polypeptide antibiotics, as well as the subsequent in-vivo detection to discover promising cyanobacteria as production strains. In the first instance, the screening is focusing on the intern cyanobacteria strain collection of the TU Kaiserslautern. Subsequently the new strains will be cultivated as biofilms in an aerosol reactor and the resulting extracellular polymeric substances can be analysed for their antimicrobial activity.

 

This project is financially supported by Ministry of Science, Further Education and Culture of Rhineland-Palatinate (mwwk.rlp) (iProcess intelligent process development – from modelling to product).