Cell-free protein synthesis of secondary metabolite ribosomal peptides

Abstract

Recently, there has been an increased emergence of antimicrobial resistant organisms and a decrease in antibiotic discovery, together producing a public health threat. To overcome this problem, novel antimicrobial scaffolds require development or existing structures need diversifying. Natural products are a group of diverse chemicals many of which display antimicrobial activity. The biosynthetic gene clusters encoding these products are often termed ‘cryptic’ due to not being expressed under normal laboratory conditions. Thus, making isolation of novel products difficult. Cell-free protein synthesis has recently been explored for its potential application in facilitating heterologous expression of natural product clusters as well as peptide diversification via incorporation of non-canonical amino acids. As it’s non-living, directly manipulatable nature lends itself to expression and alteration of toxic products. In this thesis we studied the potential of E. coli cell-free protein synthesis for expression of 3 putative lasso peptides, a class of natural product characterised by their lasso structure, isolated from Streptomyces genomes. The natural product clusters were amplified from their respective genomes using an optimized PCR protocol for the amplification of high GC sequences and cloned into plasmids for expression in cell-free systems. However, further experiments are required to allow for their expression. Moreover, steps were taken towards producing a E. coli cell-free system for incorporation of tryptophan analogues into peptides with a direct read out of successful incorporation. A deCFP marker was produced to allow for identification of ncAA incorporation, however supporting mutations within its sequence are required to increase quantum yields to suitable levels. Furthermore, amino acid depletion steps are required during cell-free lysate production to allow for efficient incorporation of ncAAs. Finally, bioinformatic investigation of RiPP biosynthetic gene cluster intergenic regions was carried out to probe for the presence of intrinsic terminators involved in their regulation. This identified secondary structures present within the intergenic regions of all RiPP clusters tested, par one. Furthermore, MSA of intergenic regions of clusters homologous to the 3 clusters of interest suggested that these secondary structures may be conserved across lasso peptides.