THESIS
2023
1 online resource (xii, 171 pages) : illustrations (chiefly color)
Abstract
Lasso peptides are a recently categorized class of ribosomally synthesized and post-translationally
modified peptides (RiPPs) in the field of natural product drug discovery. The
interesting lasso-like topology of these peptides may confer stability and bioactivity, and
could help in the ongoing fight against antibiotic-resistant microbes. We have previously
identified three putative lasso peptide BGCs from actinobacteria using genome mining with
antiSMASH, the strains being Nocardiopsis synnemataformans (DSM 44143), Nonomuraea
coxensis (DSM 45129), and Kutzneria albida / Streptosporangium albidum (DSM 43870).
In this work, we aimed to induce expression of these three putative biosynthetic gene
clusters (BGCs) in in E. coli BL21 (DE3) heterologous host, engineering various gene
construct...[
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Lasso peptides are a recently categorized class of ribosomally synthesized and post-translationally
modified peptides (RiPPs) in the field of natural product drug discovery. The
interesting lasso-like topology of these peptides may confer stability and bioactivity, and
could help in the ongoing fight against antibiotic-resistant microbes. We have previously
identified three putative lasso peptide BGCs from actinobacteria using genome mining with
antiSMASH, the strains being Nocardiopsis synnemataformans (DSM 44143), Nonomuraea
coxensis (DSM 45129), and Kutzneria albida / Streptosporangium albidum (DSM 43870).
In this work, we aimed to induce expression of these three putative biosynthetic gene
clusters (BGCs) in in E. coli BL21 (DE3) heterologous host, engineering various gene
constructs and screening bacteria fermentation conditions for those suitable to lasso
peptide production. For culture conditions, low nutrient – low growth were predominantly
trialled, and for extraction we mainly used crude extraction of cell pellet with methanol and
freeze-thaw lysis. Liquid chromatography – mass spectrometry (LC-MS) detection of peptide
products from crude extracts was conducted. To prospectively speed up the product
detection process, we trialled using reporter EGFP fluorescence in whole cell culture as
proxy for protein expression of our constructs. Although we detected significant
fluorescence in certain EGFP-fusion construct screens, to date, no conclusive lasso peptide
production has been detected in our study. Nonetheless, the content of this thesis could
hopefully provide a reference for the possibilities and challenges one may encounter if one
tackles a similar project in the future. We also discovered a semi-novel cloning method
which allowed assembly of gene fragments without in vitro restriction endonuclease
digestion of insert fragments, but unfortunately the method is still currently undergoing
troubleshooting.
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