6 research outputs found
The Mode of Action of Cyclic Monoterpenes (ā)-Limonene and (+)-Ī±-Pinene on Bacterial Cells
A broad spectrum of volatile organic compoundsā (VOCsā) biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCsāthe cyclic monoterpenes (ā)-limonene and (+)-Ī±-pineneāon bacteria was studied in this work. We used genetically engineered Escherichia coli bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the luxCDABE genes of Photorhabdus luminescens. We showed that (ā)-limonene induces the PkatG and PsoxS promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (ā)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-Ī±-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (ā)-limonene completely inhibits the DnaKJEāClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both E. coli wild type and mutant ĪibpB strains. (+)-Ī±-Pinene partially inhibits refolding only in ĪibpB mutant strain
A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering
Abstract CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8āmg/L homogentisic acid