Cabral, A. J. | Search bioRxiv

bioRxiv2024

A portable and dual-inducible control system for multistep biosynthetic pathways in Gram-negative bacteria

BackgroundThe successful production of industrially relevant natural products hinges on two key factors: the cultivation of robust microbial chassis capable of synthesizing the desired compounds, and the availability of reliable genetic tools for expressing target genes. The development of versatile and portable genetic tools offers a streamlined pathway to efficiently produce a variety of compounds in well-established chassis organisms. The {sigma}70 lac and tet expression systems - adaptations of the widely used lac and tet regulatory systems developed in our laboratory - have shown effective regulation and robust expression of recombinant proteins in various Gram-negative bacteria. Understanding the strengths and limitations of these regulatory systems in controlling recombinant protein production is essential for progress in this area. ResultsTo assess their capacity for combinatorial control, both the {sigma}70 lac and tet expression systems were combined into a single plasmid and assessed for their performance in producing fluorescent reporters as well as the terpenoids lycopene and {beta}-carotene. We thoroughly characterized the induction range, potential for synergistic effects, and metabolic costs of our dual {sigma}70 lac and tet expression system in the well-established microorganisms Escherichia coli, Pseudomonas putida, and Vibrio natriegens using combinations of fluorescent reporters. The dynamic range and basal transcriptional control of the {sigma}70 expression systems were further improved through the incorporation of translational control mechanisms via toehold switches. This improvement was assessed using the highly sensitive luciferase reporter system. The improvement in control afforded by the integration of the toehold switches enabled the accumulation of a biosynthetic intermediate (lycopene) in the {beta}-carotene synthesis pathway. ConclusionThis study presents the development and remaining challenges of a set of versatile genetic tools that are portable across well-established gammaproteobacterial chassis and capable of controlling the expression of multigene biosynthetic pathways. The enhanced {sigma}70 expression systems, combined with toehold switches, facilitate the biosynthesis and study of enzymes, recombinant proteins, and natural products, thus providing a valuable resource for producing a variety of compounds in microbial cell factories. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/598700v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@be801forg.highwire.dtl.DTLVardef@cda5deorg.highwire.dtl.DTLVardef@144b729org.highwire.dtl.DTLVardef@550011_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

The Enigma of UV Stress Granules: Research Challenges and New Perspectives

Stress granules (SGs) are non-membrane bound cytoplasmic condensates that form in response to a variety of different stressors. Canonical SGs are thought to have a cytoprotective role, reallocating cellular resources during stress by activation of the integrated stress response (ISR) to inhibit translation and avoid apoptosis. However, different stresses result in compositionally distinct, non-canonical SG formation that is likely pro-apoptotic, though the exact function(s) of both SGs subtypes remain unclear. A unique non-canonical SG subtype is triggered upon exposure to ultraviolet (UV) radiation. While it is generally agreed that UV SGs are bona fide SGs due to their dependence upon the core SG nucleating protein Ras GTPase-activating protein-binding protein 1 (G3BP1), the localization of other key components of UV SGs are unknown or under debate. Further, the dynamics of UV SGs are not known, though unique properties such as cell cycle dependence have been observed. This Perspective compiles the available information on SG subtypes and on UV SGs in particular in an attempt to understand the formation, dynamics, and function of these mysterious stress-specific complexes. We identify key gaps in knowledge related to UV SGs, and examine the unique aspects of their formation. We propose that more thorough knowledge of the distinct properties of UV SGs will lead to new avenues of understanding of the function of SGs, as well as their roles in disease.

cell biology↗