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Biosynthesis in bacteria is a fundamental anabolic process that generates essential macromolecules, including proteins, nucleic acids, lipids, and polysaccharides. These macromolecules are critical for cellular growth, replication, and function. The process is tightly regulated and energetically linked to catabolic pathways to ensure optimal resource utilization.Biosynthetic pathways begin with precursor metabolites such as pyruvate, acetyl-CoA, and glucose-6-phosphate derived from glycolysis,...
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Gene expression in prokaryotes is governed by constitutive and regulated systems, allowing cells to balance the production of essential proteins with adaptive responses to environmental changes.Constitutive Gene ExpressionConstitutive, or housekeeping, genes are continuously expressed as they encode proteins vital for fundamental cellular processes. These include enzymes for glycolysis, ribosomal components for protein synthesis, and proteins involved in DNA replication. Their constant...
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A dual-inducible control system for multistep biosynthetic pathways.

Andrés Felipe Carrillo Rincón1,2, Alexandra J Cabral1, Andras Gyorgy2

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|November 20, 2024
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Summary

This study developed enhanced genetic tools, the sigma70 (σ70) lac and tet expression systems, for microbial cell factories. These systems, combined with toehold switches, improve control over gene expression for natural product biosynthesis.

Keywords:
Escherichia coliPseudomonas putidaVibrio natriegensDual expressionInducible promotersLuciferaseLycopeneReporter systemβ-carotene

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Area of Science:

  • Synthetic biology
  • Metabolic engineering
  • Microbial biotechnology

Background:

  • Industrial natural product synthesis requires robust microbial chassis and effective genetic tools.
  • Versatile and portable genetic tools streamline compound production in established microorganisms.
  • Sigma70 (σ70) lac and tet expression systems offer regulated recombinant protein expression in Gram-negative bacteria.

Purpose of the Study:

  • To develop and assess portable genetic tools for controlling multigene biosynthetic pathways.
  • To enhance the sigma70 (σ70) lac and tet expression systems for improved recombinant protein production.
  • To evaluate the combinatorial control and metabolic impact of these systems in diverse microbial chassis.

Main Methods:

  • Combined sigma70 (σ70) lac and tet expression systems on a single plasmid.
  • Assessed performance using fluorescent reporters, lycopene, and β-carotene production.
  • Incorporated toehold switches for translational control and enhanced gene expression regulation.
  • Utilized Escherichia coli, Pseudomonas putida, and Vibrio natriegens as microbial chassis.

Main Results:

  • Dual sigma70 (σ70) lac and tet expression systems demonstrated combinatorial control capabilities.
  • Toehold switches significantly improved dynamic range and basal transcriptional control.
  • Enhanced systems enabled accumulation of the biosynthetic intermediate lycopene.
  • Metabolic costs and synergistic effects were characterized across different microbial hosts.

Conclusions:

  • Developed versatile, portable genetic tools for controlling multigene pathways in gammaproteobacteria.
  • Enhanced sigma70 (σ70) expression systems with toehold switches facilitate enzyme and natural product biosynthesis.
  • These tools provide a valuable resource for microbial cell factory applications and natural product discovery.