bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.06.12.598700

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

Rincon, A. F. C.·Cabral, A. J.·Gyorgy, A.·Farny, N. G.
View original source ↗Export metadata ↓

Abstract

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

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Rincon, A. F. C., Cabral, A. J., Gyorgy, A., Farny, N. G.. 2024-06-12. A portable and dual-inducible control system for multistep biosynthetic pathways in Gram-negative bacteria. https://doi.org/10.1101/2024.06.12.598700

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Engineering phenotypic heterogeneity for functional organization in microbial populations

Engineering microbial populations to perform complex functions requires programming not only cellular behaviour but also the functional organization through which biological activities are distributed across populations. While phenotypic heterogeneity is often regarded as variability to suppress, it can also serve as a foundation for organizing specialized functions within genetically homogeneous microbial systems. Here, we introduce PROMETEO, a modular genetic-circuit framework that programs population composition through architecture-encoded regulatory and translational asymmetries. Using a library of 27 asymmetric bistable circuits, we demonstrate that circuit architecture reproducibly specifies phenotypic distributions spanning a broad range of population compositions without continuous external induction. Programmed population structures remained stable over serial propagation and were qualitatively conserved across Escherichia coli and Pseudomonas putida. Stochastic and deterministic modelling accurately predicted architecture-dependent population compositions and hysteresis regimes, providing a quantitative framework for rational design. We further show that programmable population composition supports multiple modes of functional organization, including stable parallel specialization, inducible temporal redistribution of cellular states, and spatial ecological compartmentalization through biofilm-associated and planktonic subpopulations. As a demonstration of these capabilities, architecture-programmed organization enabled distributed Congo Red biotransformation through coordinated reductive and oxidative activities, achieving up to 98% dye removal in spatially compartmentalized populations. Together, these results establish population composition as a programmable property of genetic circuit design and provide a general strategy for engineering distributed functions within genetically homogeneous microbial populations.

synthetic biology↗
bioRxiv2026

Carboxysome-Inspired Protein Coacervates for Light-Driven CO2 Reduction and H2 Evolution

Efficient catalysis often requires high local concentrations of reactants and catalysts, which cells achieve through compartmentalization within organelles, such as carboxysomes, that increase the efficiency of bacterial carbon fixation. Here, we engineered a photocatalytic reaction compartment that concentrated an artificial metalloenzyme, carbon dioxide, and a photosensitizer by liquid-liquid phase separation triggered by a cationic polypeptide, deca(L-arginine) (R10). At low R10 concentrations, CoPPIX binding increases the alpha-helical structure of the otherwise disordered protein, supercharged cytochrome b5622(-22). At higher concentrations, electrostatic complexation produces spherical droplets that enrich the protein and cobalt cofactor and recruit the photosensitizer [Ru(bpy)3]2+. Under illumination, coacervation increased hydrogen evolution 1.9-fold and CO formation from carbon dioxide; 1.3-fold relative to the corresponding solution-phase protein system. Co-encapsulation of carbonic anhydrase changed the product distribution specifically in the condensed phase: CO production increased 3.2-fold, hydrogen evolution decreased from 1.51 to 0.70 mol, and CO selectivity among the detected two-electron products rose from 33% to 77%. These results demonstrate that bioinspired coacervates can stabilize reactive intermediates, enrich local substrate concentrations, and integrate multiple catalytic functions, providing a generalizable framework for programmable, light-driven synthetic organelles.

synthetic biology↗
bioRxiv2026

Biocatalytic Production of Galantamine in Yeast through Cytochrome P450 Optimization

Galantamine is a pharmaceutically relevant Amaryllidaceae alkaloid used for the treatment of Alzheimer's disease. Its structural complexity and low abundance in plants motivate the development of alternative manufacturing routes. Here, we established the first engineered yeast platform for the biocatalytic production of galantamine from 4OMe-norbelladine. Heterologous expression of the downstream pathway enzymes NtCYP96T6, NtNMT1, and NtAKR1 from Narcissus cv. Tete-a-Tete in Saccharomyces cerevisiae was complemented by optimization of cultivation temperature, carbon source, and medium pH enabling the first demonstration of galantamine production in yeast. Systematic screening of cytochrome P450 reductase and cytochrome b partners to boost NtCYP96T6 activity led to the identification of a novel reductase mined from the Narcissus pseudonarcissus transcriptome, NpCPR, which supported the highest pathway flux and yielded 7.0 {+/-} 0.6 mg/L galantamine, corresponding to a 7.6 % molar yield from 250 M 4OMe-norbelladine and an approximately 173-fold improvement over the parental strain. We further exploited this yeast cell factory for the precursor-directed biosynthesis of 7F-galantamine, highlighting the potential of pathway enzyme promiscuity to access new-to-nature GAL analogues that may be challenging to produce through conventional chemical synthesis. Together, this work establishes a foundation for microbial galantamine production and biosynthetic diversification of its pharmaceutically relevant scaffold.

synthetic biology↗