ABSTRACT Cyanobacteria have garnered interest as promising biological platforms for producing renewable biofuel, chemical feedstock, and bioactive molecules. For biotechnology applications, robust, well-characterized genetic tools are required for genetically modifying cyanobacteria, but these tools are often developed for specific model strains. Here, we used broad host-range RSF1010-based plasmids to characterize a set of orthogonal constitutive promoters in diverse cyanobacterial strains. The promoters are random variants of the synthetic Escherichia coli PconII promoter. A library of PconII promoters driving a fluorescent reporter gene was first evaluated in Synechococcus elongatus and found to have a wide range of gene expression levels. A set of 25 promoter variants with graded strengths was selected after characterization in S. elongatus and three additional model cyanobacterial strains. To demonstrate the utility of these promoters, we isolated new genetically tractable cyanobacterial strains with high salt and alkalinity tolerance, and transferred the subset of promoters into one of these newly isolated strains. Similar to the results with model strains, the subset of promoters had a wide range of expression levels in the non-model strain. These characterized promoters expand the genetic tools available for genetic engineering of model and non-model cyanobacterial strains. IMPORTANCE The use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications. The use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications.
Results identify P SD13 as a phage-derived promoter exhibiting strong activity in both Streptomyces and E. coli, suggesting its potential as a useful genetic element for Streptomyces engineering and heterologous gene expression.
Nana Lu· Applied Microbiology and Bio...· 0 citations
Predictable control of gene expression is essential for building genetic circuits and improving metabolic pathways, but conventional promoter libraries often behave unpredictably when genes are combined. Here we develop CRISPR-Activated Promoter-based Orthogonal expression (CAPO), a quantitative platform for controlling multiple genes in yeast. CAPO uses synthetic CRISPR-activated promoters that remain silent until matching guide RNAs recruit dCas9-VPR. We tune each gene by varying guide RNA abundance with defined T7 promoters, while keeping regulatory channels orthogonal. CAPO reaches expression levels comparable to strong native yeast promoters, maintains low background activity, and preserves promoter-strength order across different genes. We apply CAPO to program broad fluorescence color outputs and to rapidly optimize lycopene and 3-hydroxypropionic acid biosynthesis. These results establish CAPO as a scalable platform for predictable engineering of eukaryotic gene networks. Efficient bioproduction using eukaryotes, such as engineered Saccharomyces cerevisiae, requires precise control over gene expression. Here, authors develop CAPO, a CRISPR-guided system that tunes gene activity in yeast and enables multiplex colour generation and faster optimization of metabolic pathways.
Cyanobacteria offer a sustainable and environmentally friendly method of light-driven biosynthesis of chemicals with minimal environmental impact, making them an extremely attractive proposition in alleviating society’s dependency on fossil fuels. To fully exploit cyanobacteria as a chassis to meet society’s demands for energy and chemical production, genetic modification is a crucial step in improving productivity through the expression of heterologous and novel biosynthetic pathways. Genome mining was carried out with the aim of identifying potential recE and recT homologues to improve the efficiency of genetic manipulation. Two promising candidate homologues of recT and cas4 (recE homologue) were identified from the cyanobacterium Aphanocapsa feldmanni. Expression of these candidates increased recombination efficiency 3-fold, generating mutants with the expected genotypes and phenotypes, which were comparable to mutants created with conventional knockout protocols using suicide vectors. These results indicate that cyanobacteria possess recombination homologues that can be re-purposed to improve the efficiency of genome engineering to accelerate cyanobacteria for strain development.
Sean Craig, Daniel Green, Christopher A. Martin et al.· SynBio· 0 citations
This review systematically summarizes the features of Bacillus subtilis expression systems, the regulatory effects of various promoters on heterologous expression efficiency, and rational modification strategies of promoter core regions, offering valuable references for the rational development of high-efficiency promoter engineering strategies and heterologous gene expression optimization.
Man-Fei Li, Meng Zheng, Peng-Yuan Wang et al.· Journal of Microbiological M...· 0 citations
This work provides well-characterized K. phaffii strong promoter candidates, supporting future biotechnological applications and identifying and characterize promoters from the most highly transcribed endogenous genes across diverse culture conditions as strong promoter candidates to expand the genetic toolbox for this yeast.
Karla B. Fernández-Cano, J. M. Viader-Salvadó, M. Guerrero-Olazarán· PeerJ· 0 citations
Acetogenic bacteria are promising platforms for converting gaseous and liquid one-carbon (C1) substrates such as carbon monoxide, carbon dioxide (CO2), formate and methanol into fuels and chemicals, but their application is limited by sparse genetic tools and poor control over metabolic fluxes. Here, we developed a modular plasmid assembly platform and a suite of characterized native, heterologous and methanol-inducible promoters for the model acetogen Acetobacterium woodii, enabling rapid and tunable control of gene expression together with CRISPR-based genome editing. Using an anaerobe-compatible fluorescent reporter (pFAST), promoter strength was quantified, from medium to high expression levels. Subsequently, a minimal promoter replacement at the native adhE (bifunctional aldehyde-alcohol dehydrogenase) locus along with deletion of the neighboring LysR-type regulator was done employing the newly characterized promoters, demonstrating that a single promoter replacement in the genome was sufficient to shift A. woodii from a primarily acetogenic toward a partly ethanologenic phenotype. The engineered strain was able to produce ethanol not only from fructose but also from the C1 substrates methanol + CO2 and formate + syngas. Together with the promoter replacement strategy, these findings underline that ethanol formation in A. woodii is constrained both by energy conservation and by endogenous transcriptional control. Relieving energetic bottlenecks through the choice of energetically favorable C1 substrates and decoupling adhE expression from its native promoter suffices to enable ethanol production from methanol + CO2 and formate + syngas, directly linking the Wood-Ljungdahl pathway to ethanol formation. These results illustrate how small, targeted genome edits can significantly reshape carbon flux in acetogens and positions A. woodii as a viable chassis for C1-based bioproduction.
Renaud Eynard, Sephora Justine, Rémi Hocq et al.· Biotechnology for Biofuels a...· 0 citations