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1.
Nucleic Acids Res ; 50(22): 12790-12808, 2022 12 09.
Article in English | MEDLINE | ID: mdl-36533444

ABSTRACT

In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5'/3' gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.


Subject(s)
DNA Topoisomerases , Promoter Regions, Genetic , Synechocystis , Cell Division/genetics , Plasmids/genetics , Promoter Regions, Genetic/genetics , Synechocystis/enzymology , Synechocystis/genetics , Transcriptional Activation , DNA Topoisomerases/genetics , DNA Topoisomerases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism
2.
Methods Mol Biol ; 2379: 67-79, 2022.
Article in English | MEDLINE | ID: mdl-35188656

ABSTRACT

Genetic engineering of cyanobacteria is currently limited to genomic integration via homologous recombination and RSF1010-based conjugative vector systems. Here, we introduce a rationally designed conjugative vector with two BioBrick-based cloning sites which enables facilitated and modular cloning. This streamlined vector is suitable for a variety of synthetic biology applications, such as expression of multiple enzymes from metabolic pathways for the production of biofuels or secondary metabolites, or screening of modular parts such as promoters, further facilitating applications to improve crop plants using synthetic biology. Finally, we present a general approach to cloning of constructs, as well as detailed protocols for conjugation and culturing of strains carrying said constructs.


Subject(s)
Cyanobacteria , Genetic Vectors , Cyanobacteria/genetics , Genetic Engineering/methods , Genetic Vectors/genetics , Metabolic Engineering/methods , Plasmids , Synthetic Biology/methods
3.
Metab Eng Commun ; 13: e00178, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34466381

ABSTRACT

Cyanobacteria are extremely adaptable, fast-growing, solar-powered cell factories that, like plants, are able to convert carbon dioxide into sugar and oxygen and thereby produce a large number of important compounds. Due to their unique phototrophy-associated physiological properties, i.e. naturally occurring isoprenoid metabolic pathway, they represent a highly promising platform for terpenoid biosynthesis. Here, we implemented a carefully devised engineering strategy to boost the biosynthesis of commercially attractive plant sequiterpenes, in particular valencene. Sesquiterpenes are a diverse group of bioactive metabolites, mainly produced in higher plants, but with often low concentrations and expensive downstream extraction. In this work we successfully demonstrate a multi-component engineering approach towards the photosynthetic production of valencene in the cyanobacterium Synechocystis sp. PCC 6803. First, we improved the flux towards valencene by markerless genomic deletions of shc and sqs. Secondly, we downregulated the formation of carotenoids, which are essential for viability of the cell, using CRISPRi on crtE. Finally, we intended to increase the spatial proximity of the two enzymes, ispA and CnVS, involved in valencene formation by creating an operon construct, as well as a fusion protein. Combining the most successful strategies resulted in a valencene production of 19 mg/g DCW in Synechocystis. In this work, we have devised a useful platform for future engineering steps.

4.
ACS Synth Biol ; 9(4): 843-855, 2020 04 17.
Article in English | MEDLINE | ID: mdl-32134640

ABSTRACT

Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli, the available variety of tunable promoter parts for phototrophic cyanobacteria is still limited. Commonly used synthetic and semisynthetic promoters show weak dynamic ranges or no regulation at all in cyanobacterial models. Well-controlled alternatives such as native metal-responsive promoters, however, pose the problems of inducer toxicity and lacking orthogonality. Here, we present the comparative assessment of dose-response functions of four different inducible promoter systems in the model cyanobacterium Synechocystis sp. PCC 6803. Using the novel bimodular reporter plasmid pSHDY, dose-response dynamics of the re-established vanillate-inducible promoter PvanCC was compared to the previously described rhamnose-inducible Prha, the anhydrotetracycline-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages regarding dynamic range and strength of each promoter, also in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in cyanobacteria, facilitating the scalability and robustness of synthetic regulatory network designs and of complex metabolic pathway engineering strategies.


Subject(s)
Gene Expression Regulation, Bacterial/genetics , Metabolic Engineering/methods , Promoter Regions, Genetic/genetics , Synechocystis/genetics , Synthetic Biology/methods , Plasmids/genetics , Synechocystis/metabolism , Vanillic Acid/metabolism
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