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Abstract
Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.
Highlights
Cyanobacteria are a promising platform for production of renewable chemicals and fuels
Cyanobacterial replicons E. coli origin for knockout plasmids E. coli origin to be assembled with a cyanobacterial replicon
Standard biological parts and assembly schemes (e.g. BioBrick and BglBrick) have been designed for the construction of synthetic genetic systems. These systems are often centered on protein expression and operate in only a few widely used organisms such as E. coli, B. subtilis, or yeast
Summary
Cyanobacteria are a promising platform for production of renewable chemicals and fuels. Extensive genetic tools have been developed for a select group of cyanobacterial strains, including autonomously replicating vectors, integration sites, selection markers, reporter genes and promoters [1,2]. These molecular tools were originally developed to study fundamental cellular processes, but there is an increasing interest in using cyanobacteria as cell factories for the production of biomolecules. Several strains have already been genetically engineered to produce fuel molecules, including ethanol, isobutyraldehyde, isobutanol, 2,3-butanediol and free fatty acids [3,4,5] These experiments were carried out in a few genetically manipulable model strains, primarily Synechococcus elongatus PCC7942 and Synechocystis sp. Depending on the growth conditions and the products to be made, different production strains and compatible wellsuited advanced genetic tools will be needed
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