Abstract
Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.
Highlights
Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential
In E. coli, CRP binds to promoters containing specific binding sites when the concentration of cAMP is high, for example when glucose is scarce and other carbon sources must be metabolized for growth
The sequence of the CRP-binding site in Synechocystis differs by only one nucleotide to the CRP-binding site sequence found in the E. coli rhaBAD promoter, which suggests SYCRP1 might bind to this heterologous promoter sequence.[61,62]
Summary
Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. PCC 7002 have been successfully engineered to produce 2,3butanediol,[1,2] lactate,[3] isobutanol,[4] plant terpenoids[5] and ethanol,[6−9] and to allow the utilization of xylose.[10] Cyanobacteria, Synechocystis, are used as model organisms for fundamental studies of important processes such as photosynthesis,[11−16] circadian rhythms[17−19] and carbon-concentrating mechanisms.[20−23] Due to specific challenges, genetic modification of cyanobacteria is more difficult than genetic modification of model heterotrophic microorganisms such as Escherichia coli and Saccharomyces cerevisiae These challenges include polyploidy,[24,25] which makes the isolation of segregated recombinant strains slow and laborious, genetic instability of heterologous genes,[26] and limited synthetic biology tools and parts such as promoters and expression systems. One promising candidate is the Lrhamnose-inducible rhaBAD promoter system of E. coli, which naturally has almost all of the ideal properties described above.[53−55] Recently this system was optimized in E. coli by constitutive expression of the activating transcription factor RhaS, obviating the need to induce the native regulatory rhaSR operon; and identification of L-mannose as a nonmetabolisable inducer, allowing sustained expression rather than transient expression.[56]
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