Abstract
Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than those of model heterotrophic organisms, and the suite of well-characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and to ‘insulate’ genes and operons from one another. Insulating gene expression can be particularly important when heterologous or synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium, Synechocystis sp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally–integrated, rhamnose-inducible rhaBAD expression system from hypothesised ‘read-through’ from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.
Highlights
Cyanobacteria are important photosynthetic model organisms and potential photosynthetic microbial factories
We recently described the successful introduction of a rhamnose-inducible rhaBAD expression system into the cyanobacterium Synechocystis sp
Rho-independent terminators were identified from the literature including twelve strong, natural terminators from E. coli [8], four synthetic terminators that showed excellent transcriptional termination in E. coli [8], one putative cyanobacterial terminator [11] and T21 and M13 bacteriophage terminators [12,13] (Table 1)
Summary
Cyanobacteria are important photosynthetic model organisms and potential photosynthetic microbial factories. Predictable engineering of cyanobacteria remains challenging for many reasons, one of which is a shortage of well-characterised genetic parts such as promoters, ribosome-binding sites and transcriptional terminators [1,2]. This expression system has superior properties to many other previously reported inducible promoter systems, including low basal expression in the absence of inducer, a photostable and non-toxic inducer and a linear response to inducer concentration [3]. When introduced into the Synechocystis chromosome adjacent to the ndhB gene, we observed a non-zero basal level of expression, which we attributed to transcriptional read-through from promoter(s) neighbouring the integration site used. Chromosomal integration is important for the stability of expression constructs, but in cases where extremely low or zero basal expression is required, transcriptional read-through can result in unpredictable gene expression, growth defects, toxicity and genetic instability
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