Abstract
Short and well defined promoters are essential for advancing cyanobacterial biotechnology. The heterocyst of Nostoc sp. is suggested as a microbial cell factory for oxygen sensitive catalysts, such as hydrogenases for hydrogen production, due to its microoxic environment. We identified and predicted promoter elements of possible significance through a consensus strategy using a pool of heterocyst-induced DIF+ promoters known from Anabaena sp. PCC 7120. To test if these conserved promoter elements were crucial for heterocyst-specific expression, promoter-yfp reporter constructs were designed. The characterization was accomplished by replacing, -35 and -10 regions and the upstream element, with well described elements from the trc promoter of Escherichia coli, which is also functional in Nostoc sp. From the in vivo spatial fluorescence of the different promoter-yfp reporters in Nostoc punctiforme ATCC 29133, we concluded that both the consensus -35 and extended -10 regions were important for heterocyst-specific expression. Further that the promoter strength could be improved by the addition of an upstream element. We designed a short synthetic promoter of 48 nucleotides, PsynDIF, including a consensus DIF1 sequence, a 17 base pair stretch of random nucleotides and an extended consensus -10 region, and thus generated the shortest promoter for heterocyst-specific expression to date.
Highlights
Cyanobacteria are promising as platforms for biological production of fuels and other chemicals
We propose that the conserved heterocyst enhanced expression pattern from the DIF+ class promoters should be an effect of conserved regions in the promoter sequences
In this work we use consensus sequences of core promoter elements to build a minimal synthetic promoter for heterocyst-specific expression. This promoter gives an approximate 10 time increase in expression 12 hours after the start of heterocyst differentiation. This well characterized promoter expands the potential for cyanobacterial biotechnology and is of special importance for expression of oxygen intolerant enzymes in a photosynthetic host organism
Summary
Cyanobacteria are promising as platforms for biological production of fuels and other chemicals. Their fast growing photosynthetic nature makes them ideal from a sustainable production perspective. Within the vast and diverse cyanobacterial phylum, heterocyst forming cyanobacteria is a group of fascinating multicellular photosynthetic organisms that are able to differentiate a subset of their cells into specialized compartments dedicated to fixation of atmospheric nitrogen, called heterocysts. The interior of the heterocyst is kept micro-aerobic to let the oxygen sensitive nitrogenase, the enzyme-complex responsible for the nitrogen fixation, operate. The development of the nitrogen fixing cells is a complex process where environmental stimuli.
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