Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

Abstract

Cyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942, Leptolyngbya sp. strain BL0902, Anabaena sp. strain PCC 7120, and Synechocystis sp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-␤-D-thiogalactopyranoside induction of a lacI q -P trc promoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.

A tightly inducible riboswitch system in <i>Synechocystis</i> sp. PCC 6803

“…It extends previous work on the applicability of this riboswitch in cyanobacteria (Ma et al, 2014;Nakahira et al, 2013). In addition, our data imply an issue about this system: the effect of the sequence around the riboswitch.…”

“…PCC 7120, Leptolyngbya sp. BL0902 and Synechocystis WHSyn (Ma et al, 2014;Nakahira et al, 2013). Here, we describe the construction of a conjugative expression vector using a theophylline-responsive riboswitch and examine the resulting expression levels and its regulation using a green fluorescence protein (GFP) reporter in Synechocystis.…”

NOT Gate Genetic Circuits to Control Gene Expression in Cyanobacteria

“…Therefore, we used riboswitches B and C, which have high induction ratios but very low levels of basal expression in the absence of theophylline. 21 …”

“…19 A set of synthetic theophylline-responsive riboswitches that control translation, originally developed and evaluated in eight different bacterial species by Topp et al, 18 have since been shown to provide robust control of gene expression in several phylogenetically diverse cyanobacteria. 20–21 …”

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

“…high-light-responsive p sbA promoter, copper-regulated p etE promoter, nitrate/nitrite-inducible P nirA promoter and the nickel-regulated nrsA promoter) have been assessed in cyanobacteria, induction of these regulatory systems could concurrently affect the endogenous cognate regulon and result in unwanted effects [1, 31, 32]. To evaluate appropriate promoters useful for driving CRISPRi in PCC 7942, we compared different inducible promoters (P smt , P LtetO1 , P ConII-ribo , P trc , P LlacO1 and P BAD ) derived from E. coli or cyanobacteria (Additional file 1: Figure S5).…”