Abstract
The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter Ptrc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, PA1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.
Highlights
Tractable cyanobacteria are gaining attention as a host for the direct photosynthetic conversion of sunlight and CO2 into chemical energy
Given the reported genetic instability of Synechococcus elongatus PCC 7942 transformed with ethylene-forming enzyme (EFE) [3], we first confirmed that the addition of ethylene, at a level sufficient to influence a signaling event (1% (v/ v) ethylene), to the headspace of closed cultures of Synechocystis did not influence the growth and absorption spectra (400–750 nm) over a 3 day period
The addition of 10 mM iron tripled the rate of ethylene synthesis in E. coli, no such effect was observed in cyanobacteria
Summary
Tractable cyanobacteria are gaining attention as a host for the direct photosynthetic conversion of sunlight and CO2 into chemical energy. The complete genome of .50 cyanobacteria species is available and comprehensive stoichiometric reconstructions have been developed [9]. Until now there are few reports of comprehensive metabolic engineering of multi-step pathways [8,10]. In order to enable economically sustainable biological conversion of solar energy, H2O and CO2 into fuel it is necessary to engineer the catalytic hosts for the intended biotechnological purpose. This requires a molecular toolbox for metabolic engineering including promoters for user-regulated protein expression
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