Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803

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.

Zn 2+ -Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor

“…2B). These results are similar to those of Guerrero et al (13), who showed that the P smtA7002 promoter from Synechococcus sp. strain PCC 7002 was not controlled by the endogenous ziaR repressor of Synechocystis sp.…”

“…strain PCC 7002 was tested in Synechocystis sp. strain PCC 6803 for its ability to control ethylene production with similarly limited success (13).…”

Ethylene Regulates the Physiology of the Cyanobacterium Synechocystis sp. PCC 6803 via an Ethylene Receptor

“…Unlike plants and some bacteria, Synechocystis is unable to biosynthesize ethylene (Guerrero et al, 2012;Ungerer et al, 2012). Thus, it must encounter ethylene in the environment from nearby organisms that biosynthesize ethylene or from abiotic sources.…”

Enhancing photosynthetic production of ethylene in genetically engineered Synechocystis sp. PCC 6803

“…have been tested using a shuttle vector but failed to produce a high ethylene yield. 21 The E. coli-originated Trc promoter and Lac promoter variant (P A1lacO-1 ) were also investigated, and it was found that the control of the Lac-derived promoter became progressively more relaxed as the cyanobacterial culture grew denser. 21 Thus, it is important to select an appropriate promoter for effective EFE expression to achieve higher ethylene productivity.…”

“…21 The E. coli-originated Trc promoter and Lac promoter variant (P A1lacO-1 ) were also investigated, and it was found that the control of the Lac-derived promoter became progressively more relaxed as the cyanobacterial culture grew denser. 21 Thus, it is important to select an appropriate promoter for effective EFE expression to achieve higher ethylene productivity. Eight identified strong promoters, including PcpcB ( promoter of the cpcBAC operon, which encodes the protein phycocyanin 40 ), Prbc ( promoter of the rbc operon, which encodes ribulose-1,5-bisphosphate carboxylase/oxygenase 41 ) and PpsbD ( promoter of the psbD gene, which encodes the photosystem II D2 protein 42 ) from Synechocystis sp.…”