Abstract
BackgroundSynechocystis sp. PCC 6803 is a photosynthetic bacterium that has been genetically modified to produce industrially relevant chemicals, yet efflux mechanisms have not been well elucidated. These photosynthetic organisms live in environments that are often nutrient limited; therefore, the genome of these organisms encodes far fewer proteins used for efflux of chemicals when compared to members of the Enterobacteriaceae family. Understanding efflux mechanisms can lead to a greater efficiency of chemical production within the cyanobacterial cell.ResultsBoth sll0180 and slr2131 genes that encode the Sll0180 and Slr2131 proteins, respectively, were removed from Synechocystis sp. PCC 6803 and SD277, a high fatty acid-producing Synechocystis-based strain, to test the hypothesis that Sll0180 and Slr2131 contribute to the efflux of chemicals out of Synechocystis sp. PCC 6803 and SD277. The mutant Synechocystis sp. PCC 6803 and SD277 strains with either sll0180 or slr2131 removed from the chromosome had significantly decreased half maximal inhibitory concentrations to various antibiotics. The free fatty acid (FFA) concentration of the SD277 mutant strains increased intracellularly yet decreased extracellularly indicating that Sll0180 and Slr2131 have a role in FFA efflux. E. coli wild-type gene acrA (a homolog to sll0180) was added on a plasmid to the respective mutant strains lacking the sll0180 gene. Similarly, the E. coli wild-type gene acrB (a homolog to slr2131) was added to the respective mutant strains lacking the slr2131 gene. The tolerance to chloramphenicol of each mutant strain containing the wild-type E. coli gene was restored when compared to the parent stains. The extracellular FFA concentration of SD277 Δslr2131 with E. coli acrB increased significantly compared to both SD277 and SD277 Δslr2131.ConclusionsTwo proteins involved in the transportation of antibiotics and FFAs out of the Synechocystis sp. PCC 6803 cell were identified. In an effort to alleviate costs associated with mechanically or chemically separating the cells from the FFAs, the combination of genome editing of SD277 and the addition of exogenous transport gene increased extracellular concentrations of FFAs. This understanding of active transportation is critical to improving the production efficiency for all industrially relevant chemicals produced in Synechocystis sp. PCC 6803.
Highlights
Sixteen strains based on either SD100 or SD277, the high free fatty acid (FFA) synthesizing strain, were created to investigate the roles of putative transporters in SD100 and SD277 and the ability to express homologous E. coli genes encoding the AcrA and AcrB proteins in both SD100 and SD277
The removal of slr2131 or sll0180 generally decreases tolerance to antibiotics After deleting either slr2131 or sll0180 from both SD100 and SD277, the maximal half inhibitory concentration (IC50) was determined when each strain was exposed to the antibiotics ampicillin (Amp), chloramphenicol (Cm), or Erythromycin (Ery) at concentrations between 10 ng/ mL and 100 mg/mL (Table 3)
When SD100 Δslr2131 was treated with Amp, there was no significant difference when compared to wild-type SD100
Summary
PCC 6803 is a photosynthetic bacterium that has been genetically modified to produce industrially relevant chemicals, yet efflux mechanisms have not been well elucidated. These photosynthetic organisms live in environments that are often nutrient limited; the genome of these organisms encodes far fewer proteins used for efflux of chemicals when compared to members of the Enterobacteriaceae family. PCC 6803 was shown to be naturally transformable [3] and later, able uptake plasmid DNA through conjugation [4]. The combination of these findings led to the use of Synechocystis sp.
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