Increased sensitivity of heavy metal bioreporters in transporter deficient Synechocystis PCC6803 mutants

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni2+; Co2+ and Zn2+ detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium.

Advanced Metabolic Engineering Approaches and Renewable Energy to Improve Environmental Benefits of Algal Biofuels: LCA of Large-scale Biobutanol Production with Cyanobacteria Synechocystis PCC6803

With modern genetic engineering tools, microorganisms can become resilient green cell factories to produce sustainable biofuels directly. Compared to non-engineered algae and cyanobacteria, the photon conversion efficiency can be significantly increased. Furthermore, simplified harvesting processes are feasible since the novel microorganisms are excreting the biofuels or their precursors continuously and directly into the cultivation media. Along with higher productivity and direct product harvesting, it is expected that environmental benefits can be achieved, especially for climate protection. A life cycle assessment (LCA) for biobutanol production with the genetically engineered cyanobacteria Synechocystis PCC6803 is performed to test this hypothesis. A prospective and upscaled approach was applied to assess the environmental impacts at large-scale production (20 ha plant) for better comparability with conventional butanol production. The LCA results show that the engineering of microorganisms can improve the environmental impact, mainly due to the higher productivity compared to non-engineered cyanobacteria. However, the nevertheless high electricity demand required for the cultivation and harvesting process overcompensates this benefit. According to the scenario calculations, a more favourable climate gas balance can be achieved if renewable electricity is used. Then, greenhouse gas emissions are reduced to 3.1 kg CO2 eq/kg biobutanol, corresponding to 20% more than the fossil reference: (2.45 kg CO2 eq./kg 1-butanol). The results indicate the importance of genetic engineering and the energy transition towards renewable electricity supply to take full advantage of the environmental potential of microorganisms as future green cell factories for sustainable biofuel production. Besides, the necessity of developing different scenarios for perspective and upscaled LCA for a fairer comparison with mature reference technologies is demonstrated.

Differential Labeling of Chemically Modified Peptides and Lipids among Cyanobacteria Planktothrix and Microcystis

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labeling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labeled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labeling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed increased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labeling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labeled peptides was not increased suggesting a reduced spatial overlap.

Advanced Metabolic Engineering Approaches and Renewable Energy to Improve Environmental Benefits of Algal Biofuels: LCA of Large-Scale Biobutanol Production with Cyanobacteria Synechocystis PCC6803

Microalgae have the potential to serve as sustainable biocatalysts for direct sun-to-bioproduct approaches, e.g. for the synthesis of biofuels. Genetic engineered mutants with a higher photon conversion efficiency of sunlight into biofuels of interest are capable to serve as powerful green cell factories. These advanced metabolic engineering approaches are expected to have environmental benefits, especially for climate protection.The Life Cycle Assessment (LCA) on these novel technologies for the continuous production of algal biobutanol are based on unique and until now unpublished data from a pilot plant. Applying an upscaling approach the environmental impacts of algal biobutanol production at large-scale (20 ha plant) are presented for different scenarios. The results of the prospective LCA show that the higher productivity of the genetically engineered cyanobacteria Synechocystis PCC6803 and its specific feature of discharging the product biobutanol into the medium has a positive impact on the environment. However, electricity demand required for algae cultivation and product harvest overcompensates this advantage. The scenario calculations show that a positive climate gas balance can only be achieved if renewable energy is used.These results indicate the importance of genetic engineering and the energy transition for a fully renewable electricity supply to take full advantage of their environmental potential. Besides, the importance of applying upscaling approaches in LCA for a fairer comparison with mature reference technologies is demonstrated.