Dataset Information

Photosynthetic Conversion of CO₂ Into Pinene Using Engineered Synechococcus sp. PCC 7002.

ABSTRACT: Metabolic engineering of cyanobacteria has received much attention as a sustainable strategy to convert CO₂ to various longer carbon chain fuels. Pinene has become increasingly attractive since pinene dimers contain high volumetric energy and have been proposed to act as potential aircraft fuels. However, cyanobacteria cannot directly convert geranyl pyrophosphate into pinene due to the lack of endogenous pinene synthase. Herein, we integrated the gene encoding Abies grandis pinene synthase into the model cyanobacterium Synechococcus sp. PCC 7002 through homologous recombination. The genetically modified cyanobacteria achieved a pinene titer of 1.525 ± 0.l45 mg L^-1 in the lab-scale tube photobioreactor with CO₂ aeration. Specifically, the results showed a mixture of α- and β-pinene (∼33:67 ratio). The ratio of β-pinene in the product was significantly increased compared with that previously reported in the engineered Escherichia coli. Furthermore, we investigated the photoautotrophic growth performances of Synechococcus overlaid with different concentrations of dodecane. The work demonstrates that the engineered Synechococcus is a suitable potential platform for β-pinene production.

SUBMITTER: Yang R

PROVIDER: S-EPMC8718756 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundFuture sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms, which are engineered to synthesize valuable products directly from CO2 and sunlight. As cyanobacteria can be cultivated in large scale on non-arable land, these phototrophic bacteria have become attractive organisms for production of biofuels. Synechococcus sp. PCC 7002, one of the cyanobacterial model organisms, provides many attractive properties for biofuel production such as tolerance of seawater and high light intensities.ResultsHere, we performed a systems analysis of an engineered ethanol-producing strain of the cyanobacterium Synechococcus sp. PCC 7002, which was grown in artificial seawater medium over 30 days applying a 12:12 h day-night cycle. Biosynthesis of ethanol resulted in a final accumulation of 0.25% (v/v) ethanol, including ethanol lost due to evaporation. The cultivation experiment revealed three production phases. The highest production rate was observed in the initial phase when cells were actively growing. In phase II growth of the producer strain stopped, but ethanol production rate was still high. Phase III was characterized by a decrease of both ethanol production and optical density of the culture. Metabolomics revealed that the carbon drain due to ethanol diffusion from the cell resulted in the expected reduction of pyruvate-based intermediates. Carbon-saving strategies successfully compensated the decrease of central intermediates of carbon metabolism during the first phase of fermentation. However, during long-term ethanol production the producer strain showed clear indications of intracellular carbon limitation. Despite the decreased levels of glycolytic and tricarboxylic acid cycle intermediates, soluble sugars and even glycogen accumulated in the producer strain. The changes in carbon assimilation patterns are partly supported by proteome analysis, which detected decreased levels of many enzymes and also revealed the stress phenotype of ethanol-producing cells. Strategies towards improved ethanol production are discussed.ConclusionsSystems analysis of ethanol production in Synechococcus sp. PCC 7002 revealed initial compensation followed by increasing metabolic limitation due to excessive carbon drain from primary metabolism.

Project description:Successful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis. The cyanobacterium Synechococcus sp. PCC 7002 embodies key attributes for an industrial host, including a fast growth rate and high salt, light, and temperature tolerances. This study addresses key limitations in the advancement of Synechococcus sp. PCC 7002 as an industrial chassis.Tools for genome integration were developed and characterized, including several putative neutral sites for genome integration. The minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 was determined to be approximately 250 bp. Three fluorescent protein reporters (hGFP, Ypet, and mOrange) were characterized for gene expression, microscopy, and flow cytometry applications in Synechococcus sp. PCC 7002. Of these three proteins, the yellow fluorescent protein (Ypet) had the best optical properties for minimal interference with the native photosynthetic pigments and for detection using standard microscopy and flow cytometry optics. Twenty-five native promoters were characterized as tools for recombinant gene expression in Synechococcus sp. PCC 7002 based on previous RNA-seq results. This characterization included comparisons of protein and mRNA levels as well as expression under both continuous and diurnal light conditions. Promoters A2520 and A2579 were found to have strong expression in Synechococcus sp. PCC 7002 while promoters A1930, A1961, A2531, and A2813 had moderate expression. Promoters A2520 and A2813 showed more than twofold increases in gene expression under light conditions compared to dark, suggesting these promoters may be useful tools for engineering diurnal regulation.The genome integration, fluorescent protein, and promoter tools developed in this study will help to advance Synechococcus sp. PCC 7002 as a cyanobacterial chassis. The long minimum homology arm length for Synechococcus sp. PCC 7002 genome integration indicates native exonuclease activity or a low efficiency of homologous recombination. Low correlation between transcript and protein levels in Synechococcus sp. PCC 7002 suggests that transcriptomic data are poor selection criteria for promoter tool development. Lastly, the conventional strategy of using promoters from photosynthetic operons as strong promoter tools is debunked, as promoters from hypothetical proteins (A2520 and A2579) were found to have much higher expression levels.

Dataset Information

Photosynthetic Conversion of CO₂ Into Pinene Using Engineered Synechococcus sp. PCC 7002.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Photosynthetic Conversion of CO2 Into Pinene Using Engineered Synechococcus sp. PCC 7002.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Photosynthetic Conversion of CO₂ Into Pinene Using Engineered Synechococcus sp. PCC 7002.