Limits...
RNA-Seq analysis and targeted mutagenesis for improved free fatty acid production in an engineered cyanobacterium.

Ruffing AM - Biotechnol Biofuels (2013)

Bottom Line: Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs.Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields.Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sandia National Laboratories, Department of Bioenergy and Defense Technologies, MS 1413, P,O, Box 5800, 87185-1413, Albuquerque, NM, USA. aruffin@sandia.gov.

ABSTRACT

Background: High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs.

Results: Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response.

Conclusions: A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.

No MeSH data available.


Related in: MedlinePlus

FFA production and physiological measurements for wild-type (7942) and FFA-producing strains (SE01 and SE02): (A) mg of FFA per gram of dry cell weight; (B) FFA concentration in mg/L; (C) percentage of cells staining positive for ROS; (D) percentage of cells with permeable membranes. Data are averages of 3 biological replicates with error bars indicating the standard deviation of these measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750487&req=5

Figure 1: FFA production and physiological measurements for wild-type (7942) and FFA-producing strains (SE01 and SE02): (A) mg of FFA per gram of dry cell weight; (B) FFA concentration in mg/L; (C) percentage of cells staining positive for ROS; (D) percentage of cells with permeable membranes. Data are averages of 3 biological replicates with error bars indicating the standard deviation of these measurements.

Mentions: ROS levels were measured in both the wild type and FFA-producing strains to determine whether these species were present for UFA degradation. As illustrated in Figure 1C, ROS generation in the wild type (7942) was negligible (< 2.56 ± 0.65%), yet both FFA-producing strains, SE01 and SE02, had intracellular ROS accumulation (up to 11.1 ± 2.3% and 58.1 ± 24%, respectively). The highest ROS levels were measured in SE02, with nearly 60% of the cell population staining positive for ROS at day 20. In general, the measurement of ROS-positive cells trended with FFA production (7942 < SE01 < SE02), and there was a statistically significant correlation between the amount of FFA on a per cell weight basis and the percentage of ROS-positive cells (R2 = 0.46, p = 0.0367) (Figure 1 A and C). However, the values of FFA concentration (mg/L) and the percentage of ROS-positive cells did not correlate (R2 = 0.13, p = 0.721) (Figure 1 B and C), suggesting that ROS generation is due to intracellular FFAs rather than extracellular. The accumulation of ROS during FFA production may indicate a potential for UFA degradation into toxic products, as described in the first proposed mechanism; however, ROS accumulation is also a hallmark of cellular stress [10]. Thus, we are presented with the ‘chicken or the egg’ dilemma, do the UFAs react with ROS to produce compounds that are toxic to the host cell, or do the FFAs themselves cause cellular stress which leads to ROS generation? The fact that there is no ROS accumulation in the wild type suggests that the latter hypothesis is correct.


RNA-Seq analysis and targeted mutagenesis for improved free fatty acid production in an engineered cyanobacterium.

Ruffing AM - Biotechnol Biofuels (2013)

FFA production and physiological measurements for wild-type (7942) and FFA-producing strains (SE01 and SE02): (A) mg of FFA per gram of dry cell weight; (B) FFA concentration in mg/L; (C) percentage of cells staining positive for ROS; (D) percentage of cells with permeable membranes. Data are averages of 3 biological replicates with error bars indicating the standard deviation of these measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750487&req=5

Figure 1: FFA production and physiological measurements for wild-type (7942) and FFA-producing strains (SE01 and SE02): (A) mg of FFA per gram of dry cell weight; (B) FFA concentration in mg/L; (C) percentage of cells staining positive for ROS; (D) percentage of cells with permeable membranes. Data are averages of 3 biological replicates with error bars indicating the standard deviation of these measurements.
Mentions: ROS levels were measured in both the wild type and FFA-producing strains to determine whether these species were present for UFA degradation. As illustrated in Figure 1C, ROS generation in the wild type (7942) was negligible (< 2.56 ± 0.65%), yet both FFA-producing strains, SE01 and SE02, had intracellular ROS accumulation (up to 11.1 ± 2.3% and 58.1 ± 24%, respectively). The highest ROS levels were measured in SE02, with nearly 60% of the cell population staining positive for ROS at day 20. In general, the measurement of ROS-positive cells trended with FFA production (7942 < SE01 < SE02), and there was a statistically significant correlation between the amount of FFA on a per cell weight basis and the percentage of ROS-positive cells (R2 = 0.46, p = 0.0367) (Figure 1 A and C). However, the values of FFA concentration (mg/L) and the percentage of ROS-positive cells did not correlate (R2 = 0.13, p = 0.721) (Figure 1 B and C), suggesting that ROS generation is due to intracellular FFAs rather than extracellular. The accumulation of ROS during FFA production may indicate a potential for UFA degradation into toxic products, as described in the first proposed mechanism; however, ROS accumulation is also a hallmark of cellular stress [10]. Thus, we are presented with the ‘chicken or the egg’ dilemma, do the UFAs react with ROS to produce compounds that are toxic to the host cell, or do the FFAs themselves cause cellular stress which leads to ROS generation? The fact that there is no ROS accumulation in the wild type suggests that the latter hypothesis is correct.

Bottom Line: Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs.Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields.Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sandia National Laboratories, Department of Bioenergy and Defense Technologies, MS 1413, P,O, Box 5800, 87185-1413, Albuquerque, NM, USA. aruffin@sandia.gov.

ABSTRACT

Background: High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs.

Results: Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response.

Conclusions: A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.

No MeSH data available.


Related in: MedlinePlus