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Improved Free Fatty Acid Production in Cyanobacteria with Synechococcus sp. PCC 7002 as Host.

Ruffing AM - Front Bioeng Biotechnol (2014)

Bottom Line: PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology.PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures.Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioenergy and Defense Technologies, Sandia National Laboratories , Albuquerque, NM , USA.

ABSTRACT
Microbial free fatty acids (FFAs) have been proposed as a potential feedstock for renewable energy. The ability to directly convert carbon dioxide into FFAs makes cyanobacteria ideal hosts for renewable FFA production. Previous metabolic engineering efforts using the cyanobacterial hosts Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have demonstrated this direct conversion of carbon dioxide into FFAs; however, FFA yields in these hosts are limited by the negative impact of FFA production on the host cell physiology. This work investigates the use of Synechococcus sp. PCC 7002 as a cyanobacterial host for FFA production. In comparison to S. elongatus PCC 7942, Synechococcus sp. PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology. The enhanced tolerance to FFA production with Synechococcus sp. PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures. Additional genetic manipulations were targeted for increased FFA production, including thioesterases and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L. This work illustrates the importance of host strain selection for cyanobacterial biofuel production and demonstrates that the FFA tolerance of Synechococcus sp. PCC 7002 can allow for high yields of excreted FFA.

No MeSH data available.


Related in: MedlinePlus

Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in Synechococcus sp. PCC 7002 strains at two temperatures: 30°C (solid lines, filled markers) and 38°C (dashed lines, open markers). Addition of IPTG is indicated by the arrows (100 h). All data are averages of at least three biological replicates with error bars indicating the standard deviation.
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Figure 2: Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in Synechococcus sp. PCC 7002 strains at two temperatures: 30°C (solid lines, filled markers) and 38°C (dashed lines, open markers). Addition of IPTG is indicated by the arrows (100 h). All data are averages of at least three biological replicates with error bars indicating the standard deviation.

Mentions: The FFA production experiments described in the previous section and the results reported in Figure 1 were conducted at a temperature of 30°C for comparison with the engineered S. elongatus PCC 7942 strains, which have an optimal growth temperature of 30°C (Golden and Sherman, 1984). However, the reported optimal growth temperature for Synechococcus sp. PCC 7002 is 38°C (Sakamoto and Bryant, 1997). Therefore, FFA production experiments were also conducted at this higher temperature for 7002, S01, and S02 (Figure 2). As expected, the higher cultivation temperature yielded a slight improvement in FFA concentrations excreted by S01 and S02 (Figure 2A), likely due to the increased thermal kinetics associated with the higher temperature. Unexpectedly, cell growth was reduced at the higher temperature for all three strains, including the wild-type (Figure 2B). To confirm the culture temperature, the aqueous temperature of the culture vessels was measured using a standard glass thermometer. The aqueous temperature readings indicated that the culture temperatures were slightly higher than the setpoints, 32°C for the 30°C setting and 39°C for the 38°C setting. The lower range light intensities used in this experiment (60 μmol photons m−2 s−1) may also influence the optimal growth temperature, and a more rigorous, multi-factorial investigation of Synechococcus sp. PCC 7002 growth is required to evaluate the optimal growth temperature of this strain. In addition to this unexpected growth response, there were significant changes in photosynthetic yields at 38°C in comparison to 30°C. At the higher temperature, the wild-type (7002) had reduced photosynthetic yields with values up to 50% lower compared to the 30°C temperature condition (Figure 2C). The FFA-producing strains showed an additional decrease in photosynthetic yield, with S02, the highest FFA producer, having the most reduced photosynthetic yields. To investigate whether these reduced photosynthetic yields were accompanied by changes in photosynthetic pigments, the absorbance spectra of the wild-type (7002) and engineered strains (S01 and S02) were measured for cultures at 38 and 30°C after significantly reduced photosynthetic yields were detected (day 16). While the absorbance spectrum for the wild-type (7002) did not show significant differences at the two temperatures, the spectrum of the S02 cultures showed reduced peaks for both chlorophyll-a (680 nm) and the phycobiliproteins (635 nm) at 38°C (Figure 3). The reduced growth, photosynthetic yields, and photosynthetic pigments that accompany FFA production at 38°C suggest that the FFA tolerance of Synechococcus sp. PCC 7002 is temperature-dependent.


Improved Free Fatty Acid Production in Cyanobacteria with Synechococcus sp. PCC 7002 as Host.

Ruffing AM - Front Bioeng Biotechnol (2014)

Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in Synechococcus sp. PCC 7002 strains at two temperatures: 30°C (solid lines, filled markers) and 38°C (dashed lines, open markers). Addition of IPTG is indicated by the arrows (100 h). All data are averages of at least three biological replicates with error bars indicating the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126656&req=5

Figure 2: Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in Synechococcus sp. PCC 7002 strains at two temperatures: 30°C (solid lines, filled markers) and 38°C (dashed lines, open markers). Addition of IPTG is indicated by the arrows (100 h). All data are averages of at least three biological replicates with error bars indicating the standard deviation.
Mentions: The FFA production experiments described in the previous section and the results reported in Figure 1 were conducted at a temperature of 30°C for comparison with the engineered S. elongatus PCC 7942 strains, which have an optimal growth temperature of 30°C (Golden and Sherman, 1984). However, the reported optimal growth temperature for Synechococcus sp. PCC 7002 is 38°C (Sakamoto and Bryant, 1997). Therefore, FFA production experiments were also conducted at this higher temperature for 7002, S01, and S02 (Figure 2). As expected, the higher cultivation temperature yielded a slight improvement in FFA concentrations excreted by S01 and S02 (Figure 2A), likely due to the increased thermal kinetics associated with the higher temperature. Unexpectedly, cell growth was reduced at the higher temperature for all three strains, including the wild-type (Figure 2B). To confirm the culture temperature, the aqueous temperature of the culture vessels was measured using a standard glass thermometer. The aqueous temperature readings indicated that the culture temperatures were slightly higher than the setpoints, 32°C for the 30°C setting and 39°C for the 38°C setting. The lower range light intensities used in this experiment (60 μmol photons m−2 s−1) may also influence the optimal growth temperature, and a more rigorous, multi-factorial investigation of Synechococcus sp. PCC 7002 growth is required to evaluate the optimal growth temperature of this strain. In addition to this unexpected growth response, there were significant changes in photosynthetic yields at 38°C in comparison to 30°C. At the higher temperature, the wild-type (7002) had reduced photosynthetic yields with values up to 50% lower compared to the 30°C temperature condition (Figure 2C). The FFA-producing strains showed an additional decrease in photosynthetic yield, with S02, the highest FFA producer, having the most reduced photosynthetic yields. To investigate whether these reduced photosynthetic yields were accompanied by changes in photosynthetic pigments, the absorbance spectra of the wild-type (7002) and engineered strains (S01 and S02) were measured for cultures at 38 and 30°C after significantly reduced photosynthetic yields were detected (day 16). While the absorbance spectrum for the wild-type (7002) did not show significant differences at the two temperatures, the spectrum of the S02 cultures showed reduced peaks for both chlorophyll-a (680 nm) and the phycobiliproteins (635 nm) at 38°C (Figure 3). The reduced growth, photosynthetic yields, and photosynthetic pigments that accompany FFA production at 38°C suggest that the FFA tolerance of Synechococcus sp. PCC 7002 is temperature-dependent.

Bottom Line: PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology.PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures.Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioenergy and Defense Technologies, Sandia National Laboratories , Albuquerque, NM , USA.

ABSTRACT
Microbial free fatty acids (FFAs) have been proposed as a potential feedstock for renewable energy. The ability to directly convert carbon dioxide into FFAs makes cyanobacteria ideal hosts for renewable FFA production. Previous metabolic engineering efforts using the cyanobacterial hosts Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have demonstrated this direct conversion of carbon dioxide into FFAs; however, FFA yields in these hosts are limited by the negative impact of FFA production on the host cell physiology. This work investigates the use of Synechococcus sp. PCC 7002 as a cyanobacterial host for FFA production. In comparison to S. elongatus PCC 7942, Synechococcus sp. PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology. The enhanced tolerance to FFA production with Synechococcus sp. PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures. Additional genetic manipulations were targeted for increased FFA production, including thioesterases and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L. This work illustrates the importance of host strain selection for cyanobacterial biofuel production and demonstrates that the FFA tolerance of Synechococcus sp. PCC 7002 can allow for high yields of excreted FFA.

No MeSH data available.


Related in: MedlinePlus