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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 two cyanobacterial hosts: S. elongatus PCC 7942 and Synechococcus sp. PCC 7002. Wild-type strains are illustrated with black circles (7942 – open, 7002 – filled). Strains with gene knockout of the FFA-recycling acyl-ACP synthetase/long-chain-fatty-acid CoA ligase (aas/fadD) are illustrated with red squares (SE01 – open, S01 – filled). Strains with gene knockout of the FFA-recycling gene and ‘tesA expression are illustrated with blue triangles (SE02 – open, S02 – filled). 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 1: Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in two cyanobacterial hosts: S. elongatus PCC 7942 and Synechococcus sp. PCC 7002. Wild-type strains are illustrated with black circles (7942 – open, 7002 – filled). Strains with gene knockout of the FFA-recycling acyl-ACP synthetase/long-chain-fatty-acid CoA ligase (aas/fadD) are illustrated with red squares (SE01 – open, S01 – filled). Strains with gene knockout of the FFA-recycling gene and ‘tesA expression are illustrated with blue triangles (SE02 – open, S02 – filled). 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: Synechococcus sp. PCC 7002 was genetically engineered for FFA production and excretion by targeting the long-chain-fatty-acid CoA ligase (fadD, SYNPCC7002_A0675) for gene knockout and overexpression of a truncated thioesterase from E. coli (‘tesA). The resulting engineered strains, S01 (ΔfadD) and S02 (ΔfadD, ‘tesA+), are analogous to the previously constructed, FFA-producing strains of S. elongatus PCC 7942, SE01 (Δaas) and SE02 (Δaas, ‘tesA+). A schematic of the engineered pathway for FFA production can be found in the Supplemental Material (Figure S1). The FFA concentrations, cell growth profiles, and photosynthetic yields of Synechococcus sp. PCC 7002, S01, and S02 are shown in Figure 1, along with the previous results from S. elongatus PCC 7942, SE01, and SE02 (Ruffing and Jones, 2012). As expected, no FFAs are excreted by the wild-type (7002), yet both S01 and S02 synthesized and excreted FFAs. The FFA concentrations produced by fadD knockout in S01 (Figure 1A) are very low (<6 mg/L), particularly in comparison to the comparably engineered S. elongatus PCC 7942 strain, SE01, which produced up to 43 mg/L of excreted FFAs. Expression of the truncated E. coli thioesterase in S02, however, increased the level of excreted FFAs, producing concentrations similar to SE01 and higher levels than the analogous S. elongatus PCC 7942 strain, SE02 (Figure 1A). While the FFA-producing strains of Synechococcus sp. PCC 7002 (S01 and S02) did not show improved FFA production in comparison to the S. elongatus PCC 7942 strains (SE01 and SE02), there were notable differences in the physiological responses of the hosts. The late exponential growth rates of S01 and S02 showed a slight decrease in comparison to the wild-type 7002 after induction at 100 h (4.17 days) (Figure 1B); this small reduction is expected as FFA production reduces the available pool of acyl-ACP for cell membrane biosynthesis. On the other hand, SE01 and SE02 showed a severe reduction in final cell concentrations, 21 and 59% lower than wild-type (Figure 1B), much greater than expected due to FFA production. Additionally, the photosynthetic yields of S01 and S02 are very similar to the wild-type 7002, while the photosynthetic yields of SE01 and SE02 were significantly reduced compared to the wild-type 7942 strain (Figure 1C). These results suggest that FFA production in Synechococcus sp. PCC 7002 does not compromise the cellular physiology of the host strain, unlike the engineered strains of S. elongatus PCC 7942, which exhibited a stress response to FFA production and excretion.


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 two cyanobacterial hosts: S. elongatus PCC 7942 and Synechococcus sp. PCC 7002. Wild-type strains are illustrated with black circles (7942 – open, 7002 – filled). Strains with gene knockout of the FFA-recycling acyl-ACP synthetase/long-chain-fatty-acid CoA ligase (aas/fadD) are illustrated with red squares (SE01 – open, S01 – filled). Strains with gene knockout of the FFA-recycling gene and ‘tesA expression are illustrated with blue triangles (SE02 – open, S02 – filled). 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

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

Figure 1: Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in two cyanobacterial hosts: S. elongatus PCC 7942 and Synechococcus sp. PCC 7002. Wild-type strains are illustrated with black circles (7942 – open, 7002 – filled). Strains with gene knockout of the FFA-recycling acyl-ACP synthetase/long-chain-fatty-acid CoA ligase (aas/fadD) are illustrated with red squares (SE01 – open, S01 – filled). Strains with gene knockout of the FFA-recycling gene and ‘tesA expression are illustrated with blue triangles (SE02 – open, S02 – filled). 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: Synechococcus sp. PCC 7002 was genetically engineered for FFA production and excretion by targeting the long-chain-fatty-acid CoA ligase (fadD, SYNPCC7002_A0675) for gene knockout and overexpression of a truncated thioesterase from E. coli (‘tesA). The resulting engineered strains, S01 (ΔfadD) and S02 (ΔfadD, ‘tesA+), are analogous to the previously constructed, FFA-producing strains of S. elongatus PCC 7942, SE01 (Δaas) and SE02 (Δaas, ‘tesA+). A schematic of the engineered pathway for FFA production can be found in the Supplemental Material (Figure S1). The FFA concentrations, cell growth profiles, and photosynthetic yields of Synechococcus sp. PCC 7002, S01, and S02 are shown in Figure 1, along with the previous results from S. elongatus PCC 7942, SE01, and SE02 (Ruffing and Jones, 2012). As expected, no FFAs are excreted by the wild-type (7002), yet both S01 and S02 synthesized and excreted FFAs. The FFA concentrations produced by fadD knockout in S01 (Figure 1A) are very low (<6 mg/L), particularly in comparison to the comparably engineered S. elongatus PCC 7942 strain, SE01, which produced up to 43 mg/L of excreted FFAs. Expression of the truncated E. coli thioesterase in S02, however, increased the level of excreted FFAs, producing concentrations similar to SE01 and higher levels than the analogous S. elongatus PCC 7942 strain, SE02 (Figure 1A). While the FFA-producing strains of Synechococcus sp. PCC 7002 (S01 and S02) did not show improved FFA production in comparison to the S. elongatus PCC 7942 strains (SE01 and SE02), there were notable differences in the physiological responses of the hosts. The late exponential growth rates of S01 and S02 showed a slight decrease in comparison to the wild-type 7002 after induction at 100 h (4.17 days) (Figure 1B); this small reduction is expected as FFA production reduces the available pool of acyl-ACP for cell membrane biosynthesis. On the other hand, SE01 and SE02 showed a severe reduction in final cell concentrations, 21 and 59% lower than wild-type (Figure 1B), much greater than expected due to FFA production. Additionally, the photosynthetic yields of S01 and S02 are very similar to the wild-type 7002, while the photosynthetic yields of SE01 and SE02 were significantly reduced compared to the wild-type 7942 strain (Figure 1C). These results suggest that FFA production in Synechococcus sp. PCC 7002 does not compromise the cellular physiology of the host strain, unlike the engineered strains of S. elongatus PCC 7942, which exhibited a stress response to FFA production and excretion.

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