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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 engineered Synechococcus sp. PCC 7002 strains. The inset image in plot A shows FFA precipitation and floatation (white precipitate) in S07 cultures after 20 days. All data are averages of at least three biological replicates with error bars indicating the standard deviation.
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Figure 4: Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in engineered Synechococcus sp. PCC 7002 strains. The inset image in plot A shows FFA precipitation and floatation (white precipitate) in S07 cultures after 20 days. All data are averages of at least three biological replicates with error bars indicating the standard deviation.

Mentions: To determine if higher yields of FFA can be produced in Synechococcus sp. PCC 7002 compared to S. elongatus PCC 7942, additional strategies for improving FFA biosynthesis were investigated. The acyl-ACP thioesterase from C. reinhardtii (fat1) was previously shown to have activity similar to ‘tesA in S. elongatus PCC 7942 (Ruffing, 2013a). Therefore, fat1 was expressed in Synechococcus sp. PCC 7002, along with fadD knockout, to form strain S03. Unexpectedly, S03 showed only a slight increase in excreted FFA concentration compared to S01, which has only the fadD knockout (Figure 4A). The fat1 gene was previously cloned from the associated C. reinhardtii mRNA transcript (Ruffing, 2013a), which likely includes a chloroplast-targeting signal for translocation of Fat1, a nuclear-encoded protein, into the chloroplast. Inclusion of the chloroplast-targeting signal may lead to either low enzyme activity or secretion of the enzyme, as the cyanobacterial membrane is evolutionarily similar to the chloroplast membrane (Giovannoni et al., 1988). To eliminate this potential source of low activity, a putative chloroplast-targeting signal was predicted using ChloroP 1.1 (Emanuelsson et al., 1999), and a new forward primer was designed to exclude this 5′ signal sequence, yielding a truncated fat1 (tfat1). Expression of tfat1 in S05 led to a nearly threefold increase FFA production and excretion compared to the fat1-expressing S03 strain (Figure 4A). However, the amount of FFA produced by S05 was still lower than that produced by the ‘tesA-expressing S02 strain. Additionally, S05 had lower growth and photosynthetic yield compared to S02 (Figures 4B,C); therefore, further strain development utilized the ‘tesA thioesterase rather than fat1 or tfat1 acyl-ACP thioesterases.


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 engineered Synechococcus sp. PCC 7002 strains. The inset image in plot A shows FFA precipitation and floatation (white precipitate) in S07 cultures after 20 days. 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 4: Comparison of extracellular FFA concentration (A), cell concentration (B), and photosynthetic yields (C) during FFA production in engineered Synechococcus sp. PCC 7002 strains. The inset image in plot A shows FFA precipitation and floatation (white precipitate) in S07 cultures after 20 days. All data are averages of at least three biological replicates with error bars indicating the standard deviation.
Mentions: To determine if higher yields of FFA can be produced in Synechococcus sp. PCC 7002 compared to S. elongatus PCC 7942, additional strategies for improving FFA biosynthesis were investigated. The acyl-ACP thioesterase from C. reinhardtii (fat1) was previously shown to have activity similar to ‘tesA in S. elongatus PCC 7942 (Ruffing, 2013a). Therefore, fat1 was expressed in Synechococcus sp. PCC 7002, along with fadD knockout, to form strain S03. Unexpectedly, S03 showed only a slight increase in excreted FFA concentration compared to S01, which has only the fadD knockout (Figure 4A). The fat1 gene was previously cloned from the associated C. reinhardtii mRNA transcript (Ruffing, 2013a), which likely includes a chloroplast-targeting signal for translocation of Fat1, a nuclear-encoded protein, into the chloroplast. Inclusion of the chloroplast-targeting signal may lead to either low enzyme activity or secretion of the enzyme, as the cyanobacterial membrane is evolutionarily similar to the chloroplast membrane (Giovannoni et al., 1988). To eliminate this potential source of low activity, a putative chloroplast-targeting signal was predicted using ChloroP 1.1 (Emanuelsson et al., 1999), and a new forward primer was designed to exclude this 5′ signal sequence, yielding a truncated fat1 (tfat1). Expression of tfat1 in S05 led to a nearly threefold increase FFA production and excretion compared to the fat1-expressing S03 strain (Figure 4A). However, the amount of FFA produced by S05 was still lower than that produced by the ‘tesA-expressing S02 strain. Additionally, S05 had lower growth and photosynthetic yield compared to S02 (Figures 4B,C); therefore, further strain development utilized the ‘tesA thioesterase rather than fat1 or tfat1 acyl-ACP thioesterases.

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