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.
Highlights
Microbial production of free fatty acids (FFAs) has recently garnered much attention as a potential feedstock for renewable energy production (Handke et al, 2011; Lennen and Pfleger, 2012)
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-acyl carrier protein (ACP) for cell membrane biosynthesis
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
Summary
Microbial production of free fatty acids (FFAs) has recently garnered much attention as a potential feedstock for renewable energy production (Handke et al, 2011; Lennen and Pfleger, 2012). S. elongatus PCC 7942 was further modified by expression of an algal thioesterase, overexpression of ribulose1,5-bisphosphate carboxylase/oxygenase (RuBisCO), expression of a chloroplastic acetyl-CoA carboxylase, and improved gene expression using strong native promoters, yet these modifications did not improve the concentration of excreted FFAs in engineered S. elongatus PCC 7942 (Ruffing, 2013a). These initial studies of cyanobacterial FFA production illustrate the feasibility of FFA biosynthesis directly from CO2 and the difficulties associated with achieving high FFA yields using these cyanobacterial hosts
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