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Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942 - PubMed

. 2012 Sep;109(9):2190-9.

doi: 10.1002/bit.24509. Epub 2012 Apr 9.

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Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942

Anne M Ruffing et al. Biotechnol Bioeng. 2012 Sep.

Abstract

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.

Copyright © 2012 Wiley Periodicals, Inc.

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Conflict of interest statement

Conflict of Interest

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1

Simplified schematic of the metabolic pathways in S. elongatus 7942. The grey, dashed X indicates gene knockout of the acyl-ACP synthetase in SE01 and SE02, and the grey, dashed arrow indicates heterologous expression of the truncated thioesterase in SE02.

Figure 2
Figure 2

(A) FFA excreted on a dry cell weight basis; (B) cell growth profiles; (C) photosynthetic yield measurements. ● wild type (7942); □ SE01; ▲ SE02; ✗ wild type (7942) with the addition of 100 μM of linolenic acid at 100 h. Error bars represent the standard deviation of three biological replicates.

Figure 3
Figure 3

Normalized absorption spectra at 500 h for the wild type (7942) and engineered strains (SE01 and SE02) during FFA synthesis and for the wild type with addition of 100 μM of linolenic acid (LA).

Figure 4
Figure 4

Localization of photosynthetic pigments determined by hyperspectral confocal fluorescence imaging with samples collected at 350 h. Pure component spectra determined from MCR analysis (A). Relative concentration plots of allophycocyanin (APC), phycocyanin (PC), chlorophyll-a (Chl-a), and carotenoid. RGB images include the relative fluorescence intensities of APC (red), Chl-a (green), and PC (blue). The scale bar in each image is 5 μm.

Figure 4
Figure 4

Localization of photosynthetic pigments determined by hyperspectral confocal fluorescence imaging with samples collected at 350 h. Pure component spectra determined from MCR analysis (A). Relative concentration plots of allophycocyanin (APC), phycocyanin (PC), chlorophyll-a (Chl-a), and carotenoid. RGB images include the relative fluorescence intensities of APC (red), Chl-a (green), and PC (blue). The scale bar in each image is 5 μm.

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References

    1. Atsumi S, Higashide W, Liao JC. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotech. 2009;27(12):1177–1180. - PubMed
    1. Bielski BH, Arudi RL, Sutherland MW. A study of the reactivity of HO2/O2- with unsaturated fatty acids. Journal of Biological Chemistry. 1983;258(8):4759–61. - PubMed
    1. Campbell D, Hurry V, Clarke AK, Gustafsson P, Oquist G. Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation. Microbiology and Molecular Biology Reviews. 1998;62(3):667–683. - PMC - PubMed
    1. de Juan A, Maeder M, Hancewicz T, Tauler R. Use of local rank-based spatial information for resolution of spectroscopic images. Journal of Chemometrics. 2008;22:291–298.
    1. Deng M-D, Coleman JR. Ethanol synthesis by genetic engineering in cyanobacteria. Applied and Environmental Microbiology. 1999;65(2):523–528. - PMC - PubMed

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