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Metabolic phenotyping of the cyanobacterium Synechocystis 6803 engineered for production of alkanes and free fatty acids

Author

Listed:
  • Hu, Ping
  • Borglin, Sharon
  • Kamennaya, Nina A.
  • Chen, Liang
  • Park, Hanwool
  • Mahoney, Laura
  • Kijac, Aleksandra
  • Shan, George
  • Chavarría, Krystle L.
  • Zhang, Chunmin
  • Quinn, Nigel W.T.
  • Wemmer, David
  • Holman, Hoi-Ying
  • Jansson, Christer

Abstract

We demonstrate a simple high-throughput single-cell approach that exploits the ultrahigh brightness and non-invasive nature of synchrotron infrared beam to characterize strains of the cyanobacterium Synechocystis 6803 (S. 6803) constructed with altered metabolic traits affecting the acyl-CoA pool. Their metabolic responses to the modified traits were phenotyped by single-cell synchrotron radiation Fourier transform infrared (SR-FTIR) spectromicroscopy and multivariate analysis. SR-FTIR difference spectra and cluster vector plots segregated the strains as phenotypic populations based on signals in the hydrocarbon and biomolecular fingerprint regions, although each population incorporated a stochastic distribution of cells with different metabolic properties. All engineered strains exhibited an increase in FTIR features attributed to functional groups in hydrocarbon, fatty acid (FA), and/or FA ester chains, and a decrease in polysaccharide features. The metabolic signatures obtained by SR-FTIR were consistent with detailed qualitative and quantitative metabolic information provided in GC/MS/NMR data. A strain with extra copies of the FAR and FAD genes, encoding, respectively, the fatty acyl-ACP reductase and fatty aldehyde decarbonylase enzymes in the alkane biosynthesis pathway, showed up to a fivefold increase in the intracellular levels of heptadecane, a threefold increase in 9-heptadecene, and a significant increase in secreted 16:0 and 18:0 free FAs (FFAs). Inactivation of the AAS gene, encoding acyl-ACP synthetase, prevented re-thioesterification of FFAs generated from membrane lipid recycling and led to elevated levels and of intracellular FFAs of an altered composition, and a decrease in heptadecane and secreted FFAs. Introduction of a FatB gene, encoding a thioesterase (TE), which catalyzes the liberation of FFAs from acyl-ACP, yielded little effect in itself. However, the activity of the TE enzyme was clearly manifested in combination with AAS inactivation; A TE-containing train lacking AAS showed a dramatic (30-fold) increase in intracellular FFAs (with the majority being 16:0) and increases in heptadecane and secreted FFAs.

Suggested Citation

  • Hu, Ping & Borglin, Sharon & Kamennaya, Nina A. & Chen, Liang & Park, Hanwool & Mahoney, Laura & Kijac, Aleksandra & Shan, George & Chavarría, Krystle L. & Zhang, Chunmin & Quinn, Nigel W.T. & Wemmer,, 2013. "Metabolic phenotyping of the cyanobacterium Synechocystis 6803 engineered for production of alkanes and free fatty acids," Applied Energy, Elsevier, vol. 102(C), pages 850-859.
    • Handle: RePEc:eee:appene:v:102:y:2013:i:c:p:850-859
    DOI: 10.1016/j.apenergy.2012.08.047
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    1. Christer Jansson, 2012. "Employing Cyanobacteria for Biofuel Synthesis and CCS," Chapters, in: Radu Dan Rugescu (ed.), Solar Power, IntechOpen.
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    Cited by:

    1. Liu, Aiqiu & Zhu, Tao & Lu, Xuefeng & Song, Lirong, 2013. "Hydrocarbon profiles and phylogenetic analyses of diversified cyanobacterial species," Applied Energy, Elsevier, vol. 111(C), pages 383-393.

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