Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803
© Gao et al; licensee BioMed Central Ltd. 2012
Received: 18 November 2011
Accepted: 21 March 2012
Published: 21 March 2012
Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.
Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.
Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.
KeywordsBiofuel Fatty alcohol Fatty alkane Cyanobacteria Synechocystis sp. PCC6803 Fatty acid activation
Biofuel production from renewable sources is considered as a feasible solution to the energy and environmental problems we are facing. It is very important to explore and develop advanced biofuels alongside traditional biofuels such as bioethanol and biodiesel to ensure sufficient supply of renewable energy at a time when demand for energy is set to increase over the coming decades. Advanced biofuels possess higher energy density, hydrophobic properties and compatibility with existing liquid fuel infrastructure including fuel engines, refinery equipment and transportation/distribution pipelines, whilst serving as better alternatives to fuels produced from fossil fuels .
In terms of fuel properties the best replacement of petroleum fuels is "Petroleum Fuels". This means ideal biofuels produced from biological systems should be chemically similar to petroleum, such as fatty acid-based molecules including fatty alcohols and fatty alkanes .
As a candidate for biofuel-producing microbial systems, cyanobacteria have become more and more attractive due to their specific characteristics as photosynthetic bacteria.
Compared to generally utilized biofuel-producing microbes such as E. coli and S.cerevisiae, cyanobacteria are photosynthetic microbes, which can convert solar energy and carbon dioxide more efficiently into biofuels in one biological system. In contrast to plants and eukaryotic algae, cyanobacteria are prokaryotic microbes with the ability to grow a lot faster. Genetic engineering platforms for cyanobacteria are well established and they are highly tolerable to heterogeneous genes. So far over 40 genomic sequences of cyanobacteria strains are available, therefore genetic information on cyanobacteria are relatively robust http://genome.kazusa.or.jp/cyanobase. This makes genetic engineering toward efficiently producing biofuels in cyanobacteria to be a more realistic and feasible option [3–5].
The fatty acid molecules must be activated to fatty acyl-thioesters by fatty acyl-CoA synthetase (ACS, EC 18.104.22.168) or fatty acyl-ACP synthetase (AAS, EC 22.214.171.124) prior to the synthesis of fatty alcohols and alkanes. Based on sequence identity analysis, Synechocystis sp. PCC 6803 encodes only a single candidate gene for fatty acid activation, annotated as AAS and designated as slr1609. The slr1609-deletion cyanobacteria mutant was incapable of utilizing exogenous fatty acids and thus secreted endogenous fatty acids into the medium. The detected free fatty acids are released from membrane lipids. The data suggest a remarkable turnover of lipids and a role of AAS activity in recycling the released fatty acids .
The overall pathway of the fatty acid, fatty alcohol and alkane in wild-type or engineered Synechocystis strains are illustrated in Figure 1. Synechocystis sp. PCC6803 mutant strains with either overexpression or deletion of slr1609 gene have been constructed in this study. The results indicated that the AAS gene was metabolically crucial for production of free fatty acids and fatty acid derivatives in Synechocystis sp. PCC6803.
Results and discussion
Construction of Synechocystis sp. PCC6803 mutants with either overexpression or deletion of slr1609 gene
The amount of free fatty acids can be doubled in the Synechocystis mutant strain with slr1609 knockout
As to the contents of the pool of free fatty acids, the amount of unsaturated fatty acids with carbon chain length of C16 and C18 was significantly higher in the slr1609 knockout mutant strain compared to the wild-type strain. Double bonds can only be introduced into free fatty acid coupled to the glycerol backbone of membrane lipids by acyl-lipid-type desaturases. Indicating that unsaturated free fatty acids being released from membrane lipids of senescent or damaged cells, while unsaturated free fatty acids in AAS deletion mutant can not be recycled and incorporated to membrane lipids.
In the mutant strain GQ3 with slr1609 over-expression, there is no significant change to the production of free fatty acids compared to the wild-type strain (data not shown). It has been confirmed that free fatty acids are released from membrane lipids in Synechocystis sp. PCC6803 . Indicating free fatty acid production is not only determined by the fatty acyl-ACP pool size, but also by the biosynthesis of membranes and hydrolysis of membrane lipids which are physiologically regulated.
The production of alkanes was significantly reduced in the slr1609 deletion mutant strain
The production of alkanes was not enhanced by the over-expressing slr1609 gene alone in the GQ3 strain (0.39 ± 0.03 μg/mL/OD)(Figure 6B). Due to the activities of downstream enzymes of the alkane producing pathway, AAR and ADC, are rather low and fatty acyl-ACPs might not be efficiently converted to alkanes . Fatty acyl-ACPs are also a supplier of fatty acyl groups for biosynthesis of lipid A , phospholipids , and membrane-derived lipo-polysaccharides .
Synechocystis AAS plays an important role in fatty alcohol production
Although the native jojoba FAR has a preference for very-long-chain acyl-CoA substrate (C20, C22 and C24), assays of jojoba extracts indicated that it is capable of reducing C16:0-ACP and C18:0-ACP . It's a reductase with broad substrate specificity. It may be possible that the acyl-ACP produced by AAS can also be accepted as substrate in addition to acyl-CoA by jojoba FAR in engineered Synechocystis strains. It is also possible that the acyl-ACPs, which are synthesized by Synechocystis AAS, could be in turn transacylated to acyl-CoAs by a reverse catalysis of acetyl-CoA-ACP-transacylase (EC 126.96.36.199) type reaction.
In this study the effects of fatty acid activation functionalized by a fatty acyl-ACP synthase on the production of fatty acid-based biofuels including fatty alcohols and alkanes in a photosynthetic cyanobacterium were evaluated and analyzed. We found fatty acid activation to be essential for efficient production of alkanes and plays a key role in manipulating fatty alcohol production. The results here provide promising clues for metabolically engineering cyanobacteria to improve photosynthetic production of fatty acid-based biofuels.
Chemicals and reagents
Pentadecanol, eicosane and nonadecanoic acid were obtained from Sigma-Aldrich (USA). Other chemicals were from Merck (Germany) or Ameresco (USA). Oligo nucleotides and gene synthesis were carried out by Sangon (Shanghai, China). Taq DNA polymerases and all restriction endonucleases were from Fermentas (Canada) or Takara (Japan). The DNA ladders were from Takara (Japan). The kits used for molecular cloning were from Omega (USA) or Takara (Japan).
Construction of Synechocystis sp. PCC683 mutant strains
All primers used in this study are listed in the Additional file 1.
The slr1609 gene was amplified from the genomic DNA of Synechocystis sp. PCC6803 with the primers 1609NdeI/1609R and subcloned into Nde I/Xho I site of the plasmid pET21b (Novagen, USA) to generate the plasmid pGQ7. The gene was cloned from pGQ7 with the primers 1609XbaI/1609DraI and subcloned into Xba I/Sma I site of the plasmid pFQ20  to generate the plasmid pGQ11.
The plasmid pXT68 was constructed based on the site of psb A2 gene. Both upstream and downstream fragments of psb A2 gene were cloned from the genomic DNA of Synechocystis sp PCC6803 with the primers Pd1-2-f/Pd1-2-r and pD1-2 d-1/pD1-2 d-2 respectively and inserted into the TA cloning site of pMD18-T, to generate the plasmids pXT25 and pXT59. The kanamycin resistance (kanr) gene (ck2) cassette was excised with Eco RV and Xba I from pRL446  and inserted into the Pst I site of pXT25 with blunt ends, to generate the plasmid pXT62. The 4.5 kb fragment containing ck 2 and upstream of psb A2 was excised with Xba I and Sph I from pXT62 and inserted into Xba I site of pXT59 with blunt ends, to generate the plasmid pXT68. Then, the slr1609 gene was excised with Nde I and DraIII (blunted end) from pGQ7 and inserted into the Nde I/Sal I (blunted end) site of pXT68, to generate the plasmid pGQ49.
Plasmids constructed and used in this study
Relevant characteristicsa, b
Apr, pET21b derivative containing slr1609gene, T7 promoter
Apr, pMD18-T derivative containing upstream fragment of psbA2, T7 promoter
Apr, pMD18-T derivative containing downstream fragment of psbA2, T7 promoter
Apr, Kanr, pMD18-T derivative containing upstream fragment of psbA2 and CK2
Apr, Kanr, pMD18-T derivative containing upstream and downstream fragments of psbA2, CK2 and sacB
Apr Sper, pKW1188SL derivative containing lacZ, Prbc promoter
Apr Sper , pFQ20 derivative containing slr1609, Prbc promoter
Kanr, pXT68 derivative containing slr1609, PpsbA2 promoter
Apr, pMD18-T derivative containing upstream fragment of slr1609, T7 promoter
Apr, pMD18-T derivative containing downstream fragment of slr1609, T7 promoter
Apr, Kanr, pMD18-T derivative containing upstream fragment of slr1609 and CK2
Apr, Kanr, pMD18-T derivative containing upstream and downstream fragments of slr1609 and CK2
Apr, Cmr, Emr, pMD18-T derivative containing upstream and downstream fragments of slr1609, CCEII and sacB
Sper, pFQ20 derivative containing FAR gene (jojoba), Prbc promoter
Strains constructed and used in this study
Synechocystis sp. PCC6803 Wild-type, Glucose-tolerance
Prof. Xu X.
slr0168∷omega Prbc far (jojoba), psbA2∷CK2 PpsbA2slr1609
slr0168∷ omega Prbc far (jojoba); slr1609 ∷CK2
slr0168::Omega Prbc far (jojoba) Trbc
Cultivation of Synechocystis sp. PCC683 strains
Liquid cultures of Synechocystis sp. PCC 6803 were grown photo-autotrophically in BG 11 media  at 30°C under constant illumination at a photosynthetic photon flux density of approximately 30 μmol photons m-2 s-1 and with aeration by sterile air or in a shaker. When necessary, the following antibiotics were added: erythromycin (20 μg mL-1) and spectinomycin (20 μg mL-1). Growth was monitored by following the OD at 730 nm. The Synechocystis sp. PCC6803 wild-type strain, the mutant strains GQ8 with deletion of the slr1609 gene and GQ3 with overexpression of the slr1609 gene were respectively grown in 100 mL Erlenmeyer flask containing 50 mL of BG11 medium in a shaker for free fatty acid analysis. The Synechocystis sp. PCC6803 wild-type strain, the mutant strains Syn-XT14 with overexpression of the FAR gene, GQ6 with deletion of the slr1609 gene and GQ5 with overexpression of the slr1609 gene were respectively grown in a 500 mL Erlenmeyer flask containing 300 mL of BG11 medium with aeration by sterile air for fatty alkane or fatty alcohol analysis. The Synechocystis sp. PCC6803 wild-type strain, the mutant strains GQ8 with deletion of the slr1609 gene and GQ3 with overexpression of the slr1609 gene were respectively grown in a 500 mL Erlenmeyer flask containing 300 mL of BG11 medium with aeration by sterile air for fatty alkane analysis.
Extraction and analysis of free fatty acids, fatty alkanes and fatty alcohols
For extraction of free fatty acids, 20 mL of the culture was lysed by sonication (total 30 min with 10 s on and 5 s off intervals) when the stationary phase (about 240 h) reached. To each 20 mL aliquot, 20 mL of 2:1 (v/v) CHCl3:CH3OH were added and the resulting mixture was mixed well . For GC-MS analysis of free fatty acids, 10 μg of nonadecanoic acid was added as the internal standard. A two-phase system (top: aqueous, bottom: organic) was generated after shaking for 1 h and centrifugation at 3000 rpm at room temperature for 5 min. The bottom organic phase was collected and concentrated under a stream of nitrogen at 55°C giving a residue that was resuspended in 600 μL of hexane. Aliquots of this mixture were analyzed by using GC-MS with an Agilent 7890A-5975 C system equipped with Agilent HP-INNOWax (30 m × 250 μm × 0.25 μm). Helium (constant flow 1 mL/min) was used as the carrier gas. The temperature of the injector was 250°C and the following temperature program was applied: 100°C for 1 min, increase of 5°C min-1 to 200°C then increase of 25°C min-1 to 240°C for 15 min.
Previous work in our lab showed that fatty alcohol and alkane can not be detected in relative culture media (data not shown). For extraction of fatty alkanes, Synechocystis cells at stationary phase (about 240 h) were harvested from 200 mL of culture by centrifugation. The cells were resuspended in 10 mL of TE buffer (pH8.0) and then lysed by sonication. The lysate added with 30 μg of eciosane as internal standard was extracted for 1 h at room temperature with 10 mL of 2:1 (v/v) CHCl3:CH3OH. The same following sample preparation and GC-MS analysis methods described above were used for fatty alkane analysis.
The same extraction methods described above for fatty alkane analysis were used for fatty alcohol, except adding 20 μg of 1-pentadecanol as the internal standard. The following temperature program was applied here: 50°C for 1 min, increase of 20°C min-1 to 180°C then increase of 10°C min-1 to 240°C for 20 min.
acyl carrier protein
fatty acyl-CoA reductase
fatty acid synthase
This work was supported by grants from the National Basic Research Program of China (973: 2011CBA00907), National Science Foundation of China (30970048), Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-G-1-4), Shell Research Limited (Grant 51010653-09-OS) and the "100-Talent Program of the Chinese Academy of Sciences" foundation (Grant O91001110A).
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