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 [1
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 [2
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
, 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
Recently, the alkane biosynthetic pathway was identified in cyanobacteria with two enzyme families including an acyl carrier protein (ACP) reductase (AAR) and an aldehyde decarbonylase (ADC) [6
]. Genes associated with an alcohol-forming fatty acyl-CoA reductase (FAR) have not been reported in cyanobacteria, C16:0 and C18:0 alcohols can be produced by engineered cyanobacteria containing the FAR gene derived from jojoba [7
] or Arabidopsis thaliana
]. The overall pathway of the fatty acid, fatty alcohol and fatty alkane in wild-type or engineered Synechocystis
strains were illustrated in Figure .
Figure 1 The overall pathway of the fatty acid, fatty alcohol and fatty alkane in Synechocystis sp. PCC 6803. Dash arrow represents non-native and heterologously introduced pathway. ACP, acyl carrier protein; AAR, acyl-ACP reductase; ADC, aldehyde decarbonylase; (more ...)
The fatty acid molecules must be activated to fatty acyl-thioesters by fatty acyl-CoA synthetase (ACS, EC 126.96.36.199) or fatty acyl-ACP synthetase (AAS, EC 188.8.131.52) 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 [8
The overall pathway of the fatty acid, fatty alcohol and alkane in wild-type or engineered Synechocystis strains are illustrated in Figure . 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.