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The global increase of transportation fuels and the depleting reserves for petroleum has urged the need to develop alternatives to the existing fossil fuels. Biofuels serves as an alternative to solve the problem of fossil based fuels for gasoline, diesel and jet engines, etc. Although focus has been on developing biomass derived gasoline fuels such as ethanol and other short chain alcohols, the heavy transportation sector and aviation markets require high energy distillates as fuels (3).
Hydrocarbons are important group of heavy transportation fuels because of its properties such as high energy density, physical and combustion properties. To meet the demand of the finite reserves of transportation fuels, alternatives to sources of hydrocarbon are required. Fatty acids are produced in high amounts in the cell membrane of most microbial species and can be used to produce hydrocarbons (2). These fatty acids can be reduced to an aldehyde followed by decarbonylation or can be decarboxylated to produce long chain alkanes. Reports by Steen et al have confirmed that fatty acids could also be enzymatically converted to biofuels such as alkanes, fatty alcohols, fatty acid ethyl esters and olefins.
Currently, the most convenient and cost-effective approach for large-scale production of advanced biofuels is by engineering of microorganisms. Rapid engineering of microbial biosynthetic pathways can be done by molecular and synthetic biology to produce advanced biofuel such as alcohols, esters, alkanes and alkenes (8). Escherichia coliis an ideal organism for biodiesel production given its known genetics and the wealth of information available on its fatty acid biosynthesis and metabolism. Wild-type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and accounts for 9.7% of lipid (10). The excess of fatty acids produced are rapidly degraded by Î²- oxidation pathway. It has a short doubling time reaching a high cell density using inexpensive substrates. It also has the ability to metabolize C5 sugars (such as Xylose, arabinose, etc.) and C6 sugars (such as glucose, etc.) and is a large toolkit for genetic manipulation. This increases its likelihood for commercialization of microbial produced commodities (1,6,7).
The fatty acid production can be increased by targeting the genes involved in the metabolic pathway of E. coli. This can be done by site directed mutagenesis which involves knocking out the genes involved in fatty acid metabolism pathway leading to accumulation of free fatty acids. Also, over expressing the genes involved in fatty acid biosynthesis pathway. The theoretical maximum carbon yield is 0.29 to 0.35 g fatty acid per g glucose. According to Lu et al, the deletion of fatty acid dehydrogenase gene fadD in E.coli has shown to increase the yield of fatty acid production to 255nmol/ml over wild type strain (81nmol/ml). The deletion of fadE gene along with over expression of thioesterase gave 1.2 g/L fatty acids with 14% of the theoretical yield(9). Liu et al, engineered a strain of E. coli that produced 4.5 g/L of fatty acid by introducing four genetic changes reaching 20% of the theoretical maximum(4).
Several approaches have been carried out to reduce the acetate flux and increase the acetyl CoA pool. Deletion of the acetate kinase (ackA) and phosphotransacetylase (pta) and alcohol dehydrogenase (adhE) genes involved in the competitive pathways has shown an increase in fatty acid accumulation. The amount of fatty acid accumulated depends on the source of acyl ACP thioesterase. Several modifications such as over expression of acyl carrier protein (ACP) thioesterase gene from Diploknemabutyracea, Gossypiumhirsutum(11), Ricinuscommunis(11), Jatrophacurcas(11), Umbellulariacalifornica (BTE) (3),Cinnamonumcamphorum (5)has shown an increase in fatty acid production. The fatty acid strain overexpressing accas well as C. camphorumacyl-ACP thioesterases along with deletion of fadE gene under fed batch fermentation conditions produced about 2.5 g/L of fatty acids.
Efforts are being directed to commercialize the production of fatty acids. Identifying metabolic bottlenecks, targeting the genes by site directed mutagenesis, deleting the side pathways so as to divert all the acetyl CoA produced in fatty acid biosynthesis pathway and designing optimal cultivation strategies for producing free fatty acids from renewable carbon sources are the targets for improving yields.
The following will lay the basis of the proposed work of research for the production of fatty acids. E.coli is an ideal organism for fatty acid production based on its known genetics and metabolic pathways. Various approaches can be targeted for the production of these fatty acids which has its uses as biofuels. These involve deletion or knockout of the genes involved in the metabolic pathway of fatty acid and/ or overexpression of the genes involved in fatty acid biosynthesis pathway. The above two strategies or a combination of these would be used to yield a modified strain of E. coli capable of producing fatty acid with maximum yield.
AIMS AND OBJECTIVES
The potential for free fatty acid production in E coli can further be increased by the following approaches:
Genetic modifications by knocking out the genes involved in fatty acid metabolism
Overexpression of the enzymes involved in fatty acid biosynthesis.
Random mutagenesis for improving the desired strain.
In order of the work proposed the following methodology will be used:
Selection of a suitable wild type strain of E. coli.
Genetic modification of the selected strain of E. coli by:
Site directed mutagenesis: Knocking out the genes involved in Î²-oxidation pathway.
Over expression of the enzymes involved in fatty acid biosynthesis pathway.
Targeting the competing pathways involved in production of other by products.
To optimize the growth conditions for E. coli along with media engineering to improve the yields of the mutant strain.
Use of gas chromatography mass spectrometry (GCMS) to identify the profile of fatty acids.
The proposed work will develop a strain of E. coli that produces free fatty acids with improved yields.
WORK DONE TILL DATE
Wild type strains of E. coli were screened for the production of free fatty acids under normal conditions of growth. A suitable E.coli strain was then chosen that produced maximum free fatty acids under normal growth conditions. Genetic modifications by site directed mutagenesis were carried out on the strain and the fatty acid yields were observed and quantitated by Gas Chromatography Mass Spectroscopy.