Cyanobacteria has the capability to produce hydrocarbons such as alkane. Since the biosynthetic hydrocarbon is very similar chemically and structurally than the ones found in fossil fuels, it has the potential to be a replacement and be used as a Drop-in fuel.

The enzymes aldehyde deformylating oxygenase (ADO) and acyl-ACP reductase (AAR) in cyanobacteria have been confirmed by researchers to be responsible for catalyzing the production of bio-hydrocarbon in cyanobacteria. In order to optimize the expression of the two enzymes, expressions of other less important enzymes are inhibited. We do this by knocking out genes. Furthermore, by transforming the cyanobacteria with custom plasmids, production of branched alkane could increase significantly.


In an attempt to power the modern world more sustainably, in which we become less dependent on fossil fuels, we try to opt for biofuels. The very first biofuel humanity utilized was wood, long before the electricity was discovered [1]. The first generation of biofuel was produced from food crops, then cellulosic materials. Afterwards, there were lipids from algae. Recently, advanced biofuels could utilize garbage, animal waste, and even used cooking oils, which has been extremely useful to power households and communities [2]. Despite all of the possibilities, these biofuels alone would not be able to withstand the yearly consumption in aviation].

Since jet fuel is a mixture of large numbers of hydrocarbons, the International Air Transport Association has approved a mixture of 50/50 biofuels (also known as Drop ins) to conventional fossil-fuel, for commercial flights [3]. 


One of the components in jet fuel is alkanes [4]. Alkanes are more hydrophobic and have the tendency to carry less water than other Drop ins; such as ethanol or fatty acid methyl esters (FAME), which decrease the quality of the fuel as a whole and possibly are damaging to engines in the long-term. Nevertheless, long chain alkanes tend to precipitate due to hydrophobic interactions. A possible solution to this issue is hydrotreatment, similar to production of HEFA-SPK (Hydroprocessed Esters and Fatty Acids-Synthetic Paraffin Kerosene), a commonly used drop in. The HEFA-SPK pathway requires free fatty acids to be converted into alkane and eventually branched alkanes through hydrotreatment (i.e using heat and hydrogen as catalyst). By this hydrotreatment, alkane isoforms with more branches would be generated, thereby reducing the hydrophobic interactions in the final product [5].

Introduction of our concept

By genetically modifying a fast growing Cyanobacteria that naturally has the enzymes (ADO;aldehyde deformylating oxygenase and AAR; aceto-acyl reductase) to metabolize fatty-acyl ACP to produce alkanes [6], the first step of the HEFA pathway could be bypassed. This in theory, would not only save time but money. 

For this project, the cyanobacteria Synechococcus elongatus, specifically strain UTEX2973 is a perfect candidate. This specific strain has not only a fast growth rate, but the ability to take in plasmids very easily [7]. By genetically modifying this strain, a more carbon neutral alternative of biofuel could be produced, since cyanobacteria fixate atmospheric carbon dioxide.

The main idea of this project is to increase production of NADPH (an important cofactor to both enzymes AAR and ADO) in the bacteria, by knocking out genes. Increased levels of NADPH will increase AAR enzymatic activity. Unfortunately, ADO limits the rate of conversion of aldehyde to alkane, in the presence of oxygen and ubiquinone. As recent research shows that low levels of ROS (reactive oxygen species), especially H2O2 acts as an inhibitor to ADO, linking catalase to ADO would be a solution to increase the turnover of the enzyme [8]. In addition to that, ideally overexpression of NADPH dependent G6P dehydrogenase (G6PDH) encoded by zwf, ADO and AAR, by introduction of additional custom plasmids, encoding the three genes, would significantly increase the production of alkane. 

Proposed Methodology

To start, the genomic DNA of UTEX2973 would be isolated. Via reverse-PCR with specific primers, genes encoding the AAR, ADO and the zwf would be further isolated. These genes will be inserted into two different plasmid backbones. While the zwf will stand alone, the ADO and AAR would be inserted into a single plasmid. Each plasmid could then be amplified by E.coli transformation relying on its gene replication machinery. Once enough modified plasmids are accumulated, they would be used to transform the UTEX2973.

On the other hand, another batch of UTEX 2973 would have some of its genes knocked out, by using lambda red system, which uses a DNA sequence with homologous overhangs to the KO gene of interest to insert two antibiotic resistances. There are 7 genes to be knocked out; edd, ppsA, IdhA, aceA, pta, poxB, pflB. Mutants could then be selected by cultivating strain in antibiotic medium and eventually induce the elimination of resistance cassettes to achieve the desired knockouts, generating UTEX2973-T. 

After transforming the modified UTEX2973 with the two modified plasmids, strain should be the ideal strain to produce alkane. Once alkane is harvested, alkane will go through hydrotreatment to generate branched isoform.


Since UTEX2973 is a relatively new strain of cyanobacteria, some of the genes are not characterized yet (i.e. pathway holes). Database research for homologous genes in the cyanobacteria database in KEGG has come to a dead end. There seems to be limited to no gene homologs of edd, PoxB, pflB and  aceA.


  1. History of Biofuels – BioFuel Information. (n.d.). Biofuels. Retrieved September 14, 2022, from
  2. Biofuel. (n.d.). Wikipedia. Retrieved September 14, 2022, from
  3. Sustainable Aviation Fuels | EASA Eco. (n.d.). EASA. Retrieved September 14, 2022, from
  4. Jiménez-Díaz, L., Caballero, A., Pérez-Hernández, N., & Segura, A. (2017). Microbial alkane production for jet fuel industry: motivation, state of the art and perspectives. Microbial biotechnology, 10(1), 103–124. 
  5. Starck, L., Pidol, L., Jeuland, N., Chapus, T., Bogers, P., & Bauldreay, J. (2014). Production of Hydroprocessed Esters and Fatty Acids (HEFA) – Optimisation of Process Yield. Oil & Gas Science and Technology – Revue d’IFP Energies Nouvelles, 71(1), 10.
  6. Rahmana, Z., Sung, B. H., Yi, J. Y., Bui, l., Lee, J. H., & Kim, S. C. (2014). Enhanced production of n-alkanes in Escherichia coli by spatial organization of biosynthetic pathway enzymes. Journal of biotechnology, 192 Pt A, 187–191.
  7. Yu, J., Liberton, M., Cliften, P. et al.(2015). Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2. Sci Rep 5, 8132
  8. Andre, C., Kim, S. W., Yu, X.-H., & Shanklin, J. (2013). Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H 2 O 2 to the cosubstrate O 2. Proceedings of the National Academy of Sciences, 110(8), 3191-3196.