Identification of Two Genes Required for Long-Chain Alkane Production in Anabaena 7120

Oil reserves worldwide are limited, and as prices have risen, renewable fuels have become increasingly important.  Is there a biofactory that can convert carbon dioxide, water, and sunlight into fuels?  Yes, several species of cyanobacteria are known to produce alkanes and alkenes from carbon dioxide, water and sunlight. These liquid hydrocarbons are the major component of oil. This provides a more sustainable approach to producing biofuels than using grains or biomass that could compete for food. However, native cyanobacteria do not produce these hydrocarbons in sufficient yield for commercial deployment. This research is to understand how cyanobacteria photosynthetically produce fuels using only atmospheric gases and water. Two putative alkane genes in Anabaena 7120 were knocked out by inserting a gfp-spec cassette within the genes. The engineered genetic sequence in an integration plasmid was homologously integrated into the chromosome of Anabaena 7120. Through a double crossover, the functional alkane genes were replaced by the inactivated gene segment. The GC/MS data revealed that heptadecane (C₁₇H₃₆) produced by the wildtype was not found in the two knock-out  mutants, suggesting that one or both of the alkane genes that were inactivated are part of a biosynthetic pathway for the production of heptadecane.  The complementation experiment with reintroduction of the functional genes into the two mutants confirmed the role of these two genes.