Ethanol production using heterocyst-forming cyanobacteria

Cyanobacteria are of great biotechnological interest for their potential to produce biofuels driven by oxygenic photosynthesis. In other words, you can make biofuels from sunlight, water, and CO₂. One approach is to produce ethanol: in order to do this a couple of enzymes need to be introduced using genetic engineering to metabolize pyruvate to ethanol. Arguably, the most successful of this is that by company Algenol were they introduced pyruvate decarboxylase and alcohol dehydrogenase from the alpha-proteobacterium Zymomonas mobilis into the marine unicellular cyanobacterium Synechococcus sp. PCC 7642. This approach was patented and in the patent it is said that rates of 1.7 µmoles of ethanol / mg chlorophyll-a / hour were obtained, which in my opinion is a very modest rate. Certainly, it is below 1% of the rates of Photosystem II activity under saturating light. Algenol is now producing 10000 gallons / acre / year, what appears to be a promising yield... and if the life cycle analysis they published is correct, the energy balance is positive: in other words, there is more energy in the ethanol produced than it was invested to drive the company and purify the ethanol.

Here I want to propose an alternative approach that I think will generate better yields. This is based on quantitative proteomic results in multicellular cyanobacteria capable of differentiating heterocysts. Under nitrogen starvation multicellular filamentous cyanobacteria differentiate 5-10% of their cells into a cell type specialized in atmospheric nitrogen fixation, nitrogen-fixing cells are called heterocysts. Heterocysts contain nitrogenase and other oxygen intolerant enzymes and for that reason photosynthetic oxygen evolution is inactivated or slowed down in the heterocysts. However, the surrounding cells are still capable of oxygenic photosynthesis and they transfer reductant to the heterocysts in exchange for fixed nitrogen in the form of glutamine.

A, Filamentous cyanobacteria, the arrow points to a heterocysts. B, Isolated heterocyts.

The proteomic work by Ow et al. (2009) indicated that heterocysts from Nostoc punctifurme contained very large amounts of the enzyme pyruvate kinase which uses phosphoenolpyruvate to generate ATP and pyruvate. In this study it was shown that pyruvate kinase was at least 3.8 times more abundant in the heterocysts compared to the vegetative cells. This predicts that heterocysts might have naturally higher concentrations of pyruvate. The reason why pyruvate is in higher concentrations in heterocysts is because it is the precursor of 2-oxoglutarate, which is the precursor of glutamate. In heterocysts glutamate reacts with ammonia (the product of atmospheric nitrogen reduction by nitrogenase) to produce glutamine.

My idea is to express pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas or Saccharomyces in the heterocysts of a multicellular cyanobacterium, such as the fresh water Nostoc sp. PCC 7120, Nostoc punctiforme or Anabaena variabilis or a marine version like Anabaena sp. 90. The technology to do this is already in existence. Since the metabolism of heterocysts is super-ramped up to provide nitrogen for 90-95% of the cells, I am pretty sure that the yields of ethanol could be pretty high.

It does not come without challenges because probably the diversion of pyruvate to generate ethanol might impair to certain extent nitrogen-fixation, however it has been shown that heterocysts can compensate a loss of reducing equivalents by boosting up cyclic photosynthesis and probably their own metabolism. It might be that a lack of pyruvate could enhance carbon-fixation, a bonus. In any case, we will not know if this is a sound idea until we try it out.