## 2 Short Chain Alcohols  
### Ethanol  
Some cyanobacteria naturally produce small amounts of ethanol under fermentative conditions [66]. However, genetic engineering was necessary to increase the productivity and enable production under photosynthetic conditions. Ethanol production from PCC 7942 [14] has been demonstrated as early as the late 20 th century. While the productivity (54 nmol/L/d/OD\({}_{730}\)) is relatively low, it is the first alcohol produced from genetically modified cyanobacteria. Ten years later, an ethanol production titer of 550 mg/L with a productivity of 5.2 mmol/L/d/OD\({}_{730}\) from PCC 6803 [12] was achieved using photobioreactor. In both PCC 7942 and PCC 6803, ethanol was produced using the pyruvate decarboxylation pathway from _Zymomonas mobilis_ (Figure 2), employing pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh). Pyruvate decarboxylation is an irreversible reaction and therefore provides a strong driving force for acetaldehyde formation. Decarboxylation is a good example of a powerful driving force for product formation because the gaseous CO\({}_{2}\) leaves the system, pulling the carbon flux towards the product. It is uncertain why the first ethanol report using PCC 7942 was significantly less successful than using PCC 6803. However, it is possible that the gene expression was more stable in the PCC 6803 study as the two genes were introduced into the genome rather than using a plasmid based system.  
To further increase the titer of ethanol production, additional optimization was carried out [11]. In this study, PCC 6803 endogenous alcohol dehydrogenase was expressed instead of that from _Z. mobilis_. In addition, the expression of multiple copies of the _pdc_ and _adh_ increased the gene dosage.  
Figure 2: Schematics of pyruvate derived bioproducts. Dashed lines represent multiple enzymatic steps. Evolution of CO\({}_{2}\) serves as effective driving force for product formation.  
_In situ_ ethanol removal using gas stripping further improved ethanol productivity. As ethanol is continuously removed from the culture, equilibrium further favors to push carbon flux towards ethanol. Through both genetic and cultivation optimizations, ethanol production reached a titer of 5.5 g/L in 26 days, representing the highest biochemical production titer demonstrated in the cyanobacterial engineering literature.  
Alternatively, ethanol can also be produced from acetyl-CoA (Figure 3). Ethanol production from acetyl-CoA is mediated by a bifunctional aldehyde/alcohol dehydrogenase AdhE in a reversible reaction. AdhE is known to be oxygen sensitive, and therefore its functional expression in cyanobacteria would be difficult. To bypass AdhE, the expression of separate oxygen tolerant CoA-acylating aldehyde dehydrogenase and alcohol dehydrogenase effectively converts acetyl-CoA to ethanol via acetaldehyde under aerobic conditions [13]. By expressing PduP, an oxygen tolerant CoA-acylating aldehyde dehydrogenase from the 1,2-propanediol degradation pathway from _Salmonella enterica_, and YqhD, a NADPH dependent alcohol dehydrogenase, PCC 7942 was engineered to produce ethanol from acetyl-CoA. The resulting strain produced 182 mg/L of ethanol in 10 days. It is worth noting that this pathway requires two reducing cofactors. While the consumption of two reducing cofactors drives the synthesis of ethanol, these results potentially indicate that decarboxylation serves as a stronger driving force for ethanol production.  
Figure 3: Schematics of acetyl-CoA derived bioproducts. Dashed lines represent multiple enzymatic steps.