Cyanobacteria are predicted to be the first microorganisms to develop the capability of oxygenic photosynthesis, some 2.7 billion years ago [112]. Similar to algae, cyanobacteria have a great range of diverse morphologies, cellular functions, and genetics, presumably due to their long evolutionary history and their diverse habitats. As discussed previously, the ASP initially deemed cyanobacteria unfit for fuel production due to their lack of natural TAG accumulation. Since they are amenable to genetic manipulation, however, cyanobacteria can be engineered to produce a range of biofuel products (Table 1). As prokaryotes, cyanobacteria are subject to the traditional methods employed for engineering other well-developed bacterial hosts like E. coli. Some strains of cyanobacteria are even naturally transformable, uptaking exogenous DNA from their environment without the use of cell permeablization techniques [113]. As progenitors of the algal chloroplast, cyanobacteria also integrate DNA into their chromosomes using homologous recombination. Moreover, cyanobacteria do not possess the cellular components for gene silencing. The genetic tools for engineering some model strains of cyanobacteria are well developed and have been used to genetically modify cyanobacteria for several decades [113]. Another advantage of using cyanobacteria as the microbial host for hydrocarbon-based fuel production is that they have been shown to excrete potential fuel precursors such as FFAs [73]. Fuel excretion enables a continuous production process, eliminating the cost associated with harvesting the algal biomass and the time and nutrients needed to repeatedly grow new batches of algae for fuel production. The advantages of straightforward genetic manipulation and fuel excretion make cyanobacteria contenders for large-scale biofuel production despite the disadvantage of low natural lipid yields.

After the initial demonstration of engineering cyanobacteria for ethanol production [97], the production of hydrocarbon-based fuels in engineered cyanobacteria has expanded to include isoprene, FFAs, FAEEs, fatty alcohols, and alkanes/alkenes (Table 1). Isoprene biosynthesis was established in the model cyanobacterium, Synechocystis sp. PCC 6803, through expression of the isoprene synthase (ispS) from kudzu [78]. Codon optimization of ispS and the use of a strong promoter (psbA2) increased isoprene production. Engineering strategies targeting the upstream MEP pathway for isoprenoid biosynthesis, as described in Section 2.2 of this chapter, will likely further improve isoprene productivity. The remaining four hydrocarbon-based fuels are all derived from the fatty acid biosynthesis pathway. Common strategies for im­proving FFA production (see Section 2.1) have proven successful in cyanobacteria [74—76]. Eliminating non-essential, competing pathways such as polyhydroxybutyrate (PHB), cyano — phycin, and acetate biosynthesis also improved FFA production [74]. Liu and colleagues engineered a more permeable peptidoglycan layer to improve FFA excretion in Synechocystis sp., yet this weakened cell membrane resulted in slower growth rates and may also make the engineered cyanobacterium more susceptible to external predators and toxins that may be present in large-scale cultivations. Initial engineering attempts for fatty alcohol and alkane/ alkene production entail expression of a heterologous FAR and overexpression of AAR and ADC, respectively [23, 26]. Alkane/alkene synthesis was also observed with ACC overexpres­sion and native AAR and ADC activities in cyanobacteria [23]. Despite being derived from fatty acids, the synthesis of fatty alcohols and alkanes/alkenes is up to 1000-fold lower than that observed with FFA production (Table 1), suggesting that the conversion of acyl-ACP to the final fuel product is rate limiting. These inaugural proof-of-concept reports illustrate the potential of cyanobacteria as hosts for autotrophic biofuel production, but additional metabolic engineering will be required to achieve the fuel titers necessary for large-scale synthesis.