In addition to improving hydrocarbon-based fuel synthesis, metabolic engineering strategies can also be applied to address other factors affecting large-scale production. Two main issues will be addressed in this section: product toxicity and industrial strain robustness.

Product toxicity was shown to be a limiting factor in the production of first generation biofuels like ethanol. Since the interest in hydrocarbon-based fuels has developed only during the past decade, the toxicities of these fuels have not been fully explored, particularly with respect to autotrophic hosts. Fortunately, interest in hydrocarbon inhibition of microbial growth dates back almost a century [118], and we can capitalize on this wealth of information to engineer improved product tolerance in microbial hosts. Most fatty acid derived fuel molecules have shown some antimicrobial activity. FFAs, with a diverse range of carbon chain lengths and degrees of unsaturation, impart inhibitory effects on organisms including algae, Gram­negative and Gram-positive bacteria, fungi, protozoans, and various cell types of multicellular organisms [119]. Medium chain fatty alcohols such as pentanol, hexanol, heptanol, and octanol inhibited the biological activity of several algal and cyanobacterial strains, including fuel­relevant hosts C. reinhardtii and Dunaliella salina [120]. Interestingly, long-chain fatty alcohols (>C14) did not exhibit inhibitory effects on yeasts, suggesting that targeting longer chain fatty alcohols may eliminate the toxicity concern [121]. Similarly, medium-length alkanes (hexane, heptane, and isooctane) were toxic to microalgae while long-chain alkanes (C12-C16) elicited no effect [120, 121]. Microbial TAG and FAEE toxicities have not been reported. However, the phospholipid membrane surrounding algal TAGs may mask potential inhibitory effects, and FAEE production has been linked to the toxic effects of alcohol consumption in humans [122]. Isoprenoid-based fuel molecules have also illustrated inhibitory effects. Cyclic terpenes, such as pinene and limonene (Figure 2), inhibited the growth of bacteria and S. cerevisiae [123, 124], while branched isoprenoids, such as farnesyl hexanoate and geranyl acetate, were shown to be toxic to E. coli [125]. In fact, E. coli’s tolerance to isoprenoid-derived biodiesels and bioavi­ation fuels only ranged from 0.025 — 1% (v/v) [125]. Based on these previous studies, product toxicity is a major limiting factor and should be integrated into the metabolic engineering strategy.

A variety of strategies can be adopted to address product toxicity. The easiest way to avoid complications from product toxicity is to select non-toxic fuel targets. Toxicity studies can be conducted for each potential host organism, and generally, fatty alcohols longer than C14, alkanes longer than C9, and alkenes longer than C12 have shown minimal microbial inhibition [120, 121]. Alternatively, metabolic engineering techniques can be applied to allow for a more diverse range of hydrocarbon fuel targets. Many cellular modifications have been shown to improve microbial solvent tolerance: changes in membrane lipid composition; altered enzymatic activities of membrane repair and energy transduction enzymes; solvent expulsion via efflux pump activity; and cellular stress responses including heat shock, phage shock, and general stress responses [118, 125, 126]. These natural mechanisms offer a range of engineering targets: expression of a cis-trans isomerase to alter lipid composition; overexpression of enzymes involved in membrane repair and energy transduction; expression of efflux pumps such as tolC, mar, rob, soxS, and acrAB; and overexpression of stress-induced enzymes such as phage shock protein, heat shock proteins, catalases, and superoxide dismutases [125, 126]. While few metabolic engineering efforts have focused on enhancing product tolerance, a recent study explored improving hydrocarbon-based fuel tolerance in E. coli by testing a library of 43 efflux pumps [127]. This work identified efflux pumps that improved tolerance to five potential isoprenoid derived fuels. This preliminary success at engineering solvent tolerance should inspire additional efforts to improve the microbial production of both fatty acid and isoprenoid derived fuels.

In addition to product tolerance, other host traits are desirable for industrial biofuel production, particularly for autotrophic microorganisms. As discussed in the previous section, light availability is often a growth limiting factor in microalgal cultures. Microal­gae construct light harvesting complexes (LHC) to capture the available light for use in photosynthesis, and natural species actually absorb more light than is needed for photosyn­thesis under light intensities > 400 |omol photons m-2 s-1 [128]. As the sun can generate light intensities as high as 2,000 |omol photons m-2 s-1 during peak hours, it is estimated that as much as 80% of light absorbed by microalgae is ‘wasted’ as re-emitted fluorescence and heat [129]. In addition to this loss of energy, the excess energy can also cause cellular damage, known as photoinhibition [128]. In nature, this over-absorption of light will give the microalga a competitive advantage, but from a biofuel production perspective, this excess light harvesting will lead to lower culture cell densities and therefore lower biofuel productivities. Thus, there have been many attempts to engineer microalgae to absorb only the amount of light needed for photosynthesis. These efforts target genes of the light harvesting antenna complexes. Most LHC mutants were generated using random mutagen­esis techniques including chemical, UV, and transposon mutagenesis [128, 130—134]. Many of these studies focus on the model alga C. reinhardtii, but other microalgal species, such as the diatom Cyclotella sp. and the cyanobacterium Synechocystis sp., have been mutated to reduce the size of their photosynthetic antennae [130, 133]. Several recent works have applied RNAi technology in C. reinhardtii to reduce the expression of targeted LHC genes in a more controlled manner [129]. In general, the antenna mutants have shown im­proved photosynthetic quantum yields, reduced photoinhibition, enhanced productivity under high light conditions, and increased light penetration within the culture [128, 129, 131—134]. While these results are promising, several questions remain to be addressed: Are the photosynthetic antenna mutants genetically stable, or will they revert back to their more competitive and less efficient forms over time? And are these mutants less fit and there­fore more susceptible to predators and competitors in open pond systems?

Open pond systems are subject to a variety of changing environmental conditions, and as such, the optimal autotrophic host will have the necessary cellular mechanisms to adapt to these changing conditions. Desirable host traits may include temperature tolerance, salt tolerance, and resistance to predators. Open ponds are exposed to both daily and season­al temperature fluctuations which often exceed the normal temperature ranges for opti­mal cell growth and may even cause cell death. Engineering efforts have successfully altered the temperature tolerance of cyanobacteria though either gene knockout or heterologous overexpression of desaturases which influence the viscosity of both the cell and photosyn­thetic membranes [135]. Alternatively, microalgae with different temperature optima can be rotated seasonally in the open ponds, similar to seasonal crop rotations in agricultural practices. As mentioned previously, open pond systems are complicated by evaporative water loss, particularly for the sunny, arid regions that are ideal for microalgal biofuel production. Evaporation can lead to fluctuations in the salt concentration within the pond culture, and many have proposed to utilize marine or brackish water sources to reduce the cost associated with freshwater systems. Moreover, high salt and saturated salt systems will have lower evaporative water loss compared to freshwater cultures. Naturally salt-toler­ant microalgae, such as those isolated from marine or even hypersaline environments, may be selected as host for biofuel production, or efficient fuel-producing hosts can be engi­neered for increased salt tolerance. For example, the cyanobacterium Synechococcus elongatus PCC 7942, modified with expression of a A12 acyl-lipid desaturase (desA), showed improved resistance to salt and osmotic stress compared to the wildtype [136]. Lastly, pond crash due to microalgal predators like rotifers and chytrids is a major problem for open pond biofuel production systems. While there have not been any reported attempts at engineering predator-resistant microalgae, there have been reports of natural defense mechanisms such as palmelloid formation by C. reinhardtii, which produces non-motile cell aggregates that are simply too large to be consumed by grazing rotifers [137]. Once the genetic mecha­nism responsible for palmelloid formation is deciphered, it may be possible to transfer this resistance mechanism to other microalgae using genetic engineering techniques. When devising a metabolic engineering strategy for biofuel production, it is essential to consid­er the entire genomic landscape and the natural diversity of genetically-driven traits to design the optimal host for the specific industrial constraints.