The microbial production of drop-in replacement fuels faces unprecedented challenges. The sheer quantity of hydrocarbon product required to meet the world’s ever increasing demand for energy dwarfs the supply of any current microbially synthesized product. Moreover, both second (lignocellulosic feedstock) and third (microalgal feedstock) generation bio­fuels ultimately rely on sunlight and photosynthesis to supply the energy and carbon feedstocks necessary for production. This requires the development of new technology and infrastructure to facilitate the construction of this new supply chain. Finally, the low value of the final fuel product places additional financial restrictions on the development of large — scale biofuel production processes. For example, previous reports include the addition of exogenous metabolic precursors like mevalonate for isoprenoid production or FFA for FAEE biosynthesis [18, 50]. While these exogenous metabolites boost production of the desired hydrocarbon-based product, this practice is too expensive for large-scale biofuel applica­tions. These challenges currently limit the industrial production of second and third generation biofuels.

Fortunately, new biological and technological tools are rapidly being developed and applied to overcome the obstacles in biofuel production. In addition to the metabolic engineering strategies previously described in this chapter, new global strategies are being applied to engineer microbes for biofuel production. With the affordability of next-generation DNA sequencing technologies, new microbial genomes are being reported at an unprecedented rate, and this information can be used to generate metabolic models for biofuel-producing hosts. In turn, these models can be leveraged to analyze proposed metabolic engineering strategies in silico, reducing the number of costly and time-intensive strain constructions and experi­ments. This technique was shown to be successful at increasing lycopene production, an isoprenoid derivative, in E. coli [69, 138]. The advancement of synthetic DNA technology enables new engineering approaches such as multiplex automated genome engineering (MAGE) [139]. In MAGE, synthetic oligomers, consisting of degenerate DNA sequences flanked by regions homologous to the target sequences, are simultaneously transformed into E. coli, and the modified strains are screened for improvements. MAGE was used to target ribosome binding sites, for optimization of protein translation, and to inactivate genes by inserting nonsense mutations; this technique can also be applied to target promoters for improved gene transcription and enzyme active sites for enhanced activities. The technique does have some limitations, however. MAGE will likely require modification of the host organism to allow for efficient integration of the single-stranded oligonucleotides, and a high — throughput screening method is essential for screening the billions of genetic variants that are generated with MAGE. Global or systems-level technologies can also be applied to advance our fundamental understanding of genetic and regulatory mechanisms within a microbial host; this is vital to host development of non-model organisms and newly isolated strains. Omics technologies including genomics, transcriptomics, metabolomics, and proteomics provide global insight at the cellular level, which can be compared across different conditions or time points to identify the native mechanisms that control the cell metabolism. Integration of omics data can identify bottlenecks at the transcriptional, translational, and protein levels, and as such, can be applied to inform the metabolic engineering strategy for biofuel production [34]. Systems-level tools for engineering microbial hosts, including metabolic modeling, MAGE, and omics technologies, will be integral to the successful development of hosts for biofuel production.

Commercial interest in the production of second and third generation biofuels has developed rapidly in the past decade. As evidence of this, there has been a flurry of activity in patent applications regarding microbial hydrocarbon production. Companies invested in heterotro­phic hydrocarbon-based fuel production include LS9 [27, 59, 65, 66, 140, 141] and Amyris Biotechnologies [72, 142], which focus mainly on E. coli as the host, and Solazyme [143, 144], which initially focused on fuels derived from algae but has since moved toward more high — value markets, such as cosmetics and nutraceuticals. Most companies interested in algae and cyanobacteria are focused on autotrophically-produced hydrocarbon fuels. Notable compa­nies in this industry include Sapphire Energy [145, 146], Joule Unlimited [26, 77, 147], and Synthetic Genomics [68, 75]. The hydrocarbon-based fuels targeted by these companies span the entire gamut of fatty acid and isoprenoid derived fuel products. Despite this commercial interest, hydrocarbon biofuel production still remains to be demonstrated at scale and in a sustainable manner.

This chapter has described the challenges in microbial hydrocarbon production and presented metabolic engineering strategies to resolve these issues. As is evident from this discussion, microbial-based fuel production is only in the initial stages of exploration, and additional research and innovation is necessary to enable large-scale biofuel production. New metabolic engineering tools and techniques are currently being developed for engineering untraditional hosts like eukaryotic algae and cyanobacteria, and as our understanding of these new hosts matures, significant improvement in hydrocarbon yields is anticipated.

Abbreviations

Acknowledgements

This work was supported by the Harry S. Truman Fellowship in National Security Science and Engineering and the Laboratory Directed Research and Development program at Sandia National Laboratories. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corpo­ration, for the U. S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.

Author details

Anne M. Ruffing

Sandia National Laboratories, Department of Bioenergy and Defense Technologies, Albu­querque, NM, USA