The chemical composition of petroleum-based fuels: gasoline, diesel, and jet fuel, includes linear, branched, and cyclic alkanes, aromatics, and chemical additives [28]. Isoprenoid-based biofuels have the structural diversity to mimic these petroleum compounds, with up to 50,000 known isoprenoid structures including branched and cyclic hydrocarbons with varying degrees of unsaturation [29, 30]. Isoprenoids reported to be potential fuel candidates include: the hemiterpene (C5) isoprene; monoterpenes (C10): terpinene, pinene, limonene, and sabinene; the sesquiterpene (C15) farnesene, and their associated alcohols: isopentenol, terpineol, geraniol, and farnesol [12, 31]. Two metabolic pathways are capable of producing the isoprenoid building blocks isopentenyl pyrophosphate (IPP) and dimethylallyl diphos­phate (DMAPP): the mevalonate (MVA) pathway [32] and the methylerythritol phosphate (MEP) pathway, also known as the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway and the non-mevalonate pathway (Figure 3) [33]. In general, the MVA pathway is found in eukaryotes and archaea while the MEP pathway is utilized by prokaryotes. In agreement with the proposed evolutionary origin of plants, they contain both isoprenoid pathways with the MEP pathway localized in the plastid and the MVA pathway in the cytosol [34]. The MVA and MEP pathways differ with respect to their requirement for carbon, energy, and reducing equiva­lents; this is illustrated by the net balances for IPP biosynthesis from glyceraldehyde-3- phosphate (GAP):

MVA:3 GAP + 3 ADP + 4 NAD(P)+ + 2 P ® IPP + 4 CO2+ 3 ATP + 4 NAD(P)H (1)

MEP:2 GAP + ADP + CTP + P ® IPP + CO2 + ATP + CMP + PP, (2)

Based on these balances, IPP production via the MEP pathway is more efficient at carbon utilization, as only 2 GAPs are required and 1 CO2 is emitted, compared to 3 GAPs and 4 CO2 for the MVA pathway. On the other hand, IPP production via the MVA pathway is more energy efficient overall, resulting in ATP generation and yielding a net gain in reducing equivalents (NAD(P)H). These carbon, energy, and reducing equivalent requirements should be considered when designing a metabolic engineering strategy for isoprenoid biosynthesis.

The MVA pathway interfaces with the primary metabolism at the acetyl-CoA node (Figure 3), and it can be divided into two parts: the top, which involves 3 enzymatic steps to convert acetyl-CoA to MVA, and the 3 enzymatic conversions of the bottom portion to produce IPP from MVA. One novel metabolic engineering strategy compared the efficiencies of the top and bottom portions of the MVA pathway in E. coli using heterologously expressed pathways from 5 different eukaryotic sources. The most efficient top and bottom portions were combined to maximize the yield of isoprenoid building blocks [35]. Accumulation of an intermediate metabolite, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), is a known bottleneck in the top MVA pathway, and HMG-CoA was also shown to inhibit cell growth in E. coli [36]. Thus, overexpression of the HMG-CoA reductase (HMGCR) increased MEV production and synthesis of subsequent FPP-derivatives in both E. coli and S. cerevisiae [36—38]. Whole pathway expression and elimination of the HMGCR bottleneck have proven to be successful techniques for enhancing the metabolic throughput of the MVA pathway.

The MEP pathway requires two primary metabolites as precursors: GAP and pyruvate (PYR) (Figure 3). Compared to the 6 enzymatic steps of the MVA pathway, the MEP pathway is comprised of 7 steps. Metabolic engineering strategies for the MEP pathway have primarily focused on the first two enzymatic steps. Overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (dxs), catalyzing the conversion of GAP and PYR to 1-deoxy-D-xylulose-5-phosphate (DXP), resulted in 6-10-fold increases in the final isoprenoid product [39, 40]. Targeting the next enzymatic step through overexpression of DXP reductoisomerase (dxr) was shown to have little effect on isoprenoid production using the native gene; however, expression of dxs and dxr from Bacillus subtilis improved isoprenoid production 2.3-fold in E. coli [41]. The final step of the MEP pathway was also shown to be rate-limiting, as heterologous expression of IPP isomerases (IPPI) enhanced isoprenoid production in E. coli [42]. Based on its rate-limiting steps, the MEP pathway is a prime candidate for a push-pull metabolic engineering strategy, whereby overexpression of the first step ‘pushes’ carbon flux into the MEP pathway and overexpression of the final step ‘pulls’ the metabolic flux towards the end product. This strategy yielded nearly 2-fold improvements in isoprenoid production in E. coli [43, 44]. Lastly, overexpression of the entire MEP pathway can increase isoprenoid biosynthesis. In fact, Leonard and colleagues demonstrated that 5 additional copies of the MEP pathway genes yielded the highest production, while further increasing the gene copy number to 10 produced lower titers [45].

While targeted gene overexpression may alleviate pathway bottlenecks, the pathway is still subject to native regulatory mechanisms which may limit isoprenoid biosynthesis from either the MVA or MEP pathways. A highly successful strategy for overcoming regulatory limitations is overexpression of the non-native isoprenoid pathway. Expression of the MVA pathway from Saccharomyces cerevisiae in E. coli has enabled higher levels of isoprenoid synthesis compared to engineering the native MEP pathway as the sole isoprenoid pathway [46—50]. The success of this strategy has made it a favorite among metabolic engineers seeking to improve isopre — noid biosynthesis. Farmer and Liao presented a clever approach for regulating the carbon flux into an engineered MEP pathway in E. coli [51]. In this work, a native regulatory circuit was used to control the carbon flux into and through the MEP pathway by regulating expression of two key enzymes: phosphoenolpyruvate synthase (PPS) and isopentenyl diphosphate isomerase (IPPI). Under excess carbon flux, expression of pps and idi was activated using the regulatory circuit, redirecting carbon flux into and through the MEP pathway, yet when the carbon flux was growth limiting, expression of these genes was reduced. This strategy allows for high isoprenoid production without negatively impacting cell growth. As evidence, the regulated pathway improved isoprenoid titers by 50%, while simply placing pps and ippi under control of strong tac promoters resulted in growth inhibition [51]. Native regulatory mecha­nisms are often obstacles limiting isoprenoid biosynthesis, yet they can also be exploited to optimize the flux balance to support both cell growth and isoprenoid production.

Additional targets for improving isoprenoid-based fuel production include precursor supply, cofactor supply, and optimization of the downstream fuel synthesis pathway. Acetyl-CoA is

the precursor for isoprenoid production via the MVA pathway. Overexpression of acetalde­hyde dehydrogenase (ALDH) and acetyl-CoA synthetase (ACS), both of which produce acetyl — CoA, increased the acetyl-CoA supply and subsequently isoprenoid biosynthesis in S. cerevisiae [52]. On the other hand, the MEP pathway requires two precursors from the glycolysis pathway: PYR and GAP. The supply of these metabolites is complicated by the fact that PYR is derived from GAP, and consequently, the PYR/GAP balance is an important metabolic engineering target. The supply of GAP was shown to be limiting in E. coli, as modifying the conversion between PEP and PYR to redistribute the flux toward GAP synthesis increased isoprenoid production [53]. In addition to the carbon precursors, co-factors in the form of energy (ATP, CTP) and reducing equivalents (NADPH) are also required for isoprenoid synthesis. Co-factor supply is often overlooked in strategies for isoprenoid production, yet by improving the availability of NADPH in S. cerevisiae, isoprenoid synthesis through the MVA pathway increased by 85% [54]. This result emphasizes the importance of co-factor availability. Despite optimizing production of the isoprenoid building blocks, the downstream efficiency of assembling the final fuel product may still limit the overall yield. Successful strategies for improving downstream efficiency include overexpression of GPP and FPP synthases [47], overexpression and codon optimization of hemiterpene, monoterpene, and sesquiterpene synthases [41, 47, 48], fusion proteins to localize FPP synthesis and its conversion to sesqui­terpene [47], and downregulation of competing products like squalene [37, 48]. The optimized production of isoprenoid-based fuels requires strategies to address limitations throughout the metabolic pathway, from precursor and co-factor supply to end product synthesis.