## 4 Metabolic Pathway and Host Engineering for Advanced Biofuels Production
### Isoprenoid-Based Biofuels  
Isoprenoids are a group of diverse chemical compounds which includes over 50,000 compounds. Fuel molecules derived from isoprenoid pathways have branched hydrocarbon chains, which lower their freezing temperature, as well as various ring structures, which makes them potential alternatives to diesel and jet fuels [61]. These branched hydrocarbon chains are derived from two universal precursors, isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP) (Figure 6). The isoprene unit of IPP is first condensed to DMAPP and iteratively further condensed to various length of prelyl diphosphate molecules such as geranyl diphosphate (C\({}_{10}\), GPP), farnesyl diphosphate (C\({}_{15}\), FPP) and geranylgeranyl diphosphate (C\({}_{20}\), GGPP) [62]. These condensation reactions are catalyzed by prenyltransferase enzymes, of which specificity is the determining factor of hydrocarbon length of the relevant isoprenoid [63]. Finally, prenyl diphosphates are diversified to various structures of isoprenoids by terpene synthase enzymes, mostly via carbocation formation [64]. Regardless of the final structures, isoprenoids are primarily classified into different groups based on the length of the hydrocarbon backbone, and the most abundant isoprenoids belong to monoterenes (C\({}_{10}\)), sesquiterpenenes (C\({}_{15}\)) and diterpenenes (C\({}_{20}\)). Therefore, engineering efforts to produce isoprenoids-derived biofuels have been focusing on increasing production of prenyl diphosphate intermediates, particularly IPP and DMAPP, and identification and engineering of terpene synthases for desired enzymatic activity. Although two different biosynthetic pathways (the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway) have been known to produce two universal C\({}_{5}\) precursors, the MVA pathway has been more extensively exploited for production of potential diesel and jet fuel precursors such as farnesene [65], bisabolene [66, 67], pinene [27, 68] and limonene [26, 69] (Figure 6). Since isoprenoid-derived fuels are dependent on the two precursors, IPP and DMAPP, most engineering efforts are focused on optimizing pathways by balancing fluxes of metabolic intermediates, increasing the transcription level of limiting enzymes [70], improving protein expression by codon-optimization, and reducing reversibility of the reactions [71]. Among sesquiterpene-derived fuels, biological production of farnesene and bisabolene has been reported. Farnesene is converted from FPP by farnesene synthase. The previously developed FPP-overproducing _E. coli_ and yeast strains led to an initial farnesene titer of 1.1 g/L after 120 h of _E. coli_ fermentation and 728 mg/L after 72 h of yeast fermentation [72, 73]. After continuous evolution of the host strains, Amyris, a biotech company, reported farnesene production to the titer of 104.3 g/L from the engineered yeast strain [74] and commercialized trans-\(\beta\)-farnesene under the name, \"Biofene(\(\circledR\))\". Bisabolenes are another group of sesquiterpenes, of which fuel properties are qualified for diesel alternatives [67]. Initially, a titer of 400-800 mg/L of bisabolene was produced from _E. coli_ and yeast platforms engineered for FPP-overproduction [70, 75]. About 40% increase in titer was reported in _E. coli_ by applying principal component analysis of proteomics (PCAP) approach [26]. In yeast, three genes that encode two unknown functions and a transcriptional regulator, Rox1, were identified from carotenoid-based screening method, and further engineering efforts resulted in 5.2 g/L bisabolene titer in fed-batch fermentation conditions [66]. Microbial production of monoterenes was significantly improved by increasing availability of GPP and protein expression level of terpene synthases.