## 4 Metabolic Pathway and Host Engineering for Advanced Biofuels Production
### Fatty Acid-Based Biofuels  
The energy-rich hydrocarbon chains of fatty acids make them potential precursors for the production of diesel alternatives. Fatty acids are synthesized by fatty acid synthase (FAS) system, which condenses malonyl-CoAs into various lengths of fatty acyl esters with acyl-carrier protein (ACP) (Figure 5). Currently, plant oils and animal fats are chemically converted to fatty acid alkyl esters (fatty acids ethyl esters, FAEEs and fatty acids methyl esters, FAMEs) via trans-esterification. Microbial production of FAEEs was facilitated by identification of promiscuous wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT), which was first characterized in _Acinetobacter calcoaceticus_ ADP1 [44], and 1.28 g/L FAEE was produced with oleic acids feeding in _E. coli_ strain where pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) from _Z. mobilis_ were co-expressed to provide ethanol [45]. Conversion of sugars to FAEEs, fatty acid alcohols and wax esters without extracellular feeding of fatty acids or ethanol was demonstrated by expression of _pdc, adhB, tesA'_ (membrane targeting sequence truncated _E. coli_ native thioesterase) and _fadD_ (native acyl-CoA synthetase) in _E. coli_ with _fadE_ gene deletion (_AfadE_), where fatty acid metabolism was forced to produce more fatty acyl-CoA [46], an important precursor for various fatty acid-derived fuels. The introduction of a dynamic sensor-regulator system significantly increased fatty acid production several times higher in _E. coli_ by balancing substrate supply levels [28], which resulted in a FAEE titer of 1.5 g/L with 28% of maximum theoretical yield (Table 1). Figure 5: Overview of biofuel compounds derived from fatty acids pathways. ACC: acetyl-CoA carboxylase. Fatty acid-derived fuels were also produced in yeast by overexpressing all three fatty acid biosynthesis genes (ACC1, FAS1 and FAS2) in combination with the expression of downstream enzymes (diacylglycerollycerol acyltransferase, fatty acyl-CoA thioesterase, fatty acyl-CoA reductase, and wax ester synthase) [47]. Oleaginous yeast species are now extensively studied as promising hosts for FAEE production since they naturally produce and accumulate a large amount of lipids--up to 36% of their dry weights. Recent studies increased the intracellular lipid content even further, up to 40%-70%, by engineering _ex novo_ lipids biosynthesis, and it has been shown that lipids could be accumulated via tightly regulated _de novo_ synthesis pathway when acetyl-CoA carboxylase (ACC1) and diacylglycerollycerol acyltransferase (DGA1) were overexpressed [48]. Other fatty-acid-derived biofuel molecules such as fatty aldehydes, methyl ketones, alkanes and alkenes are now produced in microbial hosts (Figure 5), and production of those fatty acid-derived fuels were reviewed recently [49, 50]. The most significant progress has been made by identifying biochemical pathways and responsible enzymes to produce targeted fuel molecules from various organisms and by reconstruction of the heterologous pathway in selected hosts. For example, heterologous expression of acyl-ACP reductase and aldehyde deformylase [51] (previously known as a fatty aldehyde decarbonylase) enabled _E. coli_ to produce alkanes and alkenes [52]. Furthermore, two terminal alkene synthesis enzymes were discovered for production of \(\alpha\)-olefins; elongase decarboxylase (a homologous enzyme to type I polyketide synthase) from _Synechococcus sp_. PCC7002 [53] and fatty acid decarboxylase (a cytochrome P450, OleTJE) from _Jeotgalicoccus_ species [54], which act on acyl-ACP and fatty acids, respectively. In addition to understanding the biochemistry and structure of the identified enzymes, finding alternative enzymes to reduce fatty acids pathway intermediates to fuel molecules have been of great interest to the research community.