## 4 Metabolic Pathway and Host Engineering for Advanced Biofuels Production
### Ferrentative Pathways for 1-Butanol and Other Short Chain Alcohols  
1-butanol is naturally produced by species of Clostridia, which have innate 1-butanol fermentative pathways. The 1-butanol fermentation pathway and general aspects of Clostridia physiology (e.g., sporulation cycle and acidogenesis) were summarized along with metabolic engineering efforts to improve 1-butanol fermentation in the recent review [9]. Other specific aspects regarding 1-butanol fermentation by Clostridia have also been reviewed with a particular focus on the diversity of Clostridia strains for ABE (Acetone-Butanol-Ethanol) fermentation and 1-butanol fermentation on various substrates [31]. Metabolic engineering strategies to overcome limitations in Clostridia butanol fermentation are (i) redox balancing (e.g., regeneration of NADH via butanol fermentation, which could be increased by reducing hydrogenase activity [9]); (ii) reducing byproduct formation and improving 1-butanol productivity; and (iii) conferring host tolerance against 1-butanol [32].  
Although Clostridia species are natural 1-butanol producers, they grow slowly and their genetic manipulation is still limited. To overcome these limitations, the 1-butanol fermentation pathway was re-constructed in _E. coli_ by incorporating seven enzymes from three different species [33]. One of the key engineering strategies was substituting a flavin-dependent native Bcd/EtfAB system of Clostridia to an irreversible enoyl-CoA reductase (Ter) from _Treponema denticola_[34]. This Ter-based, synthetic pathway has been further improved by building up NADH and acetyl-CoA as a driving force for 1-butanol production under anaerobic conditions. Accumulation of NADH and acetyl-CoA could be achieved by eliminating four fermentation pathways and by expressing formate dehydrogenase (_fdh1_), and as a result, the titer could reach up to 30 g/L in _E. coli_[20]. Another study optimized transcription level of _fdh1_ to minimize redox imbalance by excessive regeneration of NADH, and it subsequently showed increased 1-butanol productivity in _E. coli_[35]. Furthermore, expression of _acrB_ efflux pump conferred tolerance of _E. coli_ to 1-butanol [36]. Not only 1-butanol, but isopropanol can be  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Product**} & \multirow{2}{*}{**Pathway**} & **\% of Apparent Theoretical Yield (from Glucose)** & **Highest Titer Reported (g/L)** & **Host** & **Reference** \\ \hline
**Gasoline** & & & & \\
1-Butanol & CoA-dependent & 41\% * & 30 & _E. coli_ & [20] \\ Isobutanol & 2-keto acids & 41\% * & 50 & _E. coli_ & [21] \\
3-methyl-1-butanol & 2-keto acids & 33\% * & 9.8 & _E. coli_ & [22] \\
3-methyl-3-butanol & MVA & 41\% * & 2.2 & _E. coli_ & [23] \\
2-methyl-1-butanol & 2-keto acids & 39\% * & 1.25 & _E. coli_ & [24] \\ \hline
**Diesel \& Jet Fuel** & & & & \\ Farnesene & MVA & 25\% * & NA & & [25] \\ Farnesene & MEP & 29\% * & NA & & [25] \\ Bisabolene & MVA & 25\% & 1.15 & _E. coli_ & [26] \\ Limonene & MVA & 25\% & 0.605 & _E. coli_ & [26] \\ Pinene & MVA & 25\% & 0.032 & _E. coli_ & [27] \\ FAEE & Fatty acids & 35\% * & 1.5 & _E. coli_ & [28] \\ Methyl ketones & Fatty acids & 33\% & 3.4 & _E. coli_ & [29] \\ \hline \hline \end{tabular}  
* [PERSON] and [PERSON], 2009 [25]; b [PERSON] and [PERSON], 2011 [30].  
\end{table}
Table 1: Production titer of various advanced biofuel molecules and theoretical yields.  
produced via fermentation by reducing acetone, one of the three fermentation products in Clostridium. The highest titer of up to 143 g/L was achieved with gas stripping via fermentation pathway [37].