Page 47 - Advances in bioenergy the sustainability challenge

Because ethanol has several undesirable fuel properties, higher chain alcohols have received
attention as possible fuels. For example, n-butanol has 20% higher energy content than ethanol.
In addition, it is more hydrophobic and thus less susceptible to inducing corrosion. 18
Traditionally, butanol is produced through refining petroleum or from acetone-butanol-ethanol
fermentation. The ability to robustly produce higher chain alcohols represents a step toward
biofuels with characteristics approaching that of gasoline.

Utilizing metabolic engineering, pathways for the production of higher chain alcohols have

been introduced into organisms such as Escherichia coli. 18,19 The use of the model bacteria
allows for the study and efficient development of novel pathways. Two pathways have recently
emerged to successfully produce C4 or C5 alcohols: coenzyme A (CoA)-mediated and
nonfermentative pathways.

The CoA-mediated pathway involves utilizing the native pathway of the butanol-producing
organism, Clostridium acetobutylicum. Introduction of five Clostridium genes into E. coli is
sufficient for production of n-butanol from acetyl-CoA. However, initial titers were at best
about 1 g/L, which is much lower than the 10 g/L typically produced by native n-butanol
producers. 19

More recently, extensive engineering of the pathway has led to insights into maximizing the
production through this pathway. Three major bottlenecks were discovered that could be
alleviated by (1) balancing the expression of upstream and downstream enzymes, (2) balancing

cofactor utilization and generation, and (3) engineering driving force to increase flux toward
the product. 20,21 Indeed, these bottlenecks seem to be recurring obstacles in many efforts to
engineer high production pathways. After addressing these bottlenecks, titers of 30 g/L could
be achieved at about 70% theoretical maximum yield. 20

The second pathway utilizes a creative nonfermentative approach, producing C4 and C5
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branched chain alcohols from intermediates in the amino acid metabolic network. The
introduction of a 2-keto-acid decarboxylase and an alcohol dehydrogenase allows the
conversion of a variety of 2-keto acid metabolites found in amino acid synthesis pathways into

their analogous branched chain alcohols: 1-propanol, isobutanol, n-butanol, 2-methyl-1-
butanol, 3-methyl-1-butanol, and 2-phenylethanol. Branched chain alcohols have higher octane
numbers than their straight-chained counterparts, making them better fuels. This process has the
advantage of avoiding CoA-mediated chemistry, while also leveraging the wealth of
understanding from decades of research on metabolic engineering for amino acid production.
As such, they were able to obtain isobutanol production of over 20 g/L at 86% of the
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theoretical maximum yield. Optimization of cofactor imbalances produced strains that
achieved 100% theoretical maximum yield, suggesting some of the same bottlenecks may exist
in this alternative pathway. 23

Fermentation of Lignocellulosic Material

In the search for improved feedstocks, the push toward cellulosic biofuels is a clear choice.
Cellulosic biomass eliminates the need to compete with food crop production as an estimated