Page 49 - Advances in bioenergy the sustainability challenge

substrates and feedstocks. It can also be used to circumvent regulatory bottlenecks to achieve
more robust feedstock utilization. Although further regulatory and metabolic understanding is
still necessary, current progress has shown great promise in enabling the production of biofuels
from lignocellulosic materials.

Production of Fatty Acid Derivatives

The use of fatty acids and their derivatives as biofuels has numerous potential benefits. These
long-chain hydrocarbon products have high energy density, are hydrophobic, and chemically
resemble their fossil fuel analogs. Termed as drop-in biofuels, these products could be quickly
integrated into existing infrastructure with little modification.

The most attractive pathway for production is the use of cytosolic thioesterase enzymes to
produce free fatty acids, which can then be converted enzymatically or catalytically into a
variety of derivative products: alkanes, alkenes, fatty acid esters, and alcohols. First
30
discovered by Cho and Cronan, expression of cytosolic thioesterase ‘tesA converts the lipid
synthesis intermediate acyl carrier protein (ACP) into its fatty acid form. Disrupting the fatty
acid synthesis pathway in this manner circumvents many of the typical fatty acid products that
are often used as triggers for feedback regulation. This engineered pathway remarkably
decouples production from growth by relieving feedback inhibition of a tightly regulated
30
pathway, providing driving force for fatty acid synthesis at all stages of the cell cycle. The
fatty acids can then be modified into a variety of derivatives: free fatty acids, alkanes, and fatty

acid ethyl esters (FAEE). 31-33 The process has even been extended into microalgae, which
34
promises biofuels production from photosynthesis. Additional engineering modifications to
increase precursor availability included overexpression of acetyl-CoA carboxylase and
coexpression of plant thioesterase. 33,35 The best reported efforts produced 4.5 g/L/day of fatty

acids through these engineering strategies. 35
In addition, this pathway has been used to advance the concept of a consolidated bioprocess

(CBP). CBP entails having one organism, which can convert feedstock to product with little to
no additional processing in between. This saves on operating costs and dramatically simplifies
the production process.

31
Steen et al. report engineering an organism that can produce FAEE from xylan. As xylan is
one of the primary constituents of hemicellulose, this process demonstrates the use of a single
organism to consolidate initial enzymatic degradation of xylan into xylose, the conversion of
xylose into fatty acids, and the transesterification of those fatty acids into ethyl esters. The
basis of this work was the discovery and utilization of a wax ester synthase that is able to
36
perform the biodiesel transesterification reaction enzymatically. Expression of xylanase and
introduction of the ethanologenic pathway allowed for the in vivo transesterification of fatty
acids to produce FAEE that can be used directly as biodiesel.

Robust control of the length and saturation of products will be a key achievement in the
development of fatty acid derivatives production as a biofuel technology. Also, because fatty
acid synthesis is tightly regulated and critical to many cellular functions, some creative