Biology Reference
In-Depth Information
Optimization of the Biofuel Pathway
The goal of pathway optimization is to maximize the efficiency of the process that
synthesizes the desired fuel from existing metabolites in the host, which is determined by
concentrations of precursors, intermediates, cofactors, and enzymes, as well as the catalytic
activities of the enzymes, which are almost always subject to regulation on multiple levels,
from gene expression to enzyme activity. This section will begin by addressing the
optimization of the fundamental pathway components: the enzymes themselves.
PROTEIN ENGINEERING TO IMPROVE FLUX
If an enzyme is found to be limiting biofuel production, one straightforward approach to
improve production is to screen a library of homologues to find a replacement for the
bottleneck enzyme. This approach relies upon the sheer numbers of homologues often
available within genetic databases that presumably span a wide range of properties, such as
stability and catalytic rates. Often, an improved enzyme can be found, as has recently been
demonstrated in work that achieved a high level of production of the biodiesel replacement
bisabolene.
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A library of five bisabolene synthases from four plant species were cloned and
screened for production titers, generating yields that varied by 100-fold, and one high-
yielding (0.5 g/L) enzyme was discovered. However, homologues may not always be
available or numerous (especially for certain classes of terpene cyclases), and high-yielding
enzymes may not always be happened upon so fortuitously, especially for enzymes that
have not evolved to produce high yields, as is often the case for secondary metabolites.
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A recent review
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argues that it is better to optimize production by adjusting the copy
number, regulation, and enzymatic properties of the components of a pathway, rather than
screening libraries of enzymes. While screening a library of enzymes might provide a coarse-
grained search over a productivity landscape, protein and metabolic engineering methods
are likely necessary to bring the pathway closer to a productivity maximum.
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The lack of biochemical characterization of the vast majority of known enzymes is a natural
consequence of the ease with which prospective genes can be found in sequence databases
and cloned, compared to the difficulties and uncertainties inherent in protein
overexpression and biochemical measurements. This is unfortunate, as knowledge of the
properties of each enzyme within a pathway can better focus pathway optimization efforts.
For instance, attempting to increase flux by increasing the copy number of a pathway
component will be ineffective if the enzyme is nonfunctional (not expressed, or lacking
necessary post-translational modifications), poorly functional (not folding properly), or
functional but permitting a lower flux than is optimal for production of the biofuel
(inhibition by a competitor or allosteric regulator). Of course, the enzymes selected for a
pathway must be compatible with the host, a property referred to as
'
functional
10
An enzyme is more likely to function in the production host if the
organism from which it is obtained shares a similar intracellular chemical environment with
the new host (such as pH or growth temperature), though the sensitivity of enzymes to
these factors likely varies widely, and often compatibility cannot be known until expression
is attempted. In particular, genes transplanted between kingdoms may suffer compatibility
problems.
14
These issues may be overcome by engineering the protein itself to improve its
'
composibility.
'
properties, a term that refers collectively to the solubility, stability, selectivity, and
activity of an enzyme.
15
in vivo
'
All enzymes need to fold into their native three-dimensional structure before they can
function. Proteins often misfold when expressed in a foreign host (or even when
overexpressed in the original host). When a pathway enzyme does not fold, it can either be
replaced or modified to improve its folding. Yoshikuni et al. found that the enzyme
γ
-humulene synthase (HUM), a sesquiterpene synthase from the gymnosperm
Abies grandis,
demonstrated low activity when transplanted into
E. coli
(15). Investigations found that very
