Biology Reference
In-Depth Information
The abrogation of feedback inhibition can be achieved not only by mutations at inhibitor-
binding allosteric sites, but also by simply relocating the enzyme to a different compartment
within the cell. For instance, removing the leader peptide sequence of an
E. coli
thioesterase
TesA prevents its export into the periplasmic space and confines it to the cytoplasm, where
it depletes the pool of fatty acid-acyl carrier protein, a repressor of fatty acid biosynthesis.
This resulted in accumulation of fatty acids.
31
Overexpression of leaderless TesA in
E. coli
provided high levels of intracellular fatty acids and accelerated the production of fatty acid
ethyl esters, a biodiesel.
32
The catalytic activity of the pathway enzymes may also be a limiting factor, as not all
enzymes exhibit a fast turnover rate, particularly when they are members of a pathway that
has not evolved to generate large amounts of its product,
12
or if the enzyme is functionally
promiscuous (generates multiple products). For instance, enzymes involved in terpene
biosynthesis generate several products from a common precursor, a property that tends to
correlate with having low catalytic activity.
33
As a result, the flux through the pathway to the
terpene products is often low. Leonard et al. screened active site mutants of the enzyme
levopimaradiene synthese (LPS) from
Gingko biloba
to improve the product titer of
diterpenoids produced by a factor of 10.
34
A colorimetric screen of random mutants of the
prenyl transferase enzyme GGPPS, which catalyzes the preceding step and provides LPS with
a substrate, generated candidate GGPPS enzymes that produced higher yields of
geranylgeranyl diphosphate (GGPP). By coupling the best-performing GPPSS mutant with
the LPS mutant, the authors succeeded in increasing the diterpenoid yield by 18-fold, a
result obtained using protein engineering alone.
INCREASING FLUX BY TAILORING ENZYME LOCALIZATION
OR COMPARTMENTALIZATION
Pathway flux can also be increased without directly engineering higher catalytic activity.
Enzymes that catalyze sequential reactions can be localized in close proximity to each other
by means of artificial scaffolds,
35
or by fusing enzymes that catalyze sequential reactions.
This approach is inspired by examples found in native organisms of multiple enzyme
activities being colocalized in one complex or organelle.
36
Because the concentration of the
product of an enzyme will be higher near its active site, placing an enzyme that reacts with
that product nearby takes advantage of the higher local concentration to increase flux
through the following reaction. Colocalization of pathway enzymes may be particularly
useful when either the enzymes within a pathway cannot be optimized further, or an
intermediate metabolite is toxic to the host cell, or is a substrate of a competing enzyme
within the cell. This approach was validated using several enzyme components of the
mevalonate pathway. The enzymes AtoB, HMGS, and HMGR were joined on an artificial
scaffold coexpressed with the pathway enzymes, and the stoichiometry of the enzymes on
the scaffold was adjusted by modifying the numbers of individual binding sites of each
enzyme on the scaffold. The best scaffold resulted in a 77-fold increase in titer over the
nonscaffolded pathway, while further enabling lower enzyme expression. Even simple
protein fusions without a scaffold have demonstrated increased production. Fusing the
enzymes FPP synthase and farnesene synthase resulted in a modest increase in farnesene
production, compared to free enzymes,
37
and eliminated formation of the side product
farnesol.
212
Colocalization of proteins within a microcompartment, a strategy used by bacteria, is a
promising avenue of research into improving pathway efficiency.
38
Carboxysomes, a
microcompartment found within cyanobacteria, increase the carbon fixation rate of RubisCo
by colocalizing carbonic anhdyrase enzymes, which produces CO
2
from biocarbonate, into
a protein shell with RubisCO, which condenses the CO
2
with ribulose 1,5-bisphosphate to
produce 3-phosphoglycerate. Because RubisCO has a low affinity for CO
2
, the increased
