Biology Reference
In-Depth Information
Biosynthesis of TAL by 2-PS
(A)
(B)
O
Malonyl-CoA
OH
OO
OOO
CoAS
Library of AraC variants
O
O
CoAS
ES
OO
CoAS
OH
(TAL)
TA L
AraC-TAL
TA L
PBAD
GFP
2-PS variant
Reporter
(lacZ)
FIGURE 2.2
Design and application of a TAL-responsive reporter based on the AraC regulatory protein. (A) An araC gene library was
constructed and expressed in E. coli, and variants induced by TAL were isolated using FACS screening. (B) TAL is produced
by the enzyme 2-PS from G. hybrida. TAL-responsive AraC variant
'
AraC-TAL
'
was used as an endogenous reporter to
screen for improved TAL production by 2-PS variants expressed in E. coli.
Auxotrophic Complementation (Selections)
Though not always possible, an alternative to screening each library member is to develop
a cellular selection system, where cell growth or survival depends on some threshold level of
function by the protein variant of interest (auxotrophic complementation). This typically
involves genetically modifying the host and/or formulating the growth medium so that the
protein function to be evolved provides an essential metabolite for its host. For very large
libraries consisting of many mutations per variant, a vast majority of library members will
have relatively little or no fitness (compared to what is being sought). A suitably developed
selection system allows for large libraries to be screened without having to physically
observe
35
library members. For example, whereas a plasmid library of 10
8
members
may be transformed into a bacterial host, subsequent plating of all transformants onto
appropriately formulated agar plates may result in formation of only 10
4
colonies,
corresponding to variants with sufficient function to provide auxotrophic complementation.
In some cases the rate of colony growth may also correlate with activity of the protein of
interest. A liquid culture-based growth selection may alternately be developed, where
the fittest members out-grow others.
'
unfit
'
In one example, Boersma and coworkers developed a selection to improve the
enantioselectivity of
Bacillus subtilis
Lipase A (LipA).
73
Here,
E. coli
s native pathways
for aspartate synthesis were deleted, and the auxotrophic strain was used to select LipA
variants that could hydrolyze the aspartate ester of the desired enantiomer
(
S
)-(
'
)-1,2-
O
-isopropylidene-sn-glycerol. A phosphonate ester of the opposite
enantiomer (
R
)-(
1
)-1,2-
O
-isopropylidene-sn-glycerol was also added to inhibit growth
in the presence of less enantioselective variants. After three rounds of selection with
increasing stringency (by increasing the phosphonate ester concentration) a mutant was
isolated with greatly improved enantioselectivity. Additional examples of using selections
for protein engineering are provided below and depicted in
Figure 2.2
.
2
APPLICATIONS OF PROTEIN ENGINEERING IN
SYNTHETIC BIOLOGY
Engineering Protein-Based Biosensors
Construction of synthetic genetic circuits and integration of novel regulatory signals are
common goals in synthetic biology. Bacterial regulatory proteins which naturally regulate
gene expression in response to small molecule inputs (
'
'
effectors
) serve as useful tools
