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243
Exploring the possibilities presented by protein engineering
John Shanklin
A combination of classical and powerful new combinatorial
genetic techniques allows the redesign of enzyme activities
and creation of proteins that are tailored to have specific
properties. These technologies have far-reaching
consequences for the future design of crop plants and the
storage compounds within them.
Addresses
Department of Biology, Building 463, 50 Bell Avenue, Brookhaven
National Laboratory, Upton, New York 11786, USA;
e-mail: [email protected]
Current Opinion in Plant Biology 2000, 3:243–248
1369-5266/00/$ — see front matter. Published by Elsevier Science Ltd.
Abbreviations
ACP
acyl-carrier protein
DIBOA 2,4-dihydroxy-1,4-benzoxazin-3-one
PCR
polymerase chain reaction
Introduction
Gene transfer into all common crop species can now be
achieved routinely. This progress raises the issue of
which genes should be introduced to convey desired
traits. Genomic sequencing efforts have provided a rich
resource of genetic material from which genes encoding
valuable traits can be selected [1]. Nevertheless, even
with this ever-increasing resource, genes with ideal
properties may not be found. For instance, transferred
genes may not perform in the transgenic plant as they
did in the plant from which they were taken, and therefore the phenotype of interest may not be conferred to
the transgenic plant. Such was the case when the 16:0∆4-desaturase gene from coriander was transferred to
tobacco. The accumulation of this unusual fatty acid was
much smaller in tobacco than in coriander [2]. For naming fatty acids, X:Y indicates that a fatty acid contains X
carbons atoms and Y number of double bonds. ∆z indicates that a double bond is located at the z carbon atom
relative to the carboxyl terminus of the fatty acid. A second problem might be that the available genes, though
exhibiting functional diversity, might not include a gene
that has ideal properties. An alternative approach to
transferring naturally occurring genes into crops is to
extract their information content and to use this to
redesign desired properties, or to use them as starting
points for combinatorial genetic manipulation to evolve
a property of interest.
The intent of this review is to bring to the attention of
plant biologists examples of approaches that have proven
successful in protein redesign. Because of the broad
scope of this review, readers are directed to a number of
excellent reviews on specific topics.
Evolution of enzymatic plasticity
In the order of 103 individual protein folds (the core
arrangement of secondary structural elements) are thought
to occur in nature, far fewer than the number of individual
proteins (which is closer to 105) [3]. This is because many
proteins share common folds. Many proteins that have the
same fold are presumed to have arisen from a common
ancestor [4]; hence enzymes with the same fold perform a
variety of reactions. An illustration of gene duplication and
functional specialization was presented by Gierl and colleagues [5,6] who reported on the biosynthetic pathway of
the plant defense compound 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA). They found that genes Bx2 through
Bx5 encode cytochrome P450-dependent monooxygenases (P450s) that catalyze four consecutive hydroxylations
and one ring expansion to form DIBOA. These P450s are
thought to have arisen though gene duplication and the
accumulation of mutations that led to a change in both
substrate- and regio-specificity (i.e., the position on the
substrate molecule that is modified). In another example
from the family of membrane-bound di-iron enzymes
[7,8•,9•], mutations affecting the desaturase reaction have
given rise to at least four distinct enzymatic activities:
hydroxylase, epoxidase, acetylenase and conjugase. New
catalytic activities are now widely thought to evolve from
pre-existing enzymes when a partial reaction, which is catalyzed by a progenitor enzyme, is retained but the
architecture of the active-site of the enzyme is modified so
as to allow the intermediate generated to be directed into
the synthesis of different end-products [10].
Comparative structure–function analysis
Gene fusions can be used to identify domains within
enzymes that control properties of interest. Once such a
region is identified, site-directed mutants can be constructed
and used to evaluate the contribution of individual aminoacid residues. Examples of the successful application of this
approach include changing an acyl-ACP (acyl-carrier protein)
Table 1
Techniques for improving enzymes and the resulting
information.
Technique
Outcome
References
Comparative
structure/function
analysis
Identification of key residues
Activity switching
Novel activities
Rational design
Structural
Computational
Novel activities
Novel activities
Directed evolution
Single gene
Multi-gene
Improvements in desired properties [29]
Novel activities
[37••]
[11]
[16••]
[12••]
[13]
[19]
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Physiology and metabolism
Figure 1
Summary of the principles of gene shuffling.
Mutations result in positive (䊊) or negative
(䊉) phenotypes.
Single gene
Random fragmentation
Pool of random
DNA fragments
Reassembly PCR
Mutagenic
Family of related
DNA sequences
Random fragmentation
Cycle
Pool of random
DNA fragments
Reassembly PCR
Mutagenic & recombinogenic
Library of
recombinant
genes
Identify clones with
combinations of
positive mutations
Screen
Eliminate clones with
negative mutations
Current Opinion in Plant Biology
thioesterase with 12:0 specificity into one specific for 14:0 by
substituting three amino acids [11], or increasing the 18:0substrate preference of an acyl-ACP thioesterase up to
13-fold [12••] by site-specific mutagenesis. In a more complex example, both the chain length- and the
regio-specificity of a 16:0-∆6-ACP desaturase was converted
into predominantly an 18:0-∆9-ACP desaturase by substituting five amino acids [13].
Key amino acids are most easily identified by this genefusion approach when the residues that contribute to the
interesting properties reside in a linear domain within the
sequence. Within the crystal structure of the 18:0-∆9-ACP
desaturase [14], the determinants of acyl-chain length
(substrate) specificity and regiospecificity can be classified
into two types: those amino acids that directly affect the
substrate binding interface and those occupying positions
remote from the binding cavity that must exert their effects
indirectly [13]. The logical gene-fusion approach is better
suited to structure–function studies than to efforts to
redesign proteins. This is because the outcome of genefusion experiments is usually to define the positions of key
residues that affect the property of interest rather than to
facilitate the design of novel activities (Table 1). It is possible, however, that information generated from gene-fusion
experiments could subsequently be used, either alone [12••]
or in conjunction with a combinatorial method [15], to
generate novel activities.
Exploring the possibilities presented by protein engineering Shanklin
A second logical approach is available when several homologous amino acid sequences are associated with two distinct
enzyme activities (paralogs). For example, Broun et al. [16••]
compared the sequences of five related oleate desaturases
with those of two independently-evolved oleate hydroxylases. Seven conserved positions within the five desaturases
were identified at which the residues differed from those at
equivalent positions in the two hydroxylases. Reciprocal
changes between pairs composed of one of the desaturases
and one of the hydroxylases were made at the seven sites,
resulting in dramatic shifts in the ratio of desaturase to
hydroxylase function, that is, away from the original enzyme
activity and towards that of the alternate activity. An advantage of this logic-based approach is that the number, as well
as the relative position, of key residues is unimportant.
Rational design
The application of protein crystallography to enzymes
brought with it the expectation that enzymes could be rationally designed to have desired properties. Although there
are examples of successful rational design, such as the engineering of a novel 16:0-∆9-ACP desaturase [13], and the
design of a thermolysin-like protease from Bacillus stearothermophilus that has improved thermostability [17•], this
method has, so far, generally failed to meet expectations.
There are many reasons for this, one being that changes in
the residues of the active-site often produce the desired
results in terms of specificity, but at a large cost to catalytic
rate. In addition, many residues that control key enzyme
properties occupy positions that are distant from the active
site and that are not readily identified by the examination of
a crystal structure. Consequently, there has been a steady
shift in the emphasis of enzyme redesign studies from ‘rational’ to ‘irrational’ (i.e., combinatorial) genetic approaches
[18]. Despite this general trend, there is also renewed interest in a specific method of rational design, pioneered by
Mayo and colleagues [19]. This method involves cycling
between new computational methods and experiments [20].
Their computational approach identifies probable functional solutions using sophisticated algorithms that eliminate
dead-end design solutions [21].
Gene shuffling: directed evolution of single
genes
It is clear that many enzymes with very different properties have evolved one from another in nature. Researchers
have therefore explored the possibility of ‘directing evolution’ or molecular breeding in the laboratory [22]. For
instance, the Arnold laboratory at Caltech [23•] and the
Stemmer group at the company Maxygen [24•] have developed powerful methodologies for mimicking the process of
natural evolution using isolated genes in the test tube.
Evolution that would take eons in nature can now occur
within days or weeks in the laboratory.
The strategy for evolving enzymes involves two steps
(Figure 1). In the first step, random mutations are introduced
into the target gene either by fragmentation followed by
245
Table 2
Appropriate methods for tiered-screen selection of the best
performing individual clones from large combinatorial
populations.
Number of individual
candidate clones
Assay method
References
>107
FACS, genetic selection,
phage display
[33••,45,46]
104–106
Solid state assays:
colorimetric or fluorescence
[29,47]
102–104
Microtiter format assays:
colorimetric or fluorescence
[29,31]
1–102
Individual high precision assays:
GC, HPLC, mass spectrometry
[30,44••]
FACS, fluorescence-activated cell sorting; GC, gas chromatography;
HPLC, high performance liquid chromatography.
reassembly or by error-prone PCR (polymerase chain reaction) [25,26]. Improved genes are then identified and
isolated and, in a second step, are genetically recombined
(i.e., shuffled), resulting in the generation of a new gene
library that contains combinations of the mutations isolated
in the first stage. Improved genes are again identified and
isolated. The cycle, of in vitro recombination followed by the
identification of improved activity is typically repeated
between five and seven times to evolve a final product. The
rationale for performing iterative cycles is that mutations are
rare (~1%), mutations that enhance the enzyme property of
interest are therefore very rare, and combinations of beneficial mutations are consequently extremely rare. The cyclical
process allows the sequential accumulation of positive mutations at the expense of negative or neutral mutations.
The process of molecular breeding of isolated genes has parallels with conventional breeding at the organismal level.
Indeed, there are molecular methods — such as backcross
PCR, which mimics the process of backcrossing in conventional breeding — that can eliminate all but the positive
amino-acid changes in much the same way as backcrossing
removes detrimental traits during classical plant breeding
[25]. One key to success in the use of single gene shuffling
is the selection of a target gene that encodes a protein with
properties as close as possible to the desired property so that
the evolutionary distance to be traversed can be minimized
[23•]. Another is to choose a selection or screening procedure that allows large numbers of recombinant genes to be
evaluated. Perhaps the best way to achieve these goals is to
create a tiered screening system that comprises a series of
assays that are successively more accurate and time consuming and that might reduce a population from 106 or more
to 104 to 102 candidates until the optimal solution is finally
identified (Table 2).
This area of technology is still in its infancy and many of
its applications are focused at present on enzymes that are
246
Physiology and metabolism
used in industry rather than in plant biotechnology; however, the principles that apply to altering the properties of
enzymes are for the most part generic. I will therefore discuss first several examples of the current application of this
technology, and then, examples of its potential application
to plant biotechnology.
Properties such as substrate- and regio-selectivity make
enzymes desirable catalysts for the industrial synthesis of
complex chemicals. Nevertheless, enzymes are not widely
used in industrial processes because they are relatively
unstable at elevated temperatures and in organic solvents.
Directed evolution experiments are ideally suited to solve
such ‘global property’ problems because selection pressures for the desired properties can be easily controlled.
For instance, the Arnold group has used the protease subtilisin E as a model enzyme for the directed evolution of
both solvent stability (in up to 60% dimethylformamide)
[22,27] and thermal stability [28]. Great advances have also
been made in the evolution of specific properties of
enzymes and proteins. In experiments focused on substrate specificity, a fucosidase was evolved from a
galactosidase [29], and with respect to stereochemical discrimination between substrate isomers, lipase
enantioselectivity was improved from 2%–81% in favor of
the S configuration at the expense of the R isomer [30].
These experiments focused on individual proteins that do
not have complex cofactor requirements. In contrast,
enzymes such as cytochrome P450s are components of
complex electron transport chains, and therefore present a
greater technical challenge to the protein engineer who
would like to exploit their ability to perform the regiospecific insertion of oxygen into complex substrates.
Recently, Arnold and colleagues [31] devised an ingenious
hydrogen-peroxide-mediated screen for the directed evolution of the Pseudomonas putida P450cam enzyme. First, they
were able to select a mutant P450 that had 20-times greater
activity than the wild-type enzyme when using peroxide as
an oxygen source, obviating the need for the complex redox
chain. Second, coexpression of this mutant P450cam along
with the enzyme horseradish peroxidase resulted in a screen
that was used to isolate a variety of novel regiospecifically
hydroxylated products [32••]. Experiments of this type will
undoubtedly lead to the evolution of monooxygenases and
dioxygenases capable of specifically hydroxylating a wide
variety of aromatic compounds.
Multi-gene shuffling: accessing a larger
portion of sequence space
Although single gene shuffling is useful for evolving a particular property by iteratively recombining point mutations, a
logical extension of this procedure, in which two or more
homologous genes are shuffled, has proven to be even more
powerful [24•,33••]. In ‘family shuffling’, genes encoding
enzymes that share the same fold, each representing a ‘successful’ but different evolutionary pathway, replace a single
gene as the starting material. The procedure can be regarded
as ‘sexual PCR’ in that related genes are amplified under
conditions that favor crossover or intergenic recombination
[33••,34–36]. As the related enzymes share a common fold,
chimeric polypeptides are likely to be functional because
they can fold appropriately. Family shuffling reaches a far
greater portion of sequence space (i.e., the number of possible combinations and permutations) than can be reached by
single gene shuffling [24•]. Family shuffling was first used to
evolve moxalactamase activity from genes encoding
cephalosporinases [33••]. Moxalactamase activity in the best
chimera was encoded by elements from three of four parents,
and was 270-times greater than that of the most active wildtype enzyme. Shuffling of two biphenol dioxygenases
produced chimeras that had substrate specificities that were
different from those of both parents; some of the chimeras
were active even on single aromatic hydrocarbons [37••].
Application to crop improvement
The ability to tailor proteins and enzymes has potential for
broad application in crop improvement with regard to both
output and input traits. For example, enzymes that control
output traits, such as fatty acid or starch composition, are
desirable targets for the improvement of crop storage compounds. Alternatively, novel enzymes and pathways could be
developed to facilitate the accumulation of new compounds
that represent new sources of valuable industrial feedstocks.
A third application might be to create enzymes that have
abnormal allosteric activation or inhibition, as has the natural
mutant of ADP glucose pyrophosphorylase that is used to
modify starch accumulation in potato tubers [38]. If appropriate amino-acid substitutions to create desirable protein
variants can be identified from in vitro experiments, new
methodologies involving chimeric RNA/DNA oligonucleotides have recently been developed to perform
site-specific mutations of the target gene in vivo [39•,40•].
For input traits, families of proteins, for example, Bacillus
thuringiensis proteins that are involved in defense against
insects, could be recombined to produce anti-insecticidal
agents that have increased selective toxicity towards targeted
pests. Proteins that detect and/or direct responses to changing environmental conditions, such as cold [41], heat and
salinity [42], could be tailored to respond to particular
thresholds for improved agronomic performance.
Conclusions
Several approaches to identify the determinants of enzyme
specificity or protein properties can be taken. Logic-based
approaches are useful for identifying the amino acids that are
responsible for controlling particular parameters, but the
changes in activity produced by altering these amino acids
often do not extend beyond the range found in the parental
genes. In directed-evolution experiments, single-gene shuffling facilitates the alteration of a parameter in a desired
direction, whereas multi-gene shuffling facilitates the creation of novel activities with respect to the parent genes.
These technologies offer the means to engineer the properties of enzymes and proteins that control the attributes of
crop plants and their storage compounds.
Exploring the possibilities presented by protein engineering Shanklin
Update
A major focus in directed evolution has been to mimic the
pathways of natural evolution as closely as possible [24•].
This involves the use of mutagenic techniques that, for the
most part, rely on point mutagenesis that results in
between four and seven of the 19 possible amino-acid substitutions. Miyazaki and Arnold [43•] have started to
explore the inclusion of non-natural substitutions to access
a greater proportion of sequence space and to enhance conventional directed evolution experiments. They found
that non-natural substitutions allowed significant improvement in the thermal stability of the protease subtilisin S41.
Although some amino acids are essential to protein function because of the chemical properties of their side
chains, others may primarily function to occupy a particular space in the structure. Thus, it seems quite logical that
presenting a larger variety of molecular shapes would present the best likelihood of achieving the most energetically favored structure. In this context, it might be
appropriate to view amino acids as ‘molecular shims’.
The past decade has seen a movement away from rational
(i.e., structure-based) design and towards the new and
extremely powerful techniques of directed evolution
[23•,24•]. Fersht and colleagues, however, recently published a landmark paper in which these two techniques
were used synergistically to engineer a new catalytic
activity, phosphoribosylanthranilate isomerase, from the
α/β-barrel-scaffold enzyme indole-3-glycerol-phosphate
synthase [44••]. Their strategy involved first performing a
'rough cut' by rational design and comparative sequence
considerations, to create an intermediate structure that
the designers thought would approximate the required
final design. This rough-cut was then subjected to gene
shuffling and improved enzymes were selected by (partial or complete) complementation of tryptophan
auxotrophy. The work is important because it shows that
rational design and directed evolution can be used as
complementary tools in protein design. Also, because the
α/β scaffold is present in approximately 10% of all proteins, Fersht’s work elegantly demonstrates that this
particularly versatile scaffold can be redesigned to have
new specifications.
Acknowledgement
This work was supported by the Office of Basic Energy Sciences of the US
Department of Energy.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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•
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This paper describes a tour de force of enzyme redesign that clearly demonstrates the power of combining the best of rational design with directed evolution. The authors also demonstrate that the TIM-barrel fold can be
redesigned to perform quite different chemistries.
45. Naki D, Paech C, Granshaw G, Schellenberger V: Selection of a
subtillisin-hyperproducing Bacillus in a highly structured
environment. Appl Microbiol Biotechnol 1998, 49:290-294.
46. Gao C, Lin CH, Lo CHL, Mao S, Wirsching P, Lerner RA, Janda KD:
Making chemistry selectable by linking it to infectivity. Proc Natl
Acad Sci USA 1997, 94:11777-11782.
47.
Moore JC, Arnold FH: Directed evolution of a para-nitrobenzyl
esterase for aqueous-organic solvents. Nat Biotechnol 1996,
14:458-467.
Exploring the possibilities presented by protein engineering
John Shanklin
A combination of classical and powerful new combinatorial
genetic techniques allows the redesign of enzyme activities
and creation of proteins that are tailored to have specific
properties. These technologies have far-reaching
consequences for the future design of crop plants and the
storage compounds within them.
Addresses
Department of Biology, Building 463, 50 Bell Avenue, Brookhaven
National Laboratory, Upton, New York 11786, USA;
e-mail: [email protected]
Current Opinion in Plant Biology 2000, 3:243–248
1369-5266/00/$ — see front matter. Published by Elsevier Science Ltd.
Abbreviations
ACP
acyl-carrier protein
DIBOA 2,4-dihydroxy-1,4-benzoxazin-3-one
PCR
polymerase chain reaction
Introduction
Gene transfer into all common crop species can now be
achieved routinely. This progress raises the issue of
which genes should be introduced to convey desired
traits. Genomic sequencing efforts have provided a rich
resource of genetic material from which genes encoding
valuable traits can be selected [1]. Nevertheless, even
with this ever-increasing resource, genes with ideal
properties may not be found. For instance, transferred
genes may not perform in the transgenic plant as they
did in the plant from which they were taken, and therefore the phenotype of interest may not be conferred to
the transgenic plant. Such was the case when the 16:0∆4-desaturase gene from coriander was transferred to
tobacco. The accumulation of this unusual fatty acid was
much smaller in tobacco than in coriander [2]. For naming fatty acids, X:Y indicates that a fatty acid contains X
carbons atoms and Y number of double bonds. ∆z indicates that a double bond is located at the z carbon atom
relative to the carboxyl terminus of the fatty acid. A second problem might be that the available genes, though
exhibiting functional diversity, might not include a gene
that has ideal properties. An alternative approach to
transferring naturally occurring genes into crops is to
extract their information content and to use this to
redesign desired properties, or to use them as starting
points for combinatorial genetic manipulation to evolve
a property of interest.
The intent of this review is to bring to the attention of
plant biologists examples of approaches that have proven
successful in protein redesign. Because of the broad
scope of this review, readers are directed to a number of
excellent reviews on specific topics.
Evolution of enzymatic plasticity
In the order of 103 individual protein folds (the core
arrangement of secondary structural elements) are thought
to occur in nature, far fewer than the number of individual
proteins (which is closer to 105) [3]. This is because many
proteins share common folds. Many proteins that have the
same fold are presumed to have arisen from a common
ancestor [4]; hence enzymes with the same fold perform a
variety of reactions. An illustration of gene duplication and
functional specialization was presented by Gierl and colleagues [5,6] who reported on the biosynthetic pathway of
the plant defense compound 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA). They found that genes Bx2 through
Bx5 encode cytochrome P450-dependent monooxygenases (P450s) that catalyze four consecutive hydroxylations
and one ring expansion to form DIBOA. These P450s are
thought to have arisen though gene duplication and the
accumulation of mutations that led to a change in both
substrate- and regio-specificity (i.e., the position on the
substrate molecule that is modified). In another example
from the family of membrane-bound di-iron enzymes
[7,8•,9•], mutations affecting the desaturase reaction have
given rise to at least four distinct enzymatic activities:
hydroxylase, epoxidase, acetylenase and conjugase. New
catalytic activities are now widely thought to evolve from
pre-existing enzymes when a partial reaction, which is catalyzed by a progenitor enzyme, is retained but the
architecture of the active-site of the enzyme is modified so
as to allow the intermediate generated to be directed into
the synthesis of different end-products [10].
Comparative structure–function analysis
Gene fusions can be used to identify domains within
enzymes that control properties of interest. Once such a
region is identified, site-directed mutants can be constructed
and used to evaluate the contribution of individual aminoacid residues. Examples of the successful application of this
approach include changing an acyl-ACP (acyl-carrier protein)
Table 1
Techniques for improving enzymes and the resulting
information.
Technique
Outcome
References
Comparative
structure/function
analysis
Identification of key residues
Activity switching
Novel activities
Rational design
Structural
Computational
Novel activities
Novel activities
Directed evolution
Single gene
Multi-gene
Improvements in desired properties [29]
Novel activities
[37••]
[11]
[16••]
[12••]
[13]
[19]
244
Physiology and metabolism
Figure 1
Summary of the principles of gene shuffling.
Mutations result in positive (䊊) or negative
(䊉) phenotypes.
Single gene
Random fragmentation
Pool of random
DNA fragments
Reassembly PCR
Mutagenic
Family of related
DNA sequences
Random fragmentation
Cycle
Pool of random
DNA fragments
Reassembly PCR
Mutagenic & recombinogenic
Library of
recombinant
genes
Identify clones with
combinations of
positive mutations
Screen
Eliminate clones with
negative mutations
Current Opinion in Plant Biology
thioesterase with 12:0 specificity into one specific for 14:0 by
substituting three amino acids [11], or increasing the 18:0substrate preference of an acyl-ACP thioesterase up to
13-fold [12••] by site-specific mutagenesis. In a more complex example, both the chain length- and the
regio-specificity of a 16:0-∆6-ACP desaturase was converted
into predominantly an 18:0-∆9-ACP desaturase by substituting five amino acids [13].
Key amino acids are most easily identified by this genefusion approach when the residues that contribute to the
interesting properties reside in a linear domain within the
sequence. Within the crystal structure of the 18:0-∆9-ACP
desaturase [14], the determinants of acyl-chain length
(substrate) specificity and regiospecificity can be classified
into two types: those amino acids that directly affect the
substrate binding interface and those occupying positions
remote from the binding cavity that must exert their effects
indirectly [13]. The logical gene-fusion approach is better
suited to structure–function studies than to efforts to
redesign proteins. This is because the outcome of genefusion experiments is usually to define the positions of key
residues that affect the property of interest rather than to
facilitate the design of novel activities (Table 1). It is possible, however, that information generated from gene-fusion
experiments could subsequently be used, either alone [12••]
or in conjunction with a combinatorial method [15], to
generate novel activities.
Exploring the possibilities presented by protein engineering Shanklin
A second logical approach is available when several homologous amino acid sequences are associated with two distinct
enzyme activities (paralogs). For example, Broun et al. [16••]
compared the sequences of five related oleate desaturases
with those of two independently-evolved oleate hydroxylases. Seven conserved positions within the five desaturases
were identified at which the residues differed from those at
equivalent positions in the two hydroxylases. Reciprocal
changes between pairs composed of one of the desaturases
and one of the hydroxylases were made at the seven sites,
resulting in dramatic shifts in the ratio of desaturase to
hydroxylase function, that is, away from the original enzyme
activity and towards that of the alternate activity. An advantage of this logic-based approach is that the number, as well
as the relative position, of key residues is unimportant.
Rational design
The application of protein crystallography to enzymes
brought with it the expectation that enzymes could be rationally designed to have desired properties. Although there
are examples of successful rational design, such as the engineering of a novel 16:0-∆9-ACP desaturase [13], and the
design of a thermolysin-like protease from Bacillus stearothermophilus that has improved thermostability [17•], this
method has, so far, generally failed to meet expectations.
There are many reasons for this, one being that changes in
the residues of the active-site often produce the desired
results in terms of specificity, but at a large cost to catalytic
rate. In addition, many residues that control key enzyme
properties occupy positions that are distant from the active
site and that are not readily identified by the examination of
a crystal structure. Consequently, there has been a steady
shift in the emphasis of enzyme redesign studies from ‘rational’ to ‘irrational’ (i.e., combinatorial) genetic approaches
[18]. Despite this general trend, there is also renewed interest in a specific method of rational design, pioneered by
Mayo and colleagues [19]. This method involves cycling
between new computational methods and experiments [20].
Their computational approach identifies probable functional solutions using sophisticated algorithms that eliminate
dead-end design solutions [21].
Gene shuffling: directed evolution of single
genes
It is clear that many enzymes with very different properties have evolved one from another in nature. Researchers
have therefore explored the possibility of ‘directing evolution’ or molecular breeding in the laboratory [22]. For
instance, the Arnold laboratory at Caltech [23•] and the
Stemmer group at the company Maxygen [24•] have developed powerful methodologies for mimicking the process of
natural evolution using isolated genes in the test tube.
Evolution that would take eons in nature can now occur
within days or weeks in the laboratory.
The strategy for evolving enzymes involves two steps
(Figure 1). In the first step, random mutations are introduced
into the target gene either by fragmentation followed by
245
Table 2
Appropriate methods for tiered-screen selection of the best
performing individual clones from large combinatorial
populations.
Number of individual
candidate clones
Assay method
References
>107
FACS, genetic selection,
phage display
[33••,45,46]
104–106
Solid state assays:
colorimetric or fluorescence
[29,47]
102–104
Microtiter format assays:
colorimetric or fluorescence
[29,31]
1–102
Individual high precision assays:
GC, HPLC, mass spectrometry
[30,44••]
FACS, fluorescence-activated cell sorting; GC, gas chromatography;
HPLC, high performance liquid chromatography.
reassembly or by error-prone PCR (polymerase chain reaction) [25,26]. Improved genes are then identified and
isolated and, in a second step, are genetically recombined
(i.e., shuffled), resulting in the generation of a new gene
library that contains combinations of the mutations isolated
in the first stage. Improved genes are again identified and
isolated. The cycle, of in vitro recombination followed by the
identification of improved activity is typically repeated
between five and seven times to evolve a final product. The
rationale for performing iterative cycles is that mutations are
rare (~1%), mutations that enhance the enzyme property of
interest are therefore very rare, and combinations of beneficial mutations are consequently extremely rare. The cyclical
process allows the sequential accumulation of positive mutations at the expense of negative or neutral mutations.
The process of molecular breeding of isolated genes has parallels with conventional breeding at the organismal level.
Indeed, there are molecular methods — such as backcross
PCR, which mimics the process of backcrossing in conventional breeding — that can eliminate all but the positive
amino-acid changes in much the same way as backcrossing
removes detrimental traits during classical plant breeding
[25]. One key to success in the use of single gene shuffling
is the selection of a target gene that encodes a protein with
properties as close as possible to the desired property so that
the evolutionary distance to be traversed can be minimized
[23•]. Another is to choose a selection or screening procedure that allows large numbers of recombinant genes to be
evaluated. Perhaps the best way to achieve these goals is to
create a tiered screening system that comprises a series of
assays that are successively more accurate and time consuming and that might reduce a population from 106 or more
to 104 to 102 candidates until the optimal solution is finally
identified (Table 2).
This area of technology is still in its infancy and many of
its applications are focused at present on enzymes that are
246
Physiology and metabolism
used in industry rather than in plant biotechnology; however, the principles that apply to altering the properties of
enzymes are for the most part generic. I will therefore discuss first several examples of the current application of this
technology, and then, examples of its potential application
to plant biotechnology.
Properties such as substrate- and regio-selectivity make
enzymes desirable catalysts for the industrial synthesis of
complex chemicals. Nevertheless, enzymes are not widely
used in industrial processes because they are relatively
unstable at elevated temperatures and in organic solvents.
Directed evolution experiments are ideally suited to solve
such ‘global property’ problems because selection pressures for the desired properties can be easily controlled.
For instance, the Arnold group has used the protease subtilisin E as a model enzyme for the directed evolution of
both solvent stability (in up to 60% dimethylformamide)
[22,27] and thermal stability [28]. Great advances have also
been made in the evolution of specific properties of
enzymes and proteins. In experiments focused on substrate specificity, a fucosidase was evolved from a
galactosidase [29], and with respect to stereochemical discrimination between substrate isomers, lipase
enantioselectivity was improved from 2%–81% in favor of
the S configuration at the expense of the R isomer [30].
These experiments focused on individual proteins that do
not have complex cofactor requirements. In contrast,
enzymes such as cytochrome P450s are components of
complex electron transport chains, and therefore present a
greater technical challenge to the protein engineer who
would like to exploit their ability to perform the regiospecific insertion of oxygen into complex substrates.
Recently, Arnold and colleagues [31] devised an ingenious
hydrogen-peroxide-mediated screen for the directed evolution of the Pseudomonas putida P450cam enzyme. First, they
were able to select a mutant P450 that had 20-times greater
activity than the wild-type enzyme when using peroxide as
an oxygen source, obviating the need for the complex redox
chain. Second, coexpression of this mutant P450cam along
with the enzyme horseradish peroxidase resulted in a screen
that was used to isolate a variety of novel regiospecifically
hydroxylated products [32••]. Experiments of this type will
undoubtedly lead to the evolution of monooxygenases and
dioxygenases capable of specifically hydroxylating a wide
variety of aromatic compounds.
Multi-gene shuffling: accessing a larger
portion of sequence space
Although single gene shuffling is useful for evolving a particular property by iteratively recombining point mutations, a
logical extension of this procedure, in which two or more
homologous genes are shuffled, has proven to be even more
powerful [24•,33••]. In ‘family shuffling’, genes encoding
enzymes that share the same fold, each representing a ‘successful’ but different evolutionary pathway, replace a single
gene as the starting material. The procedure can be regarded
as ‘sexual PCR’ in that related genes are amplified under
conditions that favor crossover or intergenic recombination
[33••,34–36]. As the related enzymes share a common fold,
chimeric polypeptides are likely to be functional because
they can fold appropriately. Family shuffling reaches a far
greater portion of sequence space (i.e., the number of possible combinations and permutations) than can be reached by
single gene shuffling [24•]. Family shuffling was first used to
evolve moxalactamase activity from genes encoding
cephalosporinases [33••]. Moxalactamase activity in the best
chimera was encoded by elements from three of four parents,
and was 270-times greater than that of the most active wildtype enzyme. Shuffling of two biphenol dioxygenases
produced chimeras that had substrate specificities that were
different from those of both parents; some of the chimeras
were active even on single aromatic hydrocarbons [37••].
Application to crop improvement
The ability to tailor proteins and enzymes has potential for
broad application in crop improvement with regard to both
output and input traits. For example, enzymes that control
output traits, such as fatty acid or starch composition, are
desirable targets for the improvement of crop storage compounds. Alternatively, novel enzymes and pathways could be
developed to facilitate the accumulation of new compounds
that represent new sources of valuable industrial feedstocks.
A third application might be to create enzymes that have
abnormal allosteric activation or inhibition, as has the natural
mutant of ADP glucose pyrophosphorylase that is used to
modify starch accumulation in potato tubers [38]. If appropriate amino-acid substitutions to create desirable protein
variants can be identified from in vitro experiments, new
methodologies involving chimeric RNA/DNA oligonucleotides have recently been developed to perform
site-specific mutations of the target gene in vivo [39•,40•].
For input traits, families of proteins, for example, Bacillus
thuringiensis proteins that are involved in defense against
insects, could be recombined to produce anti-insecticidal
agents that have increased selective toxicity towards targeted
pests. Proteins that detect and/or direct responses to changing environmental conditions, such as cold [41], heat and
salinity [42], could be tailored to respond to particular
thresholds for improved agronomic performance.
Conclusions
Several approaches to identify the determinants of enzyme
specificity or protein properties can be taken. Logic-based
approaches are useful for identifying the amino acids that are
responsible for controlling particular parameters, but the
changes in activity produced by altering these amino acids
often do not extend beyond the range found in the parental
genes. In directed-evolution experiments, single-gene shuffling facilitates the alteration of a parameter in a desired
direction, whereas multi-gene shuffling facilitates the creation of novel activities with respect to the parent genes.
These technologies offer the means to engineer the properties of enzymes and proteins that control the attributes of
crop plants and their storage compounds.
Exploring the possibilities presented by protein engineering Shanklin
Update
A major focus in directed evolution has been to mimic the
pathways of natural evolution as closely as possible [24•].
This involves the use of mutagenic techniques that, for the
most part, rely on point mutagenesis that results in
between four and seven of the 19 possible amino-acid substitutions. Miyazaki and Arnold [43•] have started to
explore the inclusion of non-natural substitutions to access
a greater proportion of sequence space and to enhance conventional directed evolution experiments. They found
that non-natural substitutions allowed significant improvement in the thermal stability of the protease subtilisin S41.
Although some amino acids are essential to protein function because of the chemical properties of their side
chains, others may primarily function to occupy a particular space in the structure. Thus, it seems quite logical that
presenting a larger variety of molecular shapes would present the best likelihood of achieving the most energetically favored structure. In this context, it might be
appropriate to view amino acids as ‘molecular shims’.
The past decade has seen a movement away from rational
(i.e., structure-based) design and towards the new and
extremely powerful techniques of directed evolution
[23•,24•]. Fersht and colleagues, however, recently published a landmark paper in which these two techniques
were used synergistically to engineer a new catalytic
activity, phosphoribosylanthranilate isomerase, from the
α/β-barrel-scaffold enzyme indole-3-glycerol-phosphate
synthase [44••]. Their strategy involved first performing a
'rough cut' by rational design and comparative sequence
considerations, to create an intermediate structure that
the designers thought would approximate the required
final design. This rough-cut was then subjected to gene
shuffling and improved enzymes were selected by (partial or complete) complementation of tryptophan
auxotrophy. The work is important because it shows that
rational design and directed evolution can be used as
complementary tools in protein design. Also, because the
α/β scaffold is present in approximately 10% of all proteins, Fersht’s work elegantly demonstrates that this
particularly versatile scaffold can be redesigned to have
new specifications.
Acknowledgement
This work was supported by the Office of Basic Energy Sciences of the US
Department of Energy.
References and recommended reading
Papers of particular interest, published within the annual period of review,
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• of special interest
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•
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unexpected and useful substrate specificities with respect to polychlorinated biphenyls (which are particularly resistant to catalytic degradation).
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