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Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 9, Number 4, May 2006
Contents
Directed Evolution Approaches for Protein Engineering
Guest Editor: Edgardo T. Farinas
Editorial Pp. 235
Optimizing a Biocatalyst for Improved
NAD(P)H Regeneration: Directed Evolution of Phosphite Dehydrogenase
Pp. 237-245
Ryan Woodyer, Wilfred A. van der Donk and Huimin Zhao
[Abstract]
Recent Advances in Biocatalysis by Directed Enzyme Evolution
Pp. 247-257
Sheryl B. Rubin-Pitel and Huimin Zhao
[Abstract]
Towards the Creation of Novel Proteins by Block
Shuffling Pp. 259-269
Toru Tsuji, Michiko Onimaru and Hiroshi Yanagawa
[Abstract]
The Diversity Challenge in Directed Protein Evolution
Pp. 271-288
Tuck Seng Wong, Daria Zhurina and Ulrich Schwaneberg
[Abstract]
A Filter Paper-Based Assay for Laboratory Evolution of Hydrolases
and Dehydrogenases Pp. 289-293
Tuck Seng Wong, Ulrich Schwaneberg, Rainer Stürmer,
Bernhard Hauer and Michael Breuer
[Abstract]
High-Throughput Selection System for Assessing
the Activity of Epoxide Hydrolases Pp. 295-299
Manfred T. Reetz and Li-Wen Wang
[Abstract]
A Bacterial One-Hybrid Selection System for Interrogating
Zinc Finger-DNA Interactions Pp. 301-311
Sundar Durai, Allen Bosley, Alice Bridgeman Abulencia,
Srinivasan Chandrasegaran and Marc Ostermeier
[Abstract]
HIV Protease-Activated Molecular Switches Based on
Beta-Glucuronidase and Alkaline Phosphatase Pp. 313-320
Taryn L. O’Loughlin and Ichiro Matsumura
[Abstract]
Fluorescence Activated Cell Sorting for Enzymatic
Activity Pp. 321-328
Edgardo T. Farinas
[Abstract]
Meet the Guest
Editor Pp. 329
Abstracts
[Back to top]
Editorial
The central aim of protein engineering is the efficient
creation of novel and practical biocatalysts and to understand
structure/function relationships. The ultimate goal would
be to create proteins designed to order on the lab bench.
An efficient protein engineering strategy is necessary to
design enzymes with improved properties. The basic strategies
for protein engineering include rational design and directed
evolution approaches. Rational design methods, such as site-directed
mutagenesis, has been met with limited success due to our
incomplete knowledge of structure/function relationships.
On the other end of the spectrum, directed evolution approaches
rely on iterative cycles of random mutagenesis and/or recombination
to create large libraries of variants that are coupled to
an efficient selection or screening strategy for identifying
mutants with improved performance. In this issue of Combinatorial
Chemistry & High Throughput Screening, we have assembled
a collection of review and research articles using directed
evolution approaches for protein engineering.
The review by Rubin-Pitel and Zhao summarizes the recent achievements
in biocatalyst engineering by directed evolution. The manuscript
focuses on altering activity, selectivity, substrate specificity,
stability, and solubility. The creation of novel enzyme activity
and products are highlighted.
Library creation is an important aspect of directed evolution.
The review by Wong et al. addresses the diversity
challenge of how to generate unbiased gene libraries by random
mutagenesis. The manuscript is a comprehensive survey that
summarizes, categorizes, and compares the methods for creating
genetic diversity. The review is particularly useful for research
labs that do not have experience with directed evolution methods.
Gilbert proposes the exon theory of genes that suggests the
first genes were composed of a combination of small polypeptide
chains or blocks. The process of creating new genes and protein
evolution is a fundamental question that may never be answered.
The review by Tsuji et al. outlines their approach
to create novel proteins by block shuffling. The authors explores
the foldability and enzyme activity of mutants created by
permutations of modules or secondary structural units. In
order to create their libraries, a new DNA recombination approach
was developed to access sequence space that is not accessible
through conventional methods such as DNA shuffling or family
shuffling. This contribution summarizes the strategy to create
proteins by block shuffling and the possible applications.
A key for a successful directed evolution experiment is often
the screening assay. Fluorescence activated cell sorting (FACS)
is powerful high-throughput screening approach to isolate
and identify mutants from large protein libraries. FACS has
been applied successfully in isolating proteins with improved
or altered binding affinity. However, FACS screening for mutants
with enhanced catalytic active has been met with limited success.
The review by Farinas focuses on the FACS screening of protein
libraries for enzymatic activity.
Creating proteins that can specifically recognize a designed
DNA sequence continues to be challenge. Duria et al.
have developed two bacterial one-hybrid systems to examine
and select for zinc-finger/DNA interactions in vivo.
The one-hybrid system is composed of a plasmid containing
the gene for the zinc-finger fused to a fragment of RNA polymerase,
and the reporter plasmid has the punitive zinc-finger binding
site upstream the reporter. The advantages and the appropriate
applications for this system are discussed.
The cofactor requirement might limit the industrial applications
for NAD(P)H-dependent oxidoreductases since the pyridine cofactors
are very expensive. Hence, cofactor regeneration systems are
viable solutions to this problem. Woodyer et al.
used a combination of directed evolution approaches and rational
design methods to optimize phosphite dehydrogenase for NAD(P)H
regeneration.
High-throughput screens for enantioselective enzymes are oftentimes
time-consuming, and eliminating inactive mutants with a pre-screen/selection
may provide a more streamlined process. Reetz and Wang have
developed a pre-selection to eliminate inactive epoxide hydroylase
mutants. The selection is based on the ability of an active
epoxide hydrolase to catalyze the hydrolysis of the toxic
epoxide substrate.
Wong et al. have developed a simple and economical
high-throughput pre-screen for hydrolase and dehydrogenase
activity that is based on the detection of aldehydes. Hydrolases
and dehydrogenases have industrial applications for the synthesis
of optically active amines and alcohols. Furthermore, the
reverse reaction also can be useful to generate amides or
esters via transfer of acyl moiety from an acyl donor
compound to an acceptor.
O’Loughlin and Matsumura have created a novel protease-activated
reporter enzyme to screen for protease activity, and β-glucuronidase
and alkaline phosphate were used as model systems. These enzymes
were engineered to contain three peptides attached to the
C-terminus of the proteins. The first contains a protease
cleavage site which activates the enzyme. The next peptide
contains an epitope to monitor expression, and the last peptide
contains a random sequence of twelve amino acids. Screening
mutant libraries of β-glucuronidase
and alkaline phosphate identified variants that are activated
upon peptide cleavage.
In conclusion, directed evolution is a reliable tool to improve
properties of proteins which can be used for industrial, pharmaceutical,
and biotechnological applications. Laboratory evolution is
also being used to elucidate complicated structure/function
relationships which will help build the “rules”
or principles for protein design. Furthermore, it is becoming
a general method to manipulate metabolic pathways, create
new screening systems, and understand the natural process
of evolution. In years to come, laboratory evolution approaches
will be used routinely in research labs, and it might eventually
be found as common experiments in undergraduate biochemistry
laboratory courses.
Edgardo T. Farinas
Department of Chemistry and Environmental Science
New Jersey Institute of Technology
University Heights
Newark, NJ 07102
USA
E-mail: Farinas@adm.njit.edu
[Back to top]
Optimizing a Biocatalyst for Improved NAD(P)H Regeneration:
Directed Evolution of Phosphite Dehydrogenase
Ryan Woodyer, Wilfred A. van der Donk and Huimin Zhao
Cofactor regeneration is an important solution to the problem
of implementing complex cofactor requiring enzymatic reactions
at the industrial scale. NAD(P)H-dependent oxidoreductases
are highly valuable biocatalysts, but the high cost of the
nicotinamide cofactors necessitates in situ cofactor
regeneration for preparative applications. Here we report
the use of directed evolution to enhance the industrially
important properties of phosphite dehydrogenase for NAD(P)H
regeneration. A two-tiered sorting method of selection and
screening was used in conjunction with random and rational
mutagenesis. Following six rounds of directed evolution, soluble
expression in E. coli was increased more than 3-fold,
while the turnover rate was increased about 2-fold, effectively
lowering the cost of the enzyme by >6-fold. Large-scale
production of the final mutant enzyme by fermentation resulted
in ~6-times higher yield (Units/Liter) than the WT enzyme.
The enhancements of PTDH were independent of expression vector
and E. coli strain utilized. The advantage of the
final mutant over the WT enzyme was demonstrated using the
industrially relevant bioconversion of trimethylpyruvate to
L-tert-leucine. The mutations discovered are discussed
in the context of a three dimensional structural model and
the resulting changes in kinetics and soluble expression.
The engineered phosphite dehydrogenase has great potential
for NAD(P)H regeneration in industrial biocatalysis.
[Back to top]
Recent Advances in Biocatalysis by Directed Enzyme
Evolution
Sheryl B. Rubin-Pitel and Huimin Zhao
Naturally occurring enzymes are remarkable biocatalysts with
numerous potential applications in industry and medicine.
However, many of their catalyst properties often need to be
further tailored to meet the specific requirements of a given
application. Within this context, directed evolution has emerged
over the past decade as a powerful tool for engineering enzymes
with new or improved functions. This review summarizes recent
advances in applying directed evolution approaches to alter
various enzyme properties such as activity, selectivity (enantio-
and regio-), substrate specificity, stability, and solubility.
Special attention will be paid to the creation of novel enzyme
activities and products by directed evolution.
[Back to top]
Towards the Creation of Novel Proteins by Block Shuffling
Toru Tsuji, Michiko Onimaru and Hiroshi Yanagawa
We have been investigating the creation of novel proteins
by means of block shuffling, where the term block refers to
an amino acid sequence that corresponds to particular features
of proteins, such as secondary structures, modules, functional
motifs, and so on. Block shuffling makes it possible to explore
the global sequence space, which is not feasible with conventional
methods, such as DNA shuffling or family shuffling. To investigate
what properties are required for the building blocks, we have
analyzed the foldability and enzymatic activity of barnase
mutants obtained by permutation of modules or secondary structure
units. This reconstructive approach indicated that secondary
structure units with mutual long-range interactions are more
suitable than modules as building blocks, at least in the
case of barnase. The results also suggested that proteins
in evolutionarily intermediate states are created by block
shuffling, and such proteins have the potential to be evolved
into mature globular proteins. For the construction of combinatorial
protein libraries, we have developed random multi-recombinant
PCR (RM-PCR), which can combine different DNA fragments without
homologous sequences. The libraries can be utilized for in
vitro selection using in vitro virus (mRNA display)
or stable (DNA display), which have also been developed in
our laboratory. In this review article, we summarize our strategy
to create novel proteins by block shuffling and review key
literature in the field. Possible applications of the block
shuffling strategy are also discussed.
[Back to top]
The Diversity Challenge in Directed Protein Evolution
Tuck Seng Wong, Daria Zhurina and Ulrich Schwaneberg
Over the past decade, we have witnessed a bloom in the field
of evolutive protein engineering which is fueled by advances
in molecular biology techniques and high-throughput screening
technology. Directed protein evolution is a powerful algorithm
using iterative cycles of random mutagenesis and screening
for tailoring protein properties to our needs in industrial
applications and for elucidating proteins’ structure
function relationships.
This review summarizes, categorizes and discusses advantages
and disadvantages of random mutagenesis methods used for generating
genetic diversity. These random mutagenesis methods have been
classified into four main categories depending on the method
employed for nucleotide substitutions: enzyme based methods
(Category I), synthetic chemistry based methods (Category
II), whole cell methods (Category III) and combined methods
(Category I-II, I-III and II-III). The basic principle of
each method is discussed and varied mutagenic conditions are
summarized in Tables and compared (benchmarked) to each other
in terms of: mutational bias, controllable mutation frequency,
ability to generate consecutive nucleotide substitutions and
subset diversity, dependency on gene length, technical simplicity/robustness
and cost-effectiveness. The latter comparison shows how highly-biased
and limited current diversity creating methods are. Based
on these limitations, strategies for generating diverse mutant
libraries are proposed and discussed (RaMuS-Flowchart; KISS
principle).
We hope that this review provides, especially for researchers
just entering the field of directed evolution, a guide for
developing successful directed evolution strategies by selecting
complementary methods for generating diverse mutant libraries.
[Back to top]
A Filter Paper-Based Assay for Laboratory Evolution
of Hydrolases and Dehydrogenases
Tuck Seng Wong, Ulrich Schwaneberg, Rainer Stürmer,
Bernhard Hauer and Michael Breuer
Industrially important enzyme classes such as hydrolases
and dehydrogenases are often not amenable to laboratory evolution
methods due to a lack of sensitive and reliable high-throughput
screening (HTS) systems. We developed a conceptually novel
and technically simple high-throughput screening system based
on detection of volatile aldehydes with the sensitive reagent
Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The
aldehyde detection takes place on a filter-paper that is pre-soaked
with Purpald and covers the microtiter plate. The filter paper-based
Purpald assay separates aldehyde detection from biocatalytical
conversion and thereby avoids interferences from biological
materials with assay components. This screening principle
allows, to our knowledge, for the first time to determine
the synthetic activity of hydrolases such as lipases and esterases
in organic solvents in a 96-well whole-cell format. Its simplicity
and cost-effectiveness make the reported HTS system suitable
as fast pre-screen in laboratory evolution experiments and
for semi-quantitative assays of improved mutants.
[Back to top]
High-Throughput Selection System for Assessing the
Activity of Epoxide Hydrolases
Manfred T. Reetz and Li-Wen Wang
Crucial to the success of directed evolution of enantioselective
enzymes for use as catalysts in synthetic organic chemistry
is the availability of high-throughput assays for determining
the enantiopurity of thousands of samples. Although several
such ee-assays are available, they entail time and
effort, which means that pre-tests for activity have been
developed to eliminate non-active mutants prior to ee-screening.
Pre-selection systems may be even more efficient and simple
to perform. In the present paper an efficient pre-selection
test for assessing the activity of epoxide hydrolases has
been developed. The bacterial (E. coli) growth on
agar plates is shown to be directly related to the presence
of active epoxide hydrolases which catalyze the detoxicating
hydrolysis of the epoxide substrates. Visual inspection of
agar plates is all that is necessary to identify positive
(active) hits in large libraries of mutant epoxide hydrolases.
[Back to top]
A Bacterial One-Hybrid Selection System for Interrogating
Zinc Finger-DNA Interactions
Sundar Durai, Allen Bosley, Alice Bridgeman Abulencia,
Srinivasan Chandrasegaran and Marc Ostermeier
We have developed two bacterial one-hybrid systems for interrogating
and selecting zinc finger-DNA interactions. Our systems utilize
two plasmids: a zinc finger-plasmid containing the gene for
the zinc finger fused to a fragment of the alpha subunit of
RNA polymerase and a reporter plasmid where the zinc finger-binding
site is located upstream of a reporter gene–either the
gene encoding the green fluorescent protein (GFP) or chloramphenicol
acetyltransferase (CAT). Binding of the zinc finger domain
to the target binding site results in a 10-fold increase in
chloramphenicol resistance with the CAT reporter and an 8-
to 22-fold increase in total cell fluorescence with the GFP
reporter. The CAT reporter allows for sequence specific zinc
fingers to be isolated in a single selection step whereas
the GFP reporter enables quantitative evaluation of libraries
using flow cytometry and theoretically allows for both negative
and positive selection. Both systems have been used to select
for zinc fingers that have affinity for the motif 5’-GGGGCAGAA-3’
from a library of approximately 2 x 105
variants. The systems have been engineered to report on zinc
finger-DNA binding with dissociation constants less than about
1 μM
in order to be most applicable for evaluating binding specificity
in an in vivo setting.
[Back to top]
HIV Protease-Activated Molecular Switches Based on
Beta-Glucuronidase and Alkaline Phosphatase
Taryn L. O’Loughlin and Ichiro Matsumura
Our long-term goal is to direct the evolution of novel protease
variants. To this end we have engineered a new type of protease-activated
reporter enzyme. Many protease-activated enzymes evolved in
nature, but the introduction of novel regulatory mechanisms
into normally unregulated enzymes poses a difficult design
challenge. Random Elongation Mutagenesis [1] was used to fuse
the p6 peptide, which is recognized and cleaved by HIV protease,
and twelve random sequence amino acids to the C-termini of
beta-glucuronidase (GUS) and alkaline phosphatase (AP). The
resulting GUS-p6-(NNN)12 and AP-p6-(NNN)12
libraries were expressed in E. coli and screened
for clones that were inactivated by the C-terminal extension
(tail). The inactivated clones were co-expressed with HIV
protease, and those that were re-activated were isolated.
The AP and GUS activities of the most responsive clones were
each >3.5-fold higher when co-expressed with HIV protease,
and this activation is correlated with in vivo proteolysis.
It should be possible to generalize this strategy to different
reporter enzymes, different target proteases, and perhaps
to other types of protein-modifying enzymes.
[Back to top]
Fluorescence Activated Cell Sorting for Enzymatic
Activity
Edgardo T. Farinas
Directed evolution is a reliable method for protein engineering
and as a tool for investigating structure/function relationships.
A key for a successful directed evolution experiment is oftentimes
the screen. Fluorescence activated cell sorting (FACS) is
powerful high-throughput screening approach to isolate and
identify mutants from large protein libraries. FACS has been
successful in isolating proteins with improved or altered
binding affinity. However, FACS screening for mutants with
enhanced catalytic activity has been met with limited success.
This review focuses on the FACS screening of protein libraries
for enzymatic activity.
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