Combinatorial Chemistry & High Throughput Screening - Volume 9, Issue 4, 2006

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.

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 &gt6-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.

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.

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.

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 costeffectiveness. 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.

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.

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.

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 1μM in order to be most applicable for evaluating binding specificity in an in vivo setting.

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 GUSp6-( 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.

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.

Dr. Edgardo T. Farinas did his undergraduate work at Loyola University of Chicago (B.S. in 1990) and pursued graduate studies in bioinorganic chemistry at the University of California at Santa Cruz (Ph.D. in 1997) under Pradip Mascharak. After postdoctoral studies with Lynne Regan at Yale University, Frances Arnold at the California Institute of Technology, and Brent Iverson and George Georgiou at University of Texas at Austin, he joined the New Jersey Institute of Technology faculty as an Assistant Professor of Chemistry. His research has focused on the interface between chemistry, biology, and engineering. His current research interests are in engineering proteins using directed evolution and rational approaches. The goals include developing high-throughput screening technologies to assay mutant enzyme libraries to discovery novel biocatalyst, combining rational and directed evolution approaches to create de novo enzymes, metabolic pathway engineering in bacteria, novel protein display technologies, and incorporation of non-natural amino acids in proteins. SELECTED PUBLICATIONS [1] Alcalde, M.; Farinas, E.T.; Arnold, F.H. J. Biomolecular Screen., 2004, 9, 141-146. [2] Farinas, E.T.; Alcalde, M.; Arnold, F.H. Tetrahedron, 2003, 60, 525-528. [3] Glieder, A.; Farinas, E.T.; Arnold, F.H. Nat. Biotechnol., 2002, 20, 1135-1139. [4] Farinas, E.T.; Schwaneberg, U.; Glieder, A.; Arnold, F.H. Advanced Synthesis and Catalysis, 2001, 343, 601-606. [5] Schwaneberg, U.; Otey, C.; Cirino, P.C.; Farinas, E.T.; Arnold, F.H. J. Biomolecular Screen., 2001, 6, 111-118. [6] Farinas, E.T.; Regan, L. Protein Science, 1998, 7, 1939-1946. [7] Farinas, E.T.; Nguyen, C.; Mascharak, P.K. Inorganica Chimica Acta, 1997, 263, 17-21. [8] Farinas, E.T.; Tan, J.D.; Mascharak, P.K. Inorg. Chem., 1996, 35, 2637. [9] Guajardo, R.J.; Chavez, F.; Farinas, E.T.; Mascharak, P.K. J. Am. Chem. Soc., 1995, 117, 3883. [10] Farinas, E.T.; Baidya, N.; Mascharak, P.K. Inorg. Chem., 1994, 33, 5970. [11] Tan, J.D.; Farinas, E.T.; David, S.S.; Mascharak, P.K. Inorg. Chem., 1994, 33, 4295. [12] Farinas, E.T.; Tan, J.D.; Baidya, N.; Mascharak, P.K. J. Am. Chem. Soc., 1993, 115, 2996.