Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.     

Directed evolution of sulfotransferases and paraoxonases by ancestral libraries

Large libraries of randomly mutated genes are applied in directed evolution experiments in order to obtain sufficient variability. These libraries, however, contain mostly inactive variants, and the very low frequency of improved variants can only be isolated by high-throughput screening. Small but efficient libraries comprise an attractive alternative. Here, we describe the application of ancestral libraries—libraries based on mutations predicted by phylogenetic analysis and ancestral inference. We designed and constructed such libraries using serum paraoxonases and cytosolic sulfotransferases (SULTs) as model enzymes. Both of these enzyme families exhibit a range of activities in drug metabolism and detoxification of xenobiotics. The ancestral serum paraoxonase and SULT libraries were screened by low-throughput means, including HPLC, using substrates and/or reactions with which all family members exhibit low activity. The libraries showed a remarkably high frequency of highly polymorphic and functionally diverse variants. Screening of as few as 300 variants enabled the isolation of mutants with up to 50-fold higher activity than the starting point enzyme. Structural and kinetic characterizations of an evolved SULT variant show how few ancestral mutations reshaped the active site and modulated the enzyme's specificity. Ancestral libraries therefore comprise a means of focusing diversity to positions and mutations that readily trigger changes in substrate and/or reaction specificity, thereby facilitating the isolation of new enzyme variants for a variety of different substrates and reactions by medium-throughput or even low-throughput screens.     

Directed Evolution of Enzymes for Biocatalytic Applications

Directed molecular evolution is a rapidly growing field revolutionizing the development of biocatalysts with improved properties. This review describes methods to create mutant libraries and assays for rapid screening or selection of desired variants. Selected examples emphasizing the evolution of enzymes for applications in biocatalysis show that it is possible to alter substrate specificity, modulate enantioselectivity and increase enzyme performance under process conditions.     

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.     

The quest for molecular quasi-species in ligand-activity space and its application to directed enzyme evolution

We propose that the proper evolving unit in enzyme evolution is not a single “fittest molecule”, but a cluster of related variants denoted a “quasi-species”. A distribution of variants provides genetic variability and thereby reduces the risk of inbreeding and evolutionary dead-ends. Different matrices of substrates or activity modulators will lead to different selection criteria and divergent evolutionary trajectories. We provide examples from our directed evolution of glutathione transferases illustrating the interplay between libraries of enzyme variants and ligand matrices in the identification of quasi-species. The ligand matrix is shown to be crucial to the outcome of the search for novel activities. In this sense the experimental system resembles the biological environment in governing the evolution of enzymes.     

Directed enzyme evolution via small and effective neutral drift libraries

Small libraries for directed evolution can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants. We describe methods for preparing such libraries, using serum paraoxonase (PON1). An optimized GFP variant fused to PON1 reported levels of soluble, functional enzyme, enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities toward several substrates, including toxic organophosphates.     

Micro-colony array based high throughput platform for enzyme library screening

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.     

A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution

Disulfide bonds provide major contributions to the conformational stability of proteins, and their cleavage often leads to unfolding. The gene-3-protein of the filamentous phage fd contains two disulfides in its N1 domain and one in its N2 domain, and these three disulfide bonds are essential for the stability of this protein. Here, we employed in vitro evolution to generate a disulfide-free variant of the N1-N2 protein with a high conformational stability. The gene-3-protein is essential for the phage infectivity, and we exploited this requirement for a proteolytic selection of stabilized protein variants from phage libraries. First, optimal replacements for individual disulfide bonds were identified in libraries, in which the corresponding cysteine codons were randomized. Then stabilizing amino acid replacements at non-cysteine positions were selected from libraries that were created by error-prone PCR. This stepwise procedure led to variants of N1-N2 that are devoid of all three disulfide bonds but stable and functional. The best variant without disulfide bonds showed a much higher conformational stability than the disulfide-containing wild-type form of the gene-3-protein. Despite the loss of all three disulfide bonds, the midpoints of the thermal transitions were increased from 48.5 degrees C to 67.0 degrees C for the N2 domain and from 60.0 degrees C to 78.7 degrees C for the N1 domain. The major loss in conformational stability caused by the removal of the disulfides was thus over-compensated by strongly improved non-covalent interactions. The stabilized variants were less infectious than the wild-type protein, probably because the domain mobility was reduced. Only a small fraction of the sequence space could be accessed by using libraries created by error-prone PCR, but still many strongly stabilized variants could be identified. This is encouraging and indicates that proteins can be stabilized by mutations in many different ways.     

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology.     

基因人工进化的分子育种技术

分子育种技术(molecular breeding technology)利用人工手段模拟生物杂交优势的分子进化模式,在体外构建含有感兴趣基因的大量突变文库,并根据基因表达产物的特性,以合适的筛选方法选择具有最佳功能的改良基因。经过近几年的快速发展,分子育种技术在创造高效随机基因突变库的方法上日臻完善,已在医学、工业和环境保护酶类研究等方面收到了很好的成效。预计未来该技术将广泛地应用于重要传染性疾病(如疟疾、艾滋病病毒等)的预防和治疗,尤其可通过DNA疫苗手段的应用而获得强大的生命力。作者全面地综述了分子育种技术的产生、发展和应用现状。     

Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries

How to explore protein sequence space efficiently and how to generate high-quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.     

Strategies and computational tools for improving randomized protein libraries

In the last decade, directed evolution has become a routine approach for engineering proteins with novel or altered properties. Concurrently, a trend away from purely 'blind' randomization strategies and towards more 'semi-rational' approaches has also become apparent. In this review, we discuss ways in which structural information and predictive computational tools are playing an increasingly important role in guiding the design of randomized libraries: web servers such as ConSurf-HSSP and SCHEMA allow the prediction of sites to target for producing functional variants, while algorithms such as GLUE, PEDEL and DRIVeR are useful for estimating library completeness and diversity. In addition, we review recent methodological developments that facilitate the construction of unbiased libraries, which are inherently more diverse than biased libraries and therefore more likely to yield improved variants.     

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.     

A colorimetric assay to quantify dehydrogenase activity in crude cell lysates

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7 degrees C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.     

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.     

Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.     

Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates

Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities.     

Screening for recombinant glutathione transferases active with monochlorobimane

A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli. The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB). This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants. Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light. The fluorescence is visible instantly. One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies. The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries. Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.     

Creating new specific ligand-receptor pairs for transgene regulation

The creation of specifically matched ligand-receptor pairs that are orthogonal to naturally present interacting pairs is essential for the development of small molecule-regulated gene expression systems for biotechnological applications. However, for many years this task has represented a significant challenge for synthetic chemists and protein engineers. Recently, Doyle and colleagues demonstrated that highly specific ligand-receptor pairs can be engineered in a rapid fashion by creating large libraries of protein variants and applying a selection scheme to identify variants with improved activation by the target synthetic ligand.     

An episomal expression vector for screening mutant gene libraries in Pichia pastoris

Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the alpha-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.