Expanding the nucleotide and sugar 1-phosphate promiscuity of nucleotidyltransferase RmlA via directed evolution

Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine- and purine-based NTPs as well as non-native D- and L-sugars (both α- and β-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.     

A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution

This protocol details the application of a high-throughput fluorescence-based screen, in conjunction with error-prone PCR/saturation mutagenesis, for altering the proficiency and/or promiscuity of a secondary metabolite glycosyltransferase (GT) via directed evolution. Given the structural and mechanistic similarities among secondary metabolite-associated GTs, this approach may provide a template for engineering other members of the GT-B superfamily.     

Eye evolution: a question of genetic promiscuity

Animal eyes have long served as a classical example of independent origin followed by convergence of structures onto a few different solutions. During the past decade this view has been challenged by the discovery of shared developmental regulatory genes. The Pax6 gene in particular is almost universally employed for eye formation in bilaterian animals, despite widely different embryological origins. The resulting controversy on the multiple or single origins of animal eyes has gradually been sharpened by continuing discoveries of further general similarities in the genetic regulatory circuits of eye development. Recent work on gene expression in specified cell types, together with comparative studies of developmental genes in cnidarians, now show some promise to a solution of the controversy.     

Modulation of the regioselectivity of a Bacillus alpha-galactosidase by directed evolution

-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.     

Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants

The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys₂–His₂ zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-finger domains enables the design of hybrid recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains. In particular, the ability to fully reprogram serine recombinase catalytic specificity has been impeded by conserved base requirements within each recombinase target site and an incomplete understanding of the factors governing DNA recognition. Here we describe an approach to complement the targeting capacity of ZFRs. Using directed evolution, we isolated mutants of the β and Sin recombinases that specifically recognize target sites previously outside the scope of ZFRs. Additionally, we developed a genetic screen to determine the specific base requirements for site-specific recombination and showed that specificity profiling enables the discovery of unique genomic ZFR substrates. Finally, we conducted an extensive and family-wide mutational analysis of the serine recombinase DNA-binding arm region and uncovered a diverse network of residues that confer target specificity. These results demonstrate that the ZFR repertoire is extensible and highlights the potential of ZFRs as a class of flexible tools for targeted genome engineering.     

Enantioselective biocatalysis optimized by directed evolution

Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.     

Improving a circularly permuted TEM-1 beta-lactamase by directed evolution

Circular permutation of proteins is a powerful technique to explore the importance of the polypeptide secondary structure order for attaining the final three-dimensional structure. Here, we designed a circular permutation of the TEM beta-lactamase in order to produce a new domain-forming amino acid arrangement in the polypeptide sequence. Closing the normal N- and C-termini with the connecting peptide GGS and creating new N- and C-termini at position 216, produces a severely impaired permuted protein. Introduction of a connector with random components allows the isolation of enzymes with better activities and indicates a selection for a potential helix-stop signal at the new super-secondary motif. We applied several directed-evolution cycles, starting from permuted enzymes with each of the two different connecting peptides, and selecting for antibiotic resistance and isolated several mutants with resistance levels close to those of the wild-type enzyme. We also analyze some of the data collected on the outcomes and paths of these evolutionary experiments. A purified sixth cycle variant with connector peptide GGS showed catalytic efficiency values approximately 8% of the natural enzyme.     

Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution

DNA polymerases recognize their substrates with exceptionally high specificity, restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3' mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates or the substitution of one with the equivalent fluorescent dye-labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.     

Improved solubility of TEV protease by directed evolution

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.     

Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.     

Enhanced enantioselectivity of a carboxyl esterase from Rhodobacter sphaeroides by directed evolution

The present work created an esterase variant from Rhodobacter sphaeroides (RspE) with enhanced selectivity in hydrolytic kinetic resolutions by directed evolution. A “model” substrate, methyl mandelate, was introduced in the high-throughput screening procedure. E values of a variant CH (Asn62Cys/Leu145His) for six different esters were 10–83, which were a relative improvement compared to 2–20 for the wild type. Our subsequent crystal structure interpretation and molecular dynamics simulations helped shed light on the source of enantioselectivity modified by directed evolution. Though mutations displayed no “direct” interaction with the substrate, they were hypothesized to strengthen the intramolecular interaction in the catalytic cavity of variant. Conformation analysis revealed that the enhanced enantioselectivity of variant CH for the seven substrates applied in this study was derived from the decrease in size of the substrate binding pocket.     

Improving the decolorization for textile dyes of a metagenome-derived alkaline laccase by directed evolution

To obtain better performing laccases for textile dyes decolorization, random mutagenesis of Lac591, a metagenome-derived alkaline laccase, was carried out. After three rounds of error-prone PCR and high-throughput screening by assaying enzymatic activity toward the phenolic substrate 2,6-dimethoxyphenol (2,6-DMP), a mutant (Lac3T93) with remarkably improved enzymatic activity was obtained. Sequence analysis revealed that four amino acid substitutions (N40S, V55A, F62L, and E316V) were accumulated in the Lac3T93. Compared to the wild-type enzyme, the specific activity of Lac3T93 toward 2,6-DMP was increased to 4.8-fold (61.22 U/mg), and its optimal temperature and pH were changed to 60°C and 8.0 from 55°C and 7.5 of the wild-type enzyme, respectively. Furthermore, the degradation ability of Lac3T93 for textile dyes was investigated, and the new variant represented improved decolorization percentage for four industrial dyes with complex phenyl structure (Basic Blue 3, Methylene Blue, Bromophenol Blue, and Crystal Violet) and higher decolorization efficiency for Indigo Carmine than that of the parent enzyme. Furthermore, the decolorization percentage of Lac3T93 for five dyes in the absence of hydroxybenzotrizole (HBT) is clearly higher than those of the wild-type enzyme with 1 mM HBT, and HBT can further improve its decolorization ability.     

Diversifying carotenoid biosynthetic pathways by directed evolution.

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products--those that could be made biosynthetically--remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these "evolved" pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed.     

Alteration of substrate specificity of aspartase by directed evolution

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.     

Optimizing the search algorithm for protein engineering by directed evolution

An in silico protein model based on the Kauffman NK-landscape, where N is the number of variable positions in a protein and K is the degree of coupling between variable positions, was used to compare alternative search strategies for directed evolution. A simple genetic algorithm (GA) was used to model the performance of a standard DNA shuffling protocol. The search effectiveness of the GA was compared to that of a statistical approach called the protein sequence activity relationship (ProSAR) algorithm, which consists of two steps: model building and library design. A number of parameters were investigated and found to be important for the comparison, including the value of K, the screening size, the system noise and the number of replicates. The statistical model was found to accurately predict the measured activities for small values of the coupling between amino acids, K 相似文献   

Significantly enhanced stability of glucose dehydrogenase by directed evolution

An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution. The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes. Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the p Trc99A expression vector and expressed in the host, Escherichia coli. In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B. megaterium IWG3, was also subjected to directed evolution by the family shuffling method. A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries. This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes. The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl. In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66 degrees C), while the half-life of isolated mutant DN-46 was 540 min at 66 degrees C, i.e., 415-fold more thermostable than mutant F20. The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.     

Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, k (cat), of the most active mutant RML protein (Q5) was increased from 10.63?±?0.80 to 71.44?±?3.20?min(-1) after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functional and propeptide domains that arise from the role played by the propeptide in the folding and maturation of the enzyme. We suggest that directed evolution of full-length proteins including their propeptides is a strategy with general validity for extending the range of conformations available to proteins, leading to the enhancement of the catalytic rates of the enzymes.     

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.     

Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE_LB400) are largely unknown. BphAE_p4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE_LB400, yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE_LB400 and BphAE_p4 and examine the biochemical properties of two BphAE_LB400 variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.     

Fusoselect: cell-cell fusion activity engineered by directed evolution of a retroviral glycoprotein

Membrane fusion plays a key role in many biological processes including vesicle trafficking, synaptic transmission, fertilization or cell entry of enveloped viruses. As a common feature the fusion process is mediated by distinct membrane proteins. We describe here 'Fusoselect', a universal procedure allowing the identification and engineering of molecular determinants for cell-cell fusion-activity by directed evolution. The system couples cell-cell fusion with the release of retroviral particles, but can principally be applied to membrane proteins of non-viral origin as well. As a model system, we chose a gamma-retroviral envelope protein, which naturally becomes fusion-active through proteolytic processing by the viral protease. The selection process evolved variants that, in contrast to the parental protein, mediated cell-cell fusion in absence of the viral protease. Detailed analysis of the variants revealed molecular determinants for fusion competence in the cytoplasmic tail (CT) of retroviral Env proteins and demonstrated the power of Fusoselect.