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PubMed1998

Analysis of pol gene heterogeneity, viral quasispecies, and drug resistance in individuals infected with group O strains of human immunodeficiency virus type 1.

Nucleotide sequences of the reverse transcriptase (RT) coding region have been compared in four new human immunodeficiency virus type 1 (HIV-1) group O isolates. Phylogenetic analysis of this pol region highlights a cluster of these four HIV-1 group O sequences with seven other group O isolates (5% intracluster nucleotide sequence diversity) similar to clusters classified as subtypes in HIV-1 group M (an average of 4.9% intrasubtype sequence diversity). Based on these analyses, this group O cluster has been designated subtype A-O. A longitudinal study of a heterosexual couple infected with group O (ESP1 and ESP2) allowed a detailed analysis of RT sequences (amino acids 28 to 219). Directed evolution and a slightly higher mutation frequency was observed in the RT sequences of patient ESP2, treated with antiretroviral drugs, than that from the untreated patient ESP1. Antiretroviral treatment also selected for specific substitutions, M184V and T215Y in the RT coding region, conferring resistance to 3'-dideoxy-3'-thiacytidine and zidovudine, respectively. A Gly98 to Glu RT substitution identified in the treated patient suggests a possible reversion of a nonnucleoside RT inhibitor-resistant phenotype. Using RT clones from this longitudinal study, both heteroduplex tracking assay and cloning-sequencing techniques were employed for an extensive genetic analysis of pol gene quasispecies. Amino acid substitutions (i.e., Phe-77 to Leu, Lys-101 to Glu, and Val-106 to Iso) associated with antiretroviral resistance were identified in RT clones from HIV-1 group O-infected patients not subjected to drug therapy or treated with unrelated drugs. Finally, phylogenetic relationships between RT clones of the treated ESP2 patient and those of the untreated ESP1 patient show how drug pressure can direct evolution of viral pol gene quasispecies independently of direct drug-resistant substitutions.

Adolescent↗
PubMed2001

Directed enzyme evolution.

Laboratory evolutionists continue to generate better enzymes for industrial and research applications. Exciting developments include new biocatalysts for enantioselective carbon-carbon bond formation and fatty acid production in plants. Creative contributions to the repertoire of evolutionary methods will ensure further growth in applications and expand the scope and complexity of biological design problems that can be addressed. Researchers are also starting to elucidate mechanisms of enzyme adaptation and natural evolution by testing evolutionary scenarios in the laboratory.

Bacterial Proteins↗
PubMed2005

Colorimetric assays for quantitative analysis and screening of epoxide hydrolase activity.

Focusing on directed evolution to tailor enzymes as usable biocatalysts for fine chemistry, we have studied in detail several colorimetric assays for quantitative analysis of epoxide hydrolase (EH) activity. In particular, two assays have been optimized to characterize variants issued from the directed evolution of the EH from Aspergillus niger. Assays described in this paper are sufficiently reliable for quantitative screening of EH activity in microtiter plates and are low cost alternatives to GC or MS analysis. Moreover, they are usable for various epoxides and not restricted to a type of substrate, such as those amenable to assay by UV absorbancy. They can be used to assay EH activity on any epoxide and to directly assay enantioselectivity when both (R) and (S) substrates are available. The advantages and drawbacks of these two methods to assay EH activity of a large number of natural samples are summarized.

Aspergillus niger↗
PubMed2006

Construction and engineering of positive feedback loops.

Artificial positive feedback loops (PFLs) have been used as genetic amplifiers for enhancing the responses of weak promoters and in the creation of eukaryotic gene switches. Here we describe the construction and directed evolution of two PFLs based on the LuxR transcriptional activator and its cognate promoter, P luxI . The wild-type PFLs are completely activated by 10 nM of 3-oxo-hexanoyl-homoserine lactone (OHHL). Directed evolution of LuxR increased the sensitivity of the feedback loops, resulting in systems that are completely activated at OHHL concentrations of 5 nM, or approximately 3 molecules per cell. The responses of the PFLs can also be modulated by adjusting inducer concentrations. These highly sensitive yet regulatable PFLs can be used to construct larger artificial genetic networks to gain understanding of the design principles of complex biological systems and are expected to find various applications in industrial fermentation and gene therapy.

Amino Acid Substitution↗
PubMed1987

Constraint, flexibility, and phylogenetic history in the evolution of direct development in sea urchins.

Development in sea urchins typically involves the production of an elaborate feeding larva, the pluteus, within which the juvenile sea urchin grows. However, a significant fraction of sea urchins have completely or partially eliminated the pluteus, and instead undergo direct development from a large egg. Direct development is achieved primarily by heterochrony, that is, by the abbreviation or elimination of larval developmental processes and the acceleration of processes involved in development of adult features. Direct development has evolved independently several times, and in several ways. These radically altered ontogenies offer remarkable opportunities for the study of the mechanisms by which early development undergoes evolutionary modification. The recent availability of monoclonal antibody and cDNA probes that recognize homologous cells in embryos of closely related typical and direct developing species makes possible an experimental analysis of the cellular and molecular bases for heterochronic changes in development.

Animals↗
PubMed2004

Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes.

Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 A. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.

Amino Acid Sequence↗
PubMed2003

A C35 carotenoid biosynthetic pathway.

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone. The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.

Alkyl and Aryl Transferases↗
PubMed2001

A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes.

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.

Alkanes↗
PubMed2004

Rational engineering of enzyme stability.

During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved.

Biotechnology↗
PubMed2007

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain.

Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.

Algorithms↗
PubMed2006

Outlook for cellulase improvement: screening and selection strategies.

Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.

Cellulase↗
PubMed2003

Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha.

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.

DNA Replication↗
PubMed2001

Combinatorial and Evolution-Based Methods in the Creation of Enantioselective Catalysts.

Combinatorial methods in the development of enantioselective homogeneous catalysts constitute a new branch of catalysis research. The goal is to prepare libraries of potential asymmetric catalysts, rather than choosing the traditional one-catalyst-at-a-time approach. Several conceptional advancements have been reported in the parallel preparation of chiral ligands. Currently the most meaningful systems constitute modularly constructed ligands on solid supports, which allow high degrees of structural diversity and thus the maximum probability of finding enantioselective catalysts or even new types of ligands for asymmetric catalysis. Search strategies have been developed which amongst other things, lead to catalysts not likely to have been discovered by traditional methods. Genuine application of such strategies involve thousands of catalysts and require high-throughput screening systems capable of assaying enantioselectivity. The first high-throughput ee-screening systems were in fact developed for use in the directed evolution of enantioselective enzymes, a process based on "evolution in the test tube" in which the appropriate methods of random mutagenesis, gene expression, and ee assays are combined. Since no screening system is likely to be universal, different approaches are necessary. Thus far these include assays based on UV/Vis, fluorescence, circular dichroism, mass spectrometry, and even modified gas chromatography as well as special forms of capillary electrophoresis. One of the most efficient systems involves the concept of the mass-spectrometric detection of deuterium-labeled pseudo-enantiomers and pseudo-prochiral compounds with which about 1000 exact ee determinations can be achieved per day, although the assay is restricted to kinetic resolution and/or reactions of prochiral compounds bearing enantiotopic groups. Super-high-throughput screening for enantioselectivity is possible in many cases by making use of chirally modified capillary array electrophoresis in a parallel step. Accordingly, 7000 to 30 000 ee determinations can be carried out per day. These and other analytical developments are expected to stimulate further research in the combinatorial search for asymmetric homogeneous catalysts and in the directed evolution of enantioselective enzymes for use in organic chemistry.

Journal Article↗
PubMed2006

Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display.

Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expected from expression levels and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing.

Amino Acid Substitution↗
PubMed2006

Optimized design and synthesis of chemical dimerizer substrates for detection of glycosynthase activity via chemical complementation.

Glycosynthases catalyze the formation of a glycosidic bond between a glycosyl fluoride donor substrate and a glycosyl acceptor substrate with high yield, thus providing a valuable approach for the synthesis of carbohydrates and glycoconjugates. Chemical complementation can be used to link glycosynthase activity to the transcription of a reporter gene in vivo, providing a selection for the directed evolution of glycosynthase enzymes with improved properties. In this approach, glycosynthase activity is detected as covalent coupling between a small molecule disaccharide acceptor substrate and a small molecule disaccharide alpha-fluoro donor substrate. Here we report the optimized design and synthesis of these small molecule substrates. These optimized substrates are shown to give a robust, glycosynthase-dependent transcriptional read-out in the chemical complementation assay. The full synthesis and characterization of these substrates are reported for the first time. These optimized chemical dimerizer substrates should allow the potential of chemical complementation for the directed evolution of glycosynthases with diverse substrate specificities and improved properties to be fully realized.

Binding Sites↗
PubMed2000

Creating hybrid genes by homologous recombination.

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution. In vitro recombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties. In vivo recombination in both E. coli and yeast is greatly enhanced by double-strand breaks; for E. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombination in vivo have distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recently in vivo recombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

Escherichia coli↗
PubMed1999

Selection of functional T cell receptor mutants from a yeast surface-display library.

The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

Animals↗
PubMed2005

High-throughput measurement of protein stability in microtiter plates.

The direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition midpoint determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removal of proteins from the 96-well plate format in which they were expressed and purified. Herein we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability. Unfolding curves generated by the serial addition of denaturant into single wells allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C(1/2) +/- 0.05 M, m(G), and DeltaG(H2O)) for cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilized with various palmitate concentrations and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability.

Animals↗