Analytical study of the effect of recombination on evolution via DNA shuffling

We investigate a multi-locus evolutionary model which is based on the DNA shuffling protocol widely applied in \textit{in vitro} directed evolution. This model incorporates selection, recombination and point mutations. The simplicity of the model allows us to obtain a full analytical treatment of both its dynamical and equilibrium properties, for the case of an infinite population. We also briefly discuss finite population size corrections

Algorithms for optimizing cross-overs in DNA shuffling

DNA shuffling generates combinatorial libraries of chimeric genes by stochastically recombining parent genes. The resulting libraries are subjected to large-scale genetic selection or screening to identify those chimeras with favorable properties (e.g., enhanced stability or enzymatic activity). While DNA shuffling has been applied quite successfully, it is limited by its homology-dependent, stochastic nature. Consequently, it is used only with parents of sufficient overall sequence identity, and provides no control over the resulting chimeric library. Results: This paper presents efficient methods to extend the scope of DNA shuffling to handle significantly more diverse parents and to generate more predictable, optimized libraries. Our CODNS (cross-over optimization for DNA shuffling) approach employs polynomial-time dynamic programming algorithms to select codons for the parental amino acids, allowing for zero or a fixed number of conservative substitutions. We first present efficient algorithms to optimize the local sequence identity or the nearest-neighbor approximation of the change in free energy upon annealing, objectives that were previously optimized by computationally-expensive integer programming methods. We then present efficient algorithms for more powerful objectives that seek to localize and enhance the frequency of recombination by producing “runs” of common nucleotides either overall or according to the sequence diversity of the resulting chimeras. We demonstrate the effectiveness of CODNS in choosing codons and allocating substitutions to promote recombination between parents targeted in earlier studies: two GAR transformylases (41% amino acid sequence identity), two very distantly related DNA polymerases, Pol X and b (15%), and beta- lactamases of varying identity (26-47%). Conclusions: Our methods provide the protein engineer with a new approach to DNA shuffling that supports substantially more diverse parents, is more deterministic, and generates more predictable and more diverse chimeric libraries

Biotecnologia, Nanobiotecnologia e Bioinformática: assuntos com acentuado desenvolvimento na Alemanha. Relatório de viagem à Alemanha.

bitstream/CENARGEN/29747/1/doc249.pd

Nature-inspired Enzyme engineering and sustainable catalysis: biochemical clues from the world of plants and extremophiles

The use of enzymes to accelerate chemical reactions for the synthesis of industrially important products is rapidly gaining popularity. Biocatalysis is an environment-friendly approach as it not only uses non-toxic, biodegradable, and renewable raw materials but also helps to reduce waste generation. In this context, enzymes from organisms living in extreme conditions (extremozymes) have been studied extensively and used in industries (food and pharmaceutical), agriculture, and molecular biology, as they are adapted to catalyze reactions withstanding harsh environmental conditions. Enzyme engineering plays a key role in integrating the structure-function insights from reference enzymes and their utilization for developing improvised catalysts. It helps to transform the enzymes to enhance their activity, stability, substrates-specificity, and substrate-versatility by suitably modifying enzyme structure, thereby creating new variants of the enzyme with improved physical and chemical properties. Here, we have illustrated the relatively less-tapped potentials of plant enzymes in general and their sub-class of extremozymes for industrial applications. Plants are exposed to a wide range of abiotic and biotic stresses due to their sessile nature, for which they have developed various mechanisms, including the production of stress-response enzymes. While extremozymes from microorganisms have been extensively studied, there are clear indications that plants and algae also produce extremophilic enzymes as their survival strategy, which may find industrial applications. Typical plant enzymes, such as ascorbate peroxidase, papain, carbonic anhydrase, glycoside hydrolases and others have been examined in this review with respect to their stress-tolerant features and further improvement via enzyme engineering. Some rare instances of plant-derived enzymes that point to greater exploration for industrial use have also been presented here. The overall implication is to utilize biochemical clues from the plant-based enzymes for robust, efficient, and substrate/reaction conditions-versatile scaffolds or reference leads for enzyme engineering

Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and A Cell-Based Oral Delivery Platform

Proteins, with their ability to perform a variety of highly specific biological functions, have emerged as an important class of therapeutics. However, to fully harness their therapeutic potential, proteins often need to be optimized by molecular engineering; therapeutic efficacy can be improved by modulating protein properties such as binding affinity/specificity, half-life, bioavailability, and immunogenicity. In this work, we first present an introductory example in which a mechanistic mathematical model was used to improve target selection for directed evolution of an aglycosylated Fc domain of an antibody to enhance phagocytosis of tumor cells. Several aspects of directed evolution experimental methods were then optimized. A model-guided ligation strategy was developed to maximize ligation yield in DNA library construction, and this design tool is freely available through a web server. Streamlined protocols for mRNA display and ribosome display, which are powerful in vitro selection methods, were also created to allow robust selection of a variety of therapeutic proteins, including monomeric Fc domains, designed ankyrin repeat proteins, a single-chain insulin analog (SCI-57), and leptin. Anti-ICAM-1 scFv antibody fragments were also optimized for ribosome display by grafting complementarity determining regions onto a stable human framework. In addition to engineering the proteins themselves, effective delivery systems are essential for maximizing the therapeutic benefit of these proteins in a clinical setting. We therefore also developed a novel oral delivery platform based on the food-grade bacterium Lactococcus lactis. SCI-57, leptin, and SCI-57-leptin fusion proteins have been successfully secreted from this host in vitro and preliminary studies in a diabetic mouse model show reduced glucose levels after oral administration of L. lactis secreting SCI-57. We then further improved the secretion potential of this host through directed evolution of a L. lactis signal peptide. In summary, our studies have provided important advances to the field of protein engineering through the development of mechanistic mathematical models, streamlined experimental methodologies, and polypeptides with improved properties. This work has also opened up the possibility of systemic delivery of protein therapeutics using live microorganisms

Génération d'une inosine-uridine nucléoside hydrolase (IU-NH) par évolution moléculaire dirigée capable de mieux fonctionner en solvant organique

L'approche d'évolution moléculaire dirigée, laquelle est un ensemble de méthodes in vitro et in vivo basées sur la génération d'une bibliothèque de chimères suivie d'une méthode rapide de sélection, a été utilisée pour modifier et améliorer diverses propriétés d'une protéine comme la thermostabilité, la solubilité, le niveau d'expression du gène et l'activité dans un environnement naturel ou non-naturel. Nous avons utilisé la mutagénèse dirigée et le"DNA shuffling", une technique d'évolution dirigée, pour tenter de convertir la nucléoside hydrolase en une enzyme capable d'opérer dans un milieu pauvre en eau, la réaction inverse de l'hydrolyse, ouvrant de ce fait la porte à la synthèse d'agents antiviraux ou antitumoraux. Cette protéine suscite beaucoup d'intérêt au niveau de la chimie médicinale. Le projet est basé sur un gène synthétique de 985 paires de bases codant pour l'inosine uridine nucléoside hydrolase (RU-NH) comparable au gène du protozoaire Crithidia fasciculata, préalablement assemblé et cloné dans Escherichia coli

Using gene shuffling to increase genetic diversity in genes involved in beta-lactam biosynthesis

The actinomycetes are gram-positive bacteria that produce more than two-thirds of the known biologically active microbial natural products, including many commercially important antibiotics, anti-cancer agents, other pharmacologically useful agents, animal health products and agrochemicals. The prevailing utilization of antibiotics continues to be the mainstay against microbial infections and a majority ofthe over six thousand antibiotics discovered thus far are from Streptomyces spp. One of the most well-characterized antibiotic biosynthetic pathway is the one involving the biosynthesis of the penicillins, cephalosporins and cephamycins. This pathway involves two initial steps which are common in filamentous fungi, lower eukaryotes and prokaryotes. The penam nucleus of penicillins and the cephem nucleus of both cephamycins andcephalosporins are formed by the condensation of the three precursor amino acids L-a-aminoadipic acid, Lcysteine and L-valine by a mechanism designated as 'non-ribosomal peptide synthesis', which involves activation and condensation of the three component amino acids and epimerization of the L- to D-valine to form a linear acyclic tripeptide called o-(L-a-aminoadipyl)-L-cysteinyl-Dvaline (ACV) by the action of a peptide synthetase. ACV is then cyclized to form isopenicillin N, an intermediate that contains an L-a-aminoadipyl side-chain attached to the penem nucleus (Fig. 1.2) by isopenicilin N synthase (IPNS or Cyclase) and this encompasses the creation of the Beta-lactam and thiazolidine rings. A broad range of ~-lactam producing Streptomyces spp were grown, the DNA extraction procedure optimised and total chromosomal DNA isolated. A bioinformatics analysis of known IPNS gene sequences allowed the synthesis of PCR primers for the iso-penicillin N synthase gene. IPNS genes and lPNS-like genes were successfully amplified from the total DNA of ten strains including two novel thermophilic strains, A. and B. Sequencing was carried out on the genes from S. hygroscopicus, S. tanashiensis and the two thermophiles A and B. This allowed development of the conditions for gene shuffiing of the IPNS gene which was carried out pairwise and resulted in the reconstitution of shuffied genes of the correct size. The resulting mixed gene sequences were cloned into the pTrcHis2-TOPO expression vector and the plasmid DNA screened and assayed for IPNS activity using HPLC which showed ten fold increase in IPNS activity as a result of the shuffiing

Simulated Annealing

The book contains 15 chapters presenting recent contributions of top researchers working with Simulated Annealing (SA). Although it represents a small sample of the research activity on SA, the book will certainly serve as a valuable tool for researchers interested in getting involved in this multidisciplinary field. In fact, one of the salient features is that the book is highly multidisciplinary in terms of application areas since it assembles experts from the fields of Biology, Telecommunications, Geology, Electronics and Medicine