Obtaining more contaminant-resistant variants from a native Chlorella vulgaris strain.

Cildáñez stream (in Matanza-Riachuelo basin, Buenos Aires) is one of the most polluted watercourses of Argentina, containing a mixed contamination from agricultural and industrial wastes. The application of water bioremediation processes for this kind of effluent will require microorganisms with a high tolerance to contamination. In this sense, obtaining higher contaminant-resistant microalgae lines is widely desired. In this study, adaptive laboratory evolution (ALE) and random mutagenesis were used to obtain Chlorella vulgaris LMPA-40 strains adapted to grow in polluted water from the Cildáñez stream. The ALE process was performed by 22 successive subcultures under selective pressure (Cildáñez wastewater alone or with the addition of phenol or H2O2) while random mutagenesis was performed with UV-C radiation at 275nm. Not all the cell lines obtained after ALE could adapt enough to overcome the stress caused by the Cildáñez wastewater, indicating that the process is quite random and depends on the stressor used. The best results were obtained for the Cildáñez wastewater adapted cells (Cild 3 strain) that were more resistant than the original strain. The concentration of protein, Chlorophyll A, Chlorophyll B, and carotenoids in the Cild 3 ALE evolved strain was higher than that of the control strain. However, this strain exhibited half of the lipid content compared to the same control strain. Interestingly, these alterations and the acquired tolerance may be reversed over time during storage. These findings suggest that the acquisition of novel cell lines could not be permanent, a fact that must be considered for future trials.

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

mSphere of Influence: Experimental Evolution of RNA Viruses.

Gonzalo Moratorio works in the field of experimental evolution of viruses. In this mSphere of Influence article, he reflects on how the papers "Virus attenuation by genome-scale changes in codon pair bias" by Coleman et al. (Science 320:1784-1787, 2008, https://doi.org/10.1126/science.1155761) and "Codon usage determines the mutational robustness, evolutionary capacity, and virulence of an RNA virus" by Lauring et al. (Cell Host Microbe 12:623-632, 2012, https://doi.org/10.1016/j.chom.2012.10.008) made an impact on his thinking about how to employ synthetic biology to study experimental evolution of viruses.

Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

Tailoring Proteins to Re-Evolve Nature: A Short Review.

Proteins are key biomolecules for most biological processes, their function is related to their conformation that is also dictated by their sequence of amino acids. Through evolution, nature has produced an immense variety of enzymatic tools of high efficiency and selectivity, and thanks to the understanding of the molecular basis of life and the technological advances, scientists have learned to introduce mutations and select mutant enzymes, to optimize and control their molecular fitness characteristics mainly for industrial, medical and environmental applications. The relationship between protein structure and enzymatic functionality is essential, and there are various experimental and instrumental techniques for unravelling the molecular changes, activities and specificities. Protein engineering applies computational tools, in hand with experimental tools for mutations, like directed evolution and rational design, along with screening methods to obtain protein variations with the desired properties under a short time frame. With innovations in technology, it is possible to fine tune properties in proteins and reach new frontiers in their applications. The present review will briefly discuss these points and methods, with a glimpse on their strengths and pitfalls, while giving an overview of the versatility of synthetic proteins and their huge potential for biotechnological and biomedical fields.

The Influence of Polyploidy on the Evolution of Yeast Grown in a Sub-Optimal Carbon Source.

Polyploidization events have occurred during the evolution of many fungi, plant, and animal species and are thought to contribute to speciation and tumorigenesis, however little is known about how ploidy level contributes to adaptation at the molecular level. Here we integrate whole genome sequencing, RNA expression analysis, and relative fitness of ∼100 evolved clones at three ploidy levels. Independent haploid, diploid, and tetraploid populations were grown in a low carbon environment for 250 generations. We demonstrate that the key adaptive mutation in the evolved clones is predicted by a gene expression signature of just five genes. All of the adaptive mutations identified encompass a narrow set of genes, however the tetraploid clones gain a broader spectrum of adaptive mutations than haploid or diploid clones. While many of the adaptive mutations occur in genes that encode proteins with known roles in glucose sensing and transport, we discover mutations in genes with no canonical role in carbon utilization (IPT1 and MOT3), as well as identify novel dominant mutations in glucose signal transducers thought to only accumulate recessive mutations in carbon limited environments (MTH1 and RGT1). We conclude that polyploid cells explore more genotypic and phenotypic space than lower ploidy cells. Our study provides strong evidence for the beneficial role of polyploidization events that occur during the evolution of many species and during tumorigenesis.

One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T5015 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T5015. The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity.

Avaliação da acurácia de metástases implantadas em camundongos nude através da ultrassonografia / Ultrasonography accuracy of liver metastasis xenograft in nude mice

A pesquisa translacional é pilar central no entendimento do câncer e na sua progressão. Camundongo imunodeficiente é o modelo animal comumente empregado no estudo de tumores humanos transplantados ortotopicamente. O exame ultrassonográfico emerge como método viável na mensuração de tumores implantados na cavidade abdominal. O objetivo desse estudo foi avaliar achados ultrassonográficos de tumores hepáticos implantados em camundongos, utilizando transdutor de 13 MHz e correlacioná-los com os achados anatomopatológicos. Foram obtidas cirurgicamente, amostras tumorais de metástases hepáticas de câncer colorretal proveniente de pacientes. Um fragmento de 1mm3 foi confeccionado e implantado no parênquima hepático de camundongos nude. Ao todo foram utilizados 38 animais. Todos os camundongos foram, ultrassonograficamente, monitorados mensalmente até eutanásia. Dos 38 camundongos implantados 11 apresentaram crescimento tumoral. O exame ultrassonográfico detectou lesões nodulares nos 11 animais macroscopicamente positivos e foi capaz de identificar os padrões de tumores enxertados. A ultrassonografia é um método viável e não invasivo, para avaliar parênquima hepático de camundongos nude. Neste estudo, o método obteve 100% de sensibilidade e especificidade na detecção e caracterização de lesões nodulares em parênquima hepático (AU)

Translational research is key to understanding cancer and its progression. The immunodeficient mouse is the animal model usually employed for studying orthotopically implanted human tumors, and ultrasonography has emerged as a viable method for measuring tumors implanted into the abdominal cavity. The objective of this study was to evaluate findings from ultrasonography of hepatic tumors implanted into mice, using a 13 MHz transducer, and to correlate these findings with the results of pathological examinations. Tumor samples from hepatic metastases of colorectal cancer were obtained surgically from patients, and 1mm3 fragments were implanted into the hepatic parenchyma of nude mice. A total of 38 animals were used. All of the mice were monitored monthly by ultrasonography until sacrifice. Of the 38 mice implanted 11 showed tumor growth. Ultrasonography detected nodular lesions in the 11 macroscopically positive animals, and was able to identify the patterns of the grafted tumors. Ultrasonography is a viable, and non-invasive, method for evaluating the hepatic parenchyma of nude mice. In this study, the method showed 100% sensitivity and specificity in detecting and characterizing nodular lesions in the hepatic parenchyma (AU)

Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

Optimization of Conformational Dynamics in an Epistatic Evolutionary Trajectory.

The understanding of protein evolution depends on the ability to relate the impact of mutations on molecular traits to organismal fitness. Biological activity and robustness have been regarded as important features in shaping protein evolutionary landscapes. Conformational dynamics, which is essential for protein function, has received little attention in the context of evolutionary analyses. Here we employ NMR spectroscopy, the chief experimental tool to describe protein dynamics at atomic level in solution at room temperature, to study the intrinsic dynamic features of a metallo- Β: -lactamase enzyme and three variants identified during a directed evolution experiment that led to an expanded substrate profile. We show that conformational dynamics in the catalytically relevant microsecond to millisecond timescale is optimized along the favored evolutionary trajectory. In addition, we observe that the effects of mutations on dynamics are epistatic. Mutation Gly262Ser introduces slow dynamics on several residues that surround the active site when introduced in the wild-type enzyme. Mutation Asn70Ser removes the slow dynamics observed for few residues of the wild-type enzyme, but increases the number of residues that undergo slow dynamics when introduced in the Gly262Ser mutant. These effects on dynamics correlate with the epistatic interaction between these two mutations on the bacterial phenotype. These findings indicate that conformational dynamics is an evolvable trait, and that proteins endowed with more dynamic active sites also display a larger potential for promoting evolution.

Inactivation of the PTS as a Strategy to Engineer the Production of Aromatic Metabolites in Escherichia coli.

Laboratory and industrial cultures of Escherichia coli employ media containing glucose which is mainly transported and phosphorylated by the phosphotransferase system (PTS). In these strains, 50% of the phosphoenolpyruvate (PEP), which results from the catabolism of transported glucose, is used as a phosphate donor for its phosphorylation and translocation by the PTS. This characteristic of the PTS limits the production of industrial biocommodities that have PEP as a precursor. Furthermore, when E. coli is exposed to carbohydrate mixtures, the PTS prevents expression of catabolic and non-PTS transport genes by carbon catabolite repression and inducer exclusion. In this contribution, we discuss the main strategies developed to overcome these potentially limiting effects in production strains. These strategies include adaptive laboratory evolution selection of PTS(-) Glc(+) mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source. We discuss the benefits of using alternative glucose transport systems and describe the application of these strategies to E. coli strains with specific genetic modifications in target pathways. These efforts have resulted in significant improvements in the production of diverse biocommodities, including aromatic metabolites, biofuels and organic acids.

Evolution Is an Experiment: Assessing Parallelism in Crop Domestication and Experimental Evolution: (Nei Lecture, SMBE 2014, Puerto Rico).

In this commentary, I make inferences about the level of repeatability and constraint in the evolutionary process, based on two sets of replicated experiments. The first experiment is crop domestication, which has been replicated across many different species. I focus on results of whole-genome scans for genes selected during domestication and ask whether genes are, in fact, selected in parallel across different domestication events. If genes are selected in parallel, it implies that the number of genetic solutions to the challenge of domestication is constrained. However, I find no evidence for parallel selection events either between species (maize vs. rice) or within species (two domestication events within beans). These results suggest that there are few constraints on genetic adaptation, but conclusions must be tempered by several complicating factors, particularly the lack of explicit design standards for selection screens. The second experiment involves the evolution of Escherichia coli to thermal stress. Unlike domestication, this highly replicated experiment detected a limited set of genes that appear prone to modification during adaptation to thermal stress. However, the number of potentially beneficial mutations within these genes is large, such that adaptation is constrained at the genic level but much less so at the nucleotide level. Based on these two experiments, I make the general conclusion that evolution is remarkably flexible, despite the presence of epistatic interactions that constrain evolutionary trajectories. I also posit that evolution is so rapid that we should establish a Speciation Prize, to be awarded to the first researcher who demonstrates speciation with a sexual organism in the laboratory.

Concommitant adaptation of a GH11 xylanase by directed evolution to create an alkali-tolerant/thermophilic enzyme.

As part of an ongoing directed evolution program, the catalytic performance of the Xylanase A from Bacillus subtilis (XynA), which presents temperature and pH optima of 50°C and 6.0, respectively, has been enhanced to create a highly thermostable and alkali-tolerant enzyme. A library of random XynA mutants generated by error-prone polymerase chain reaction was screened by halo formation on agar containing xylan at pH 8.0. Two mutants showing higher catalytic activity at elevated pH in relation to the wild-type XynA were selected, and pooled with a further 5 XynA variants selected by screening thermostable XynA obtained from a previous directed evolution study for activity at alkaline pH. This pool of variants was used as a template for a further round of error-prone polymerase chain reaction and DNase fragment shuffling, with screening at pH 12.0 at 55°C. Selected mutants were subjected to further DNase shuffling, and a final round of screening at pH 12.0 and 80°C. A XynA variant containing eight mutations was isolated (Q7H/G13R/S22P/S31Y/T44A/I51V/I107L/S179C) that presented a temperature optimum of 80°C, a 3-fold increase in the specific activity compared with the wild-type enzyme at pH 8.0, and a 50% loss of activity (t50) of 60 min at 80°C (wild type <2 min). This directed evolution strategy therefore allows the concomitant adaption of increased thermostability and alkali tolerance of an endo-xylanase.

A novel human recombinant antibody fragment capable of neutralizing Mexican scorpion toxins.

Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 (KD = 25.1 × 10(-9) M) or Cn2 (KD = 8.1 × 10(-9) M), this antibody fragment neutralized one LD50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus. Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.

A roadmap to directed enzyme evolution and screening systems for biotechnological applications.

Enzymes have been long used in man-made biochemical processes, from brewing and fermentation to current industrial production of fine chemicals. The ever-growing demand for enzymes in increasingly specific applications requires tailoring naturally occurring enzymes to the non-natural conditions found in industrial processes. Relationships between enzyme sequence, structure and activity are far from understood, thus hindering the capacity to design tailored biocatalysts. In the field of protein engineering, directed enzyme evolution is a powerful algorithm to generate and identify novel and improved enzymes through iterative rounds of mutagenesis and screening applying a specific evolutive pressure. In practice, critical checkpoints in directed evolution are: selection of the starting point, generation of the mutant library, development of the screening assay and analysis of the output of the screening campaign. Each step in directed evolution can be performed using conceptually and technically different approaches, all having inherent advantages and challenges. In this article, we present and discuss in a general overview, challenges of designing and performing a directed enzyme evolution campaign, current advances in methods, as well as highlighting some examples of its applications in industrially relevant enzymes.

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

Enzymes have been long used in man-made biochemical processes, from brewing and fermentation to current industrial production of fine chemicals. The ever-growing demand for enzymes in increasingly specific applications requires tailoring naturally occurring enzymes to the non-natural conditions found in industrial processes. Relationships between enzyme sequence, structure and activity are far from understood, thus hindering the capacity to design tailored biocatalysts. In the field of protein engineering, directed enzyme evolution is a powerful algorithm to generate and identify novel and improved enzymes through iterative rounds of mutagenesis and screening applying a specific evolutive pressure. In practice, critical checkpoints in directed evolution are: selection of the starting point, generation of the mutant library, development of the screening assay and analysis of the output of the screening campaign. Each step in directed evolution can be performed using conceptually and technically different approaches, all having inherent advantages and challenges. In this article, we present and discuss in a general overview, challenges of designing and performing a directed enzyme evolution campaign, current advances in methods, as well as highlighting some examples of its applications in industrially relevant enzymes.

Automatic design of decision-tree induction algorithms tailored to flexible-receptor docking data.

BACKGROUND: This paper addresses the prediction of the free energy of binding of a drug candidate with enzyme InhA associated with Mycobacterium tuberculosis. This problem is found within rational drug design, where interactions between drug candidates and target proteins are verified through molecular docking simulations. In this application, it is important not only to correctly predict the free energy of binding, but also to provide a comprehensible model that could be validated by a domain specialist. Decision-tree induction algorithms have been successfully used in drug-design related applications, specially considering that decision trees are simple to understand, interpret, and validate. There are several decision-tree induction algorithms available for general-use, but each one has a bias that makes it more suitable for a particular data distribution. In this article, we propose and investigate the automatic design of decision-tree induction algorithms tailored to particular drug-enzyme binding data sets. We investigate the performance of our new method for evaluating binding conformations of different drug candidates to InhA, and we analyze our findings with respect to decision tree accuracy, comprehensibility, and biological relevance. RESULTS: The empirical analysis indicates that our method is capable of automatically generating decision-tree induction algorithms that significantly outperform the traditional C4.5 algorithm with respect to both accuracy and comprehensibility. In addition, we provide the biological interpretation of the rules generated by our approach, reinforcing the importance of comprehensible predictive models in this particular bioinformatics application. CONCLUSIONS: We conclude that automatically designing a decision-tree algorithm tailored to molecular docking data is a promising alternative for the prediction of the free energy from the binding of a drug candidate with a flexible-receptor.

Engineering and adaptive evolution of Escherichia coli for D-lactate fermentation reveals GatC as a xylose transporter.

Despite the abundance of xylose in nature, the production of chemicals from C5 sugars remains challenging in metabolic engineering. By deleting xylFGH genes and using adaptive evolution, an efficient E. coli strain capable of producing D-lactate from xylose was engineered. Quantitative proteomics and genome sequencing were used to understand the new phenotype and the metabolic limitations of xylose conversion to D-lactate. Proteomics identified major changes in enzyme concentration in the glycolytic and tricarboxylic acid pathways. Whole genome sequencing of the evolved strain identified a point mutation in the gatC gene, which resulted in a change from serine to leucine at position 184 of the GatC protein. The knockout of gatC in a number of strains and the insertion of the mutation in the non-evolved strain confirmed its activity as a xylose transporter and demonstrated that the mutation is responsible for the high xylose consumption phenotype in the evolved strain. The newly found xylose transporter is a candidate for future strain engineering for converting C5-C6 syrups into valuable chemicals.

Recent trends in biocatalysis engineering.

During the last 30 years the scope of biocatalysis has been expanding due to the advances in several technological fields. Diverse techniques as structural enzyme improvement (e.g. protein engineering, direct evolution), engineering approaches (e.g. ionic liquids, supercritical fluids) and physical stabilization (e.g. immobilization, CLEAS) have been developed, which in combination are powerful tools to improve biotransformation and to synthesize new products. In the present work, recent advances in biocatalysis are reviewed.

A practical teaching course in directed protein evolution using the green fluorescent protein as a model.

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.