scholarly journals Cryptic genetic variation defines the adaptive evolutionary potential of enzymes

2017 ◽  
Author(s):  
Florian Baier ◽  
Nansook Hong ◽  
Gloria Yang ◽  
Anna Pabis ◽  
Alexandre Barrozo ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Catalytic Activity ◽  
Directed Evolution ◽  
Molecular Basis ◽  
Initial Activity ◽  
Evolutionary Potential ◽  
Phenotypic Properties ◽  
Changing Environments ◽  
Protein Functions ◽  
Evolution Of Proteins

AbstractGenetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may also impact the evolutionary potential of proteins, but the molecular basis for this remains unclear. Here we perform comparative directed evolution in which four orthologous metallo-β-lactamases were evolved toward a new function. We found that genetic variation between these enzymes resulted in distinct evolutionary outcomes. The ortholog with the lower initial activity reached a 20-fold higher fitness plateau exclusively via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show that the cryptic molecular properties and conformational variation of residues in the initial genotypes cause epistasis, thereby constraining evolutionary outcomes. Our work highlights that understanding the molecular details relating genetic variation to protein functions is essential to predicting the evolution of proteins.

scholarly journals Cryptic genetic variation shapes the adaptive evolutionary potential of enzymes

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Florian Baier ◽  
Nansook Hong ◽  
Gloria Yang ◽  
Anna Pabis ◽  
Charlotte M Miton ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Catalytic Activity ◽  
Active Site ◽  
Protein Function ◽  
Molecular Basis ◽  
Initial Activity ◽  
Active Site Residues ◽  
Evolutionary Potential ◽  
Phenotypic Properties ◽  
Changing Environments

Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-β-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.

scholarly journals Laboratory-Directed Protein Evolution

2005 ◽  
Vol 69 (3) ◽  
pp. 373-392 ◽  
Author(s):  
Ling Yuan ◽  
Itzhak Kurek ◽  
James English ◽  
Robert Keenan
Keyword(s):  
Protein Evolution ◽  
Selective Pressure ◽  
Evolutionary Potential ◽  
Effective Strategies ◽  
Scientific Disciplines ◽  
Wide Range ◽  
Protein Functions ◽  
Evolution Of Proteins ◽  
Efficient Screening ◽  
New Protein

SUMMARY Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences.

scholarly journals Directed Evolution of Proteins throughIn VitroProtein Synthesis in Liposomes

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Takehiro Nishikawa ◽  
Takeshi Sunami ◽  
Tomoaki Matsuura ◽  
Tetsuya Yomo
Keyword(s):  
Protein Synthesis ◽  
Directed Evolution ◽  
Single Copy ◽  
Functional Evaluation ◽  
Future Directions ◽  
Pure System ◽  
Protein Functions ◽  
A Cell ◽  
Evolution Of Proteins

Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.”In vitrocompartmentalization (IVC) is anin vitrogene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment,in vitroprotein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized byin vitroprotein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP andβ-glucuronidase are described. We discuss the future directions for this method and its applications.

scholarly journals Fluctuating environments select for short-term phenotypic variation leading to long-term exploration

2018 ◽  
Author(s):  
Rosangela Canino-Koning ◽  
Michael J. Wiser ◽  
Charles Ofria
Keyword(s):  
Evolutionary Dynamics ◽  
Population Bottlenecks ◽  
Evolutionary Potential ◽  
Short Term ◽  
Phenotypic Properties ◽  
Mutational Landscape ◽  
Changing Environments ◽  
Digital Organisms ◽  
Mutational Step

AbstractGenetic spaces are often described in terms of fitness landscapes or genotype-to-phenotype maps, where each genetic sequence is associated with phenotypic properties and linked to other genotypes that are a single mutational step away. The positions close to a genotype make up its “mutational landscape” and, in aggregate, determine the short-term evolutionary potential of a population. Populations with wider ranges of phenotypes in their mutational neighborhood are known to be more evolvable. Likewise, those with fewer phenotypic changes available in their local neighborhoods are more mutationally robust. Here, we examine whether forces that change the distribution of phenotypes available by mutation profoundly alter subsequent evolutionary dynamics.We compare evolved populations of digital organisms that were subject to either static or cyclically-changing environments. For each of these, we examine diversity of the phenotypes that are produced through mutations in order to characterize the local genotype-phenotype map. We demonstrate that environmental change can push populations toward more evolvable mutational landscapes where many alternate phenotypes are available, though purely deleterious mutations remain suppressed. Further, we show that populations in environments with harsh changes switch phenotypes more readily than those in environments with more benign changes. We trace this effect to repeated population bottlenecks in the harsh environments, which result in shorter coalescence times and keep populations in regions of the mutational landscape where the phenotypic shifts in question are more likely to occur. Typically, static environments select solely for immediate optimization, at the expensive of long-term evolvability. In contrast, we show that with changing environments, short-term pressures to deal with immediate challenges can align with long-term pressures to explore a more productive portion of the mutational landscape.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

scholarly journals Recent Advances in the Chemical Biology of N-Glycans

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1040
Author(s):  
Asuka Shirakawa ◽  
Yoshiyuki Manabe ◽  
Koichi Fukase
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Molecular Basis ◽  
Chemical Biology ◽  
Molecular Level ◽  
Chemoenzymatic Synthesis ◽  
Complex Structures ◽  
Recent Advances ◽  
Protein Functions ◽  
Potential Applications

Asparagine-linked N-glycans on proteins have diverse structures, and their functions vary according to their structures. In recent years, it has become possible to obtain high quantities of N-glycans via isolation and chemical/enzymatic/chemoenzymatic synthesis. This has allowed for progress in the elucidation of N-glycan functions at the molecular level. Interaction analyses with lectins by glycan arrays or nuclear magnetic resonance (NMR) using various N-glycans have revealed the molecular basis for the recognition of complex structures of N-glycans. Preparation of proteins modified with homogeneous N-glycans revealed the influence of N-glycan modifications on protein functions. Furthermore, N-glycans have potential applications in drug development. This review discusses recent advances in the chemical biology of N-glycans.

scholarly journals Advances in the directed evolution of proteins

2014 ◽  
Vol 22 ◽  
pp. 129-136 ◽  
Author(s):  
Michael D Lane ◽  
Burckhard Seelig
Keyword(s):  
Directed Evolution ◽  
Evolution Of Proteins

Directed Evolution of Proteins for Device Applications

2008 ◽  
Author(s):  
Jeremy Koscielecki ◽  
Jason Hillebrecht ◽  
Robert Birge
Keyword(s):  
Directed Evolution ◽  
Device Applications ◽  
Evolution Of Proteins

scholarly journals Loci, genes, and gene networks associated with life history variation in a model ecological organism,Daphnia pulex(complex)

2018 ◽  
Author(s):  
Jacob W. Malcom ◽  
Thomas E. Juenger ◽  
Mathew A. Leibold
Keyword(s):  
Gene Expression ◽  
Genetic Variation ◽  
Life History ◽  
Molecular Basis ◽  
Life History Traits ◽  
Evolutionary Dynamics ◽  
Daphnia Pulex ◽  
Life History Trait ◽  
Life History Variation ◽  
Trait Variation

ABSTRACTBackgroundIdentifying the molecular basis of heritable variation provides insight into the underlying mechanisms generating phenotypic variation and the evolutionary history of organismal traits. Life history trait variation is of central importance to ecological and evolutionary dynamics, and contemporary genomic tools permit studies of the basis of this variation in non-genetic model organisms. We used high density genotyping, RNA-Seq gene expression assays, and detailed phenotyping of fourteen ecologically important life history traits in a wild-caught panel of 32Daphnia pulexclones to explore the molecular basis of trait variation in a model ecological species.ResultsWe found extensive phenotypic and a range of heritable genetic variation (~0 < H2< 0.44) in the panel, and accordingly identify 75-261 genes—organized in 3-6 coexpression modules—associated with genetic variation in each trait. The trait-related coexpression modules possess well-supported promoter motifs, and in conjunction with marker variation at trans- loci, suggest a relatively small number of important expression regulators. We further identify a candidate genetic network with SNPs in eight known transcriptional regulators, and dozens of differentially expressed genes, associated with life history variation. The gene-trait associations include numerous un-annotated genes, but also support several a priori hypotheses, including an ecdysone-induced protein and several Gene Ontology pathways.ConclusionThe genetic and gene expression architecture ofDaphnialife history traits is complex, and our results provide numerous candidate loci, genes, and coexpression modules to be tested as the molecular mechanisms that underlieDaphniaeco-evolutionary dynamics.

scholarly journals Intraspecific genomic variation and local adaptation in a young hybrid species

2019 ◽  
Author(s):  
Angélica Cuevas ◽  
Mark Ravinet ◽  
Glenn-Peter Sætre ◽  
Fabrice Eroukhmanoff
Keyword(s):  
Genetic Variation ◽  
Local Adaptation ◽  
Climatic Variation ◽  
Genomic Variation ◽  
Population Divergence ◽  
Parent Species ◽  
Evolutionary Potential ◽  
Variable Environment ◽  
Hybrid Species ◽  
Species Population

ABSTRACTHybridization increases genetic variation, hence hybrid species may have a strong evolutionary potential once their admixed genomes have stabilized and incompatibilities have been purged. Yet, little is known about how such hybrid lineages evolve at the genomic level following their formation, in particular the characteristics of their adaptive potential, i.e. constraints and facilitations of diversification. Here we investigate how the Italian sparrow (Passer italiae), a homoploid hybrid species, has evolved and locally adapted to its variable environment. Using restriction site-associated DNA sequencing (RAD-seq) on several populations across the Italian peninsula, we evaluate how genomic constraints and novel genetic variation have influenced population divergence and adaptation. We show that population divergence within this hybrid species has evolved in response to climatic variation. As in non-hybrid species, climatic differences may even reduce gene flow between populations, suggesting ongoing local adaptation. We report outlier genes associated with adaptation to climatic variation, known to be involved in beak morphology in other species. Most of the strongly divergent loci among Italian sparrow populations seem not to be differentiated between its parent species, the house and Spanish sparrow. Within the parental species, population divergence has occurred mostly in loci where different alleles segregate in the parent species, unlike in the hybrid, suggesting that novel combinations of parental alleles in the hybrid have not necessarily enhanced its evolutionary potential. Rather, our study suggests that constraints linked to incompatibilities may have restricted the evolution of this admixed genome, both during and after hybrid species formation.

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