scholarly journals Scalable continuous evolution of genes at mutation rates above genomic error thresholds

2018 ◽  
Author(s):  
Arjun Ravikumar ◽  
Garri A. Arzumanyan ◽  
Muaeen K.A. Obadi ◽  
Alex A. Javanpour ◽  
Chang C. Liu
Keyword(s):  
Directed Evolution ◽  
Fitness Landscape ◽  
Drug Resistant ◽  
Wild Type ◽  
New Paradigm ◽  
Wild Type Enzyme ◽  
Epistatic Interactions ◽  
Powerful Approach ◽  
Experimental Strategies

Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation1-3. However, experimental strategies for directed evolution are notoriously low-throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. Here, we report OrthoRep, a yeast orthogonal DNA polymerase-plasmid pair that stably mutates ~100,000-fold faster than the host genome in vivo, exceeding error thresholds of genomic replication that lead to single-generation extinction4. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly scalable process. Using OrthoRep, we evolved drug resistant malarial DHFRs 90 times and uncovered a more complex fitness landscape than previously realized5-9. We find rare fitness peaks that resist the maximum soluble concentration of the antimalarial pyrimethamine – these resistant variants support growth at pyrimethamine concentrations >40,000-fold higher than the wild-type enzyme can tolerate – and also find that epistatic interactions direct adaptive trajectories to convergent outcomes. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.

scholarly journals Thermostabilization of Bacterial Fructosyl-Amino Acid Oxidase by Directed Evolution

2003 ◽  
Vol 69 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Ryoichi Sakaue ◽  
Naoki Kajiyama
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Amino Acid Substitutions ◽  
Acid Oxidase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Amino Acid Oxidase ◽  
Fourth Round ◽  
Fructosyl Amino Acid Oxidase

ABSTRACT 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°C, whereas the wild-type enzyme was not stable above 37°C. The Km 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.

scholarly journals Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Hui Cai ◽  
Meisui Liu ◽  
Charles J. Russell
Keyword(s):  
Gene Expression ◽  
Influenza Virus ◽  
Virus Infection ◽  
Directed Evolution ◽  
Influenza Virus Infection ◽  
Foreign Gene ◽  
Reporter Virus ◽  
Wild Type ◽  
Bioluminescence Signal

ABSTRACTReporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus withNanoLuc luciferaseinserted into the 5′ end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we comparedin vivoreplication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations andPB2noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-likein vivofitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCEInfluenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

scholarly journals Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

2003 ◽  
Vol 47 (3) ◽  
pp. 1037-1046 ◽  
Author(s):  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Trudy H. Grossman ◽  
Paul S. Charifson ◽  
Steven Bellon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Mutant Proteins ◽  
E Coli ◽  
Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

scholarly journals Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

ISRN Oncology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-12
Author(s):  
Fei Chu ◽  
Jessica A. Naiditch ◽  
Sandra Clark ◽  
Yi-Yong Qiu ◽  
Xin Zheng ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Chemotherapy Resistance ◽  
Neuroblastoma Cells ◽  
Cytotoxic Agents ◽  
Drug Resistant ◽  
Wild Type ◽  
Cytoprotective Effect ◽  
Acquired Drug Resistance

Resistance to cytotoxic agents has long been known to be a major limitation in the treatment of human cancers. Although many mechanisms of drug resistance have been identified, chemotherapies targeting known mechanisms have failed to lead to effective reversal of drug resistance, suggesting that alternative mechanisms remain undiscovered. Previous work identified midkine (MK) as a novel putative survival molecule responsible for cytoprotective signaling between drug-resistant and drug-sensitive neuroblastoma, osteosarcoma and breast carcinoma cells in vitro. In the present study, we provide further in vitro and in vivo studies supporting the role of MK in neuroblastoma cytoprotection. MK overexpressing wild type neuroblastoma cells exhibit a cytoprotective effect on wild type cells when grown in a co-culture system, similar to that seen with doxorubicin resistant cells. siRNA knockdown of MK expression in doxorubicin resistant neuroblastoma and osteosarcoma cells ameliorates this protective effect. Overexpression of MK in wild type neuroblastoma cells leads to acquired drug resistance to doxorubicin and to the related drug etoposide. Mouse studies injecting various ratios of doxorubicin resistant or MK transfected cells with GFP transfected wild type cells confirm this cytoprotective effect in vivo. These findings provide additional evidence for the existence of intercellular cytoprotective signals mediated by MK which contribute to chemotherapy resistance in neuroblastoma.

scholarly journals Analysis of the oxidative stress regulon identifies soxS as a genetic target for resistance reversal in multi-drug resistant Klebsiella pneumoniae

2020 ◽  
Author(s):  
João Anes ◽  
Katherine Dever ◽  
Athmanya Eshwar ◽  
Scott Nguyen ◽  
Yu Cao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Similar Data ◽  
Drug Resistant ◽  
Rna Seq ◽  
Wild Type ◽  
Knock Out ◽  
Global Regulators

AbstractIn bacteria, the defense system deployd to counter oxidative stress is orchestrated by three transcriptional factors – SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data is not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH 78578 using paraquat and the corresponding oxidative stress regulon recorded using RNA-seq. The soxS gene was significantly induced during oxidative stress and a knock-out mutant was constructed, to explore its functionality. The wild-type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress regulon and soxS regulon were identified and denoted as the ‘oxidative SoxS regulon’ – these included a stringent group of genes specifically regulated by SoxS. Efflux pump encoding genes such as acrAB-tolC, acrE, and global regulators such as marRAB were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo, in a zebrafish embryo model. We conclude that the soxS gene could be considered as a genetic target against which an inhibitor could be developed in the future and used in combinatorial therapy with tetracycline to combat infections associated with multi-drug resistant K. pneumoniae.

scholarly journals Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

2006 ◽  
Vol 397 (2) ◽  
pp. 305-312 ◽  
Author(s):  
G. H. Erica Law ◽  
Olga A. Gandelman ◽  
Laurence C. Tisi ◽  
Christopher R. Lowe ◽  
James A. H. Murray
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Green Light ◽  
Firefly Luciferase ◽  
Wild Type ◽  
Photinus Pyralis ◽  
Wild Type Enzyme ◽  
Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

scholarly journals Decreased Activity of the Ghrhr and Gh Promoters Causes Dominantly Inherited GH Deficiency in Humanized GH1 Mouse Models

Endocrinology ◽  
2019 ◽  
Vol 160 (11) ◽  
pp. 2673-2691
Author(s):  
Daisuke Ariyasu ◽  
Emika Kubo ◽  
Daisuke Higa ◽  
Shinsuke Shibata ◽  
Yutaka Takaoka ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Gh Deficiency ◽  
Gene Promoter ◽  
Dominant Negative ◽  
Hormone Deficiency ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
New Paradigm ◽  
Gh Secretion

Abstract Isolated growth hormone deficiency type II (IGHD2) is mainly caused by heterozygous splice-site mutations in intron 3 of the GH1 gene. A dominant-negative effect of the mutant GH lacking exon 3 on wild-type GH secretion has been proposed; however, the molecular mechanisms involved are elusive. To uncover the molecular systems underlying GH deficiency in IGHD2, we established IGHD2 model mice, which carry both wild-type and mutant copies of the human GH1 gene, replacing each of the endogenous mouse Gh loci. Our IGHD2 model mice exhibited growth retardation along with intact cellular architecture and mildly activated endoplasmic reticulum stress in the pituitary gland, caused by decreased GH-releasing hormone receptor (Ghrhr) and Gh gene promoter activities. Decreased Ghrhr and Gh promoter activities were likely caused by reduced levels of nuclear CREB3L2, which was demonstrated to stimulate Ghrhr and Gh promoter activity. To our knowledge, this is the first in vivo study to reveal a novel molecular mechanism of GH deficiency in IGHD2, representing a new paradigm that differs from widely accepted models.

scholarly journals Crystal Structures of Pyrophosphatase from Acinetobacter baumannii: Snapshots of Pyrophosphate Binding and Identification of a Phosphorylated Enzyme Intermediate

2019 ◽  
Vol 20 (18) ◽  
pp. 4394
Author(s):  
Yunlong Si ◽  
Xing Wang ◽  
Guosong Yang ◽  
Tong Yang ◽  
Yuying Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Water Molecule ◽  
Acinetobacter Baumannii ◽  
Divalent Cations ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Catalytic Center ◽  
Favorable Reaction ◽  
Enzyme Intermediate

All living things have pyrophosphatases that hydrolyze pyrophosphate and release energy. This energetically favorable reaction drives many energetically unfavorable reactions. An accepted catalytic model of pyrophosphatase shows that a water molecule activated by two divalent cations (M1 and M2) within the catalytic center can attack pyrophosphate in an SN2 mechanism and thus hydrolyze the molecule. However, our co-crystal structure of Acinetobacter baumannii pyrophosphatase with pyrophosphate shows that a water molecule from the solvent may, in fact, be the actual catalytic water. In the co-crystal structure of the wild-type pyrophosphatase with pyrophosphate, the electron density of the catalytic centers of each monomer are different from one another. This indicates that pyrophosphates in the catalytic center are dynamic. Our mass spectroscopy results have identified a highly conserved lysine residue (Lys30) in the catalytic center that is phosphorylated, indicating that the enzyme could form a phosphoryl enzyme intermediate during hydrolysis. Mutation of Lys30 to Arg abolished the activity of the enzyme. In the structure of the apo wild type enzyme, we observed that a Na+ ion is coordinated by residues within a loop proximal to the catalytic center. Therefore, we mutated three key residues within the loop (K143R, P147G, and K149R) and determined Na+ and K+-induced inhibition on their activities. Compared to the wild type enzyme, P147G is most sensitive to these cations, whereas K143R was inactive and K149R showed no change in activity. These data indicate that monovalent cations could play a role in down-regulating pyrophosphatase activity in vivo. Overall, our results reveal new aspects of pyrophosphatase catalysis and could assist in the design of specific inhibitors of Acinetobacter baumannii growth.

scholarly journals AMaLa: analysis of Directed Evolution experiments via Annealed Mutational approximated Landscape

2021 ◽  
Author(s):  
Luca Sesta ◽  
Guido Uguzzoni ◽  
Jorge Ferndadez-de-Cossio-Diaz ◽  
Andrea Pagnani
Keyword(s):  
Directed Evolution ◽  
High Throughput Sequencing ◽  
Fitness Landscape ◽  
Fitness Landscapes ◽  
Functional Screening ◽  
Wild Type ◽  
Wild Type Sequence ◽  
Mutational Step ◽  
Antibiotic Drug ◽  
Type Sequence

We present Annealed Mutational approximated landscape (AMaLa), a new method to infer fitness landscapes from Directed Evolution experiment sequencing data. Directed Evolution experiments typically start from a single wild-type sequence, which undergoes Darwinian in vitro evolution acted via multiple rounds of mutation and selection with respect to a target phenotype. In the last years, Directed Evolution is emerging as a powerful instrument to probe fitness landscapes under controlled experimental condition and, thanks to the use of high-throughput sequencing of the different rounds, as a relevant testing ground to develop accurate statistical models and inference algorithms. Fitness landscape modeling strategies, either use as input data the enrichment of variants abundances and hence require observing the same variants at different rounds, or they simply assume that the variants at the last sequenced round are the results of a sampling process at equilibrium. AMaLa aims at leveraging effectively the information encoded in the time evolution of all sequenced rounds. To do so, on the one hand we assume statistical sampling independence between sequenced rounds, and on the other we gauge all possible trajectories in sequence space with a time-dependent statistical weight consisting of two contributions: (i) a statistical energy term accounting for the selection process, (ii) a simple generalized Jukes-Cantor model to describe the purely mutational step. This simple scheme allows us to accurately describe the Directed Evolution dynamics in a concrete experimental setup and to infer a fitness landscape that reproduces correctly the measures of the phenotype under selection (e.g. antibiotic drug resistance), notably outperforming widely used inference strategies. We assess the reliability of AMaLa by showing how the inferred statistical model could be used to predict relevant structural properties of the wild-type sequence, and to reproduce the mutational effects of large scale functional screening not used to train the model.

scholarly journals A Biocatalytic Approach to a Key Intermediate for the Synthesis of the COVID-19 Experimental Drug Molnupiravir

2021 ◽  
Author(s):  
Ashleigh Burke ◽  
William Birmingham ◽  
Ying Zhuo ◽  
Bruna Zuculoto da Costa ◽  
Rebecca Crawshaw ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
Cytidine Deaminase ◽  
Good Activity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Experimental Drug ◽  
Hydrolysis Activity

Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance the ratio of N-hydroxycytidine to uridine produced through directed evolution of CD. In particular, a T123G mutation close to the active site dramatically reduces cytidine hydrolysis activity whilst preserving desired amination activty. The approach reported provides a new route to a key intermediate for the COVID-19 experimental drug Molnupiravir 1.

Sign in / Sign up

Export Citation Format

Share Document