Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities.
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ABSTRACT:
SUBMITTER: Rix G
PROVIDER: S-EPMC7648111 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In this commentary we discuss recent progress in our understanding of adaptive protein evolution. We begin with a general introduction to proteins and their evolution, quickly focusing on the question of how natural selection produces proteins with novel functions. We then summarize the theory of latent protein adaptation advanced by the joint articles by Amitai et al. (2007), HFSP J. 1, 67-78 and Wroe et al. (2007) HFSP J. 1, 79-87, published in the first issue edition of the HFSP Journal. This theory provides a biophysical framework linking the effects of individual mutations on promiscuous protein function, neutral genetic drift, and gene duplication to the process of adaptive protein evolution.
| S-EPMC2639842 | biostudies-other
Project description:Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation. However, experimental strategies for directed evolution are notoriously labor intensive and low throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. We report OrthoRep, an orthogonal DNA polymerase-plasmid pair in yeast that stably mutates ∼100,000-fold faster than the host genome in vivo, exceeding the error threshold of genomic replication that causes single-generation extinction. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly straightforward and scalable process. Using OrthoRep, we evolved drug-resistant malarial dihydrofolate reductases (DHFRs) in 90 independent replicates. We uncovered a more complex fitness landscape than previously realized, including common adaptive trajectories constrained by epistasis, rare outcomes that avoid a frequent early adaptive mutation, and a suboptimal fitness peak that occasionally traps evolving populations. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.
| S-EPMC6343851 | biostudies-literature
Project description:A key step in metabolic pathway evolution is the recruitment of promiscuous enzymes to perform new functions. Despite the recognition that promiscuity is widespread in biology, factors dictating the preferential recruitment of one promiscuous enzyme over other candidates are unknown. Escherichia coli contains four sugar kinases that are candidates for recruitment when the native glucokinase machinery is deleted-allokinase (AlsK), manno(fructo)kinase (Mak), N-acetylmannosamine kinase (NanK), and N-acetylglucosamine kinase (NagK). The catalytic efficiencies of these enzymes are 103- to 105-fold lower than native glucokinases, ranging from 2,400 M-1 s-1 for the most active candidate, NagK, to 15 M-1 s-1 for the least active candidate, AlsK. To investigate the relationship between catalytic activities of promiscuous enzymes and their recruitment, we performed adaptive evolution of a glucokinase-deficient E. coli strain to restore glycolytic metabolism. We observed preferential recruitment of NanK via a trajectory involving early mutations that facilitate glucose uptake and amplify nanK transcription, followed by nonsynonymous substitutions in NanK that enhance the enzyme's promiscuous glucokinase activity. These substitutions reduced the native activity of NanK and reduced organismal fitness during growth on an N-acetylated carbon source, indicating that enzyme recruitment comes at a cost for growth on other substrates. Notably, the two most active candidates, NagK and Mak, were not recruited, suggesting that catalytic activity alone does not dictate evolutionary outcomes. The results highlight our lack of knowledge regarding biological drivers of enzyme recruitment and emphasize the need for a systems-wide approach to identify factors facilitating or constraining this important adaptive process.
| S-EPMC10519446 | biostudies-literature
Project description:The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.
| S-EPMC8154213 | biostudies-literature
Project description:BackgroundSome clinically important genetic variants are not easily evaluated with next-generation sequencing (NGS) methods due to technical challenges arising from high- similarity copies (e.g., PMS2, SMN1/SMN2, GBA1, HBA1/HBA2, CYP21A2), repetitive short sequences (e.g., ARX polyalanine repeats, FMR1 AGG interruptions in CGG repeats, CFTR poly-T/TG repeats), and other complexities (e.g., MSH2 Boland inversions).MethodsWe customized our NGS processes to detect the technically challenging variants mentioned above with adaptations including target enrichment and bioinformatic masking of similar sequences. Adaptations were validated with samples of known genotypes.ResultsOur adaptations provided high-sensitivity and high-specificity detection for most of the variants and provided a high-sensitivity primary assay to be followed with orthogonal disambiguation for the others. The sensitivity of the NGS adaptations was 100% for all of the technically challenging variants. Specificity was 100% for those in PMS2, GBA1, SMN1/SMN2, and HBA1/HBA2, and for the MSH2 Boland inversion; 97.8%-100% for CYP21A2 variants; and 85.7% for ARX polyalanine repeats.ConclusionsNGS assays can detect technically challenging variants when chemistries and bioinformatics are jointly refined. The adaptations described support a scalable, cost-effective path to identifying all clinically relevant variants within a single sample.
| S-EPMC9747563 | biostudies-literature
Project description:We present a fast computational method to efficiently screen enzyme activity. In the presented method, the effect of mutations on the barrier height of an enzyme-catalysed reaction can be computed within 24 hours on roughly 10 processors. The methodology is based on the PM6 and MOZYME methods as implemented in MOPAC2009, and is tested on the first step of the amide hydrolysis reaction catalyzed by the Candida Antarctica lipase B (CalB) enzyme. The barrier heights are estimated using adiabatic mapping and shown to give barrier heights to within 3 kcal/mol of B3LYP/6-31G(d)//RHF/3-21G results for a small model system. Relatively strict convergence criteria (0.5 kcal/(molÅ)), long NDDO cutoff distances within the MOZYME method (15 Å) and single point evaluations using conventional PM6 are needed for reliable results. The generation of mutant structures and subsequent setup of the semiempirical calculations are automated so that the effect on barrier heights can be estimated for hundreds of mutants in a matter of weeks using high performance computing.
| S-EPMC3524253 | biostudies-literature
Project description:The metallo-β-lactamase fold is an ancient protein structure present in numerous enzyme families responsible for diverse biological processes. The crystal structure of the hyperthermostable crenarchaeal enzyme Igni18 from Ignicoccus hospitalis was solved at 2.3 Å and could resemble a possible first archetype of a multifunctional metallo-β-lactamase. Ancestral enzymes at the evolutionary origin are believed to be promiscuous all-rounders. Consistently, Igni18´s activity can be cofactor-dependently directed from β-lactamase to lactonase, lipase, phosphodiesterase, phosphotriesterase or phospholipase. Its core-domain is highly conserved within metallo-β-lactamases from Bacteria, Archaea and Eukarya and gives insights into evolution and function of enzymes from this superfamily. Structural alignments with diverse metallo-β-lactamase-fold-containing enzymes allowed the identification of Protein Variable Regions accounting for modulation of activity, specificity and oligomerization patterns. Docking of different substrates within the active sites revealed the basis for the crucial cofactor dependency of this enzyme superfamily.
| S-EPMC7846560 | biostudies-literature
Project description:The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-continuous evolution, three new phage-assisted continuous evolution strategies for DNA binding and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer-sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediated indel formation and base editing in human cells and enabled A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.
| S-EPMC7145744 | biostudies-literature
Project description:One of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates, which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution. The molecular basis for this widespread problem remains hazy. To investigate the mechanism of inhibition at a molecular level, we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogen-deuterium exchange mass spectrometry. For AmpC beta-lactamase, binding to aggregates of the small molecule rottlerin increased the deuterium exchange of all 10 reproducibly detectable peptides, which covered 41% of the sequence of beta-lactamase. This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation. We then investigated whether enzyme-aggregate complexes were more susceptible to proteolysis than uninhibited enzyme. For five aggregators, trypsin degradation of beta-lactamase increased substantially when beta-lactamase was inhibited by aggregates, whereas uninhibited enzyme was generally stable to digestion. Combined, these results suggest that the mechanism of action of aggregate-based inhibitors proceeds via partial protein unfolding when bound to an aggregate particle.
| S-EPMC2664636 | biostudies-literature
Project description:The probability of the evolution of a character depends on two factors: the probability of moving from one character state to another character state and the probability of the new character state fixation. The more the evolution of a character is probable, the more the convergent evolution will be witnessed, and consequently, convergent evolution could mean that the convergent character evolution results as a combination of these two factors. We investigated this phenomenon by studying the convergent evolution of biochemical functions. For the investigation we used the case of β-lactamases. β-lactamases hydrolyze β-lactams, which are antimicrobials able to block the DD-peptidases involved in bacterial cell wall synthesis. β-lactamase activity is present in two different superfamilies: the metallo-β-lactamase and the serine β-lactamase. The mechanism used to hydrolyze the β-lactam is different for the two superfamilies. We named this kind of evolution an allo-convergent evolution. We further showed that the β-lactamase activity evolved several times within each superfamily, a convergent evolution type that we named iso-convergent evolution. Both types of convergent evolution can be explained by the two evolutionary mechanisms discussed above. The probability of moving from one state to another is explained by the promiscuous β-lactamase activity present in the ancestral sequences of each superfamily, while the probability of fixation is explained in part by positive selection, as the organisms having β-lactamase activity allows them to resist organisms that secrete β-lactams. Indeed, an organism that has a mutation that increases the β-lactamase activity will be selected, as the organisms having this activity will have an advantage over the others.
| S-EPMC7504333 | biostudies-literature