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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations

Abstract Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5′-MCRTRCRN4YGYAYGK-3′. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation

ABSTRACT The ability to better understand the function of proteins expressed by bacteria has typically relied upon the development of genetic mutant strains. This approach has been especially challenging for plasmid-encoded genes, as most of the previously described allelic replacement vectors are inefficient for plasmid genome mutation as they either rely on plasmid-derived counterselection toxins or depend on other strategies suchsacB,tetA, andrpsLwhich have been proven to be less efficient for mutant selection. Integrative vectors lack chromophore indicators, thus requiring laborious screening or excision of the vector’s backbone relies on the introduction of a flippase (FLP)-expressing plasmid. The allelic replacement vector, designated here as pDG1, expresses an X-Gal hydrolyzing enzyme (BgaB) that can be used for blue/white screening allowing identification of colonies that integrated and successfully removed the mutagenesis plasmid without a bias for those still carrying it. pDG1 was further improved by including the rhamnose-inducible Tse2 toxin as a potent counterselection system. The efficacy of pDG1 was validated by deleting portions of the plasmid-encoded VirB4/D4 type IV secretion system and aerobactin-synthesizing operons inSalmonella enterica. The integrative vectors, which contain an ΦC31attPsite and genes encoding ΦC31 integrase (int), can seamlessly integrate to target ΦC31attBonSalmonellaplasmids or chromosome. These vectors were improved by insertingbgaBand FLP-encoding genes so that, following integration, most of the vector’s backbone encompassingint,bgaB, and FLP genes can be excised by FLP, without the need for another FLP-expressing vector, creating white colonies carrying a stably integrated target gene. As such, we were able to integrate a 9.3-kb DNA fragment toSalmonellachromosome and flipped out most of the integrated vector in one step, leaving the target fragment in the chromosome. IMPORTANCE In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens includingin vivostudies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

Microbiology↗
DOE OSTI2022

Fast Evaluation of Viral Emerging Risks (FEVER): A computational tool for biosurveillance, diagnostics, and mutation typing of emerging viral pathogens

Viral pathogens can rapidly evolve, adapt to novel hosts, and evade human immunity. The early detection of emerging viral pathogens through biosurveillance coupled with rapid and accurate diagnostics are required to mitigate global pandemics. However, RNA viruses can mutate rapidly, hampering biosurveillance and diagnostic efforts. Here, we present a novel computational approach called FEVER (Fast Evaluation of Viral Emerging Risks) to design assays that simultaneously accomplish: 1) broad-coverage biosurveillance of an entire group of viruses, 2) accurate diagnosis of an outbreak strain, and 3) mutation typing to detect variants of public health importance. We demonstrate the application of FEVER to generate assays to simultaneously 1) detect sarbecoviruses for biosurveillance; 2) diagnose infections specifically caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and 3) perform rapid mutation typing of the D614G SARS-CoV-2 spike variant associated with increased pathogen transmissibility. These FEVER assays had a high in silico recall (predicted positive) up to 99.7% of 525,708 SARS-CoV-2 sequences analyzed and displayed sensitivities and specificities as high as 92.4% and 100% respectively when validated in 100 clinical samples. The D614G SARS-CoV-2 spike mutation PCR test was able to identify the single nucleotide identity at position 23,403 in the viral genome of 96.6% SARS-CoV-2 positive samples without the need for sequencing. This study demonstrates the utility of FEVER to design assays for biosurveillance, diagnostics, and mutation typing to rapidly detect, track, and mitigate future outbreaks and pandemics caused by emerging viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bubble lifetimes in DNA gene promoters and their mutations affecting transcription

Relative lifetimes of inherent double stranded DNA openings with lengths up to ten base pairs are presented for different gene promoters and corresponding mutants that either increase or decrease transcriptional activity, in the framework of the Peyrard-Bishop-Dauxois model. Extensive microcanonical simulations are used, with energies corresponding to physiological temperature. The bubble lifetime profiles along the DNA sequences demonstrate a significant reduction of the average lifetime at the mutation sites when the mutated promoter decreases transcription, while a corresponding enhancement of the bubble lifetime is observed in the case of mutations leading to increased transcription. The relative difference of bubble lifetimes between the mutated and the wild type promoters at the position of mutation varies from 20% to more than 30% as the bubble length is decreasing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Promise of Mutation Resistant Drugs for SARS-CoV-2 That Interdict in the Folding of the Spike Protein Receptor Binding Domain

In recent work, we proposed that effective therapeutic drugs aimed at treating the SARS-CoV-2 infection could be developed based on interdicting in the early steps of the folding pathway of key viral proteins, including the receptor binding domain (RBD) of the spike protein. In order to provide for a drug target on the protein, the earliest contact-formation event along the dominant folding pathway of the RBD spike protein was predicted employing the Sequential Collapse Model (SCM). The segments involved in the predicted earliest contact were suggested to provide optimal folding interdiction target regions (FITRs) for potential therapeutic drugs, with a focus on folding interdicting peptides (FIPs). In this paper, we extend our analysis to include 13 known single mutations of the RBD spike protein as well as the triple mutation B1.351 and the recent double mutation B1.617.2. The results show that the location of the FITR does not change in any of the 15 studied mutations, providing for a mutation-resistant drug design strategy for the RBD-spike protein.

Bergasa-Caceres, Fernando↗
DOE OSTI2022

Mutations of Omicron Variant at the Interface of the Receptor Domain Motif and Human Angiotensin-Converting Enzyme-2

The most recent Omicron variant of SARS-CoV-2 has caused global concern and anxiety. The only thing certain about this strain, with a large number of mutations in the spike protein, is that it spreads quickly, seems to evade immune defense, and mitigates the benefits of existing vaccines. Based on the ultra-large-scale ab initio computational modeling of the receptor binding motif (RBM) and the human angiotensin-converting enzyme-2 (ACE2) interface, we provide the details of the effect of Omicron mutations at the fundamental atomic scale level. In-depth analysis anchored in the novel concept of amino acid-amino acid bond pair units (AABPU) indicates that mutations in the Omicron variant are connected with (i) significant changes in the shape and structure of AABPU components, together with (ii) significant increase in the positive partial charge, which facilitates the interaction with ACE2. We have identified changes in bonding due to mutations in the RBM. The calculated bond order, based on AABPU, reveals that the Omicron mutations increase the binding strength of RBM to ACE2. Our findings correlate with and are instrumental to explain the current observations and can contribute to the prediction of next potential new variant of concern.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1994

Mutation induction in bacteria after heavy ion irradiation

From a compilation of experimental data on the mutagenic effects of heavy ions in bacteria, main conclusions have been drawn as follows: (1) The mutagenic efficacy of heavy ions in bacteria depends on physical and biological variables. Physical variables are the radiation dose, energy and charge of the ion; the biological variables are the bacterial strain, the repair genotype of bacteria, and the endpoint investigated (type of mutation, induction of enzymes related to mutagenesis); (2) The responses on dose or fluence are mainly linear or linear quadratic. The quadratic component, if found for low LET radiation, is gradually reduced with increasing LET; (3) At low values of Z and LET the cross section of mutation induction sigma m (as well as SOS response, sigma sos. and lambda phage induction, sigma lambda versus LET curves can be quite consistently described by a common function which increases up to approximately 100 keV/mu m. For higher LET values, the sigma(m) versus LET curves show the so-called 'hooks' observed also for other endpoints; (4) For light ions (Z is less than or equal to 4), the cross sections mostly decrease with increasing ion energy, which is probably related to the decrease of the specific energy departed by the ion inside the sensitive volume (cell). For ions in the range of Z = 10, sigma(m) is nearly independent on the ion energy. For heavier ions (Z is greater than or equal to 16), sigma(m) increases with the energy up to a maximum or saturation around 10 MeV/u. The increment becomes steeper with increasing atomic number of the ion. It correlates with the increasing track radius of the heavy ion; (5) The mutagenic efficiency per lethal event changes slightly with ion energy, if Z is small indicating a rough correlation between cellular lethality and mutation induction, only. For ions of higher Z this relation increases with energy, indicating a change in the 'mode' of radiation action from 'killing-prone' to 'mutation-prone'; and (6) Repair genotype substantially influences the radiation induced mutagenesis. Different mechanisms of mutation induction and/or different types of biologically significant lesions in wild type cells compared to repair deficient strains are a likely explanation.

Horneck, G.↗
NASA NTRS1997

Calculation of Heavy Ion Inactivation and Mutation Rates in Radial Dose Model of Track Structure

In the track structure model, the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated by using the dose response of the system to gamma rays and the radial dose of the ions and may be equal to unity at small impact parameters. We apply the track structure model to recent data with heavy ion beams irradiating biological samples of E. Coli, B. Subtilis spores, and Chinese hamster (V79) cells. Heavy ions have observed cross sections for inactivation that approach and sometimes exceed the geometric size of the cell nucleus. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT (hypoxanthine guanine phosphoribosyl transferase) mutations in V79 cells, and good agreement is found. Calculations show the high probability for mutation by relativistic ions due to the radial extension of ions track from delta rays. The effects of inactivation on mutation rates make it very unlikely that a single parameter such as LET (linear energy transfer) can be used to specify radiation quality for heavy ion bombardment.

Cucinotta, Francis A.↗
NASA NTRS2002

Ascorbate, added after irradiation, reduces the mutant yield and alters the spectrum of CD59- mutations in A(L) cells irradiated with high LET carbon ions

It has been reported that X-ray induced HPRT- mutation in cultured human cells is prevented by ascorbate added after irradiation. Mutation extinction is attributed to neutralization by ascorbate, of radiation-induced long-lived radicals (LLR) with half-lives of several hours. We here show that post-irradiation treatment with ascorbate (5 mM added 30 min after radiation) reduces, but does not eliminate, the induction of CD59- mutants in human-hamster hybrid A(L) cells exposed to high-LET carbon ions (LET of 100 KeV/microm). RibCys, [2(R,S)-D-ribo-1',2',3',4'-Tetrahydroxybutyl]-thiazolidene-4(R)-ca riboxylic acid] (4 mM) gave a similar but lesser effect. The lethality of the carbon ions was not altered by these chemicals. Preliminary data are presented that ascorbate also alters the spectrum of CD59- mutations induced by the carbon beam, mainly by reducing the incidence of small mutations and mutants displaying transmissible genomic instability (TGI), while large mutations are unaffected. Our results suggest that LLR are important in initiating TGI.

NASA Discipline Radiation Health↗
DOE OSTI2022

Uncovering novel mutational signatures by de novo extraction with SigProfilerExtractor

Mutational signature analysis is commonly performed in cancer genomic studies. Here, we present SigProfilerExtractor, an automated tool for de novo extraction of mutational signatures, and benchmark it against another 13 bioinformatics tools by using 34 scenarios encompassing 2,500 simulated signatures found in 60,000 synthetic genomes and 20,000 synthetic exomes. For simulations with 5% noise, reflecting high-quality datasets, SigProfilerExtractor outperforms other approaches by elucidating between 20% and 50% more true-positive signatures while yielding 5-fold less false-positive signatures. Applying SigProfilerExtractor to 4,643 whole-genome- and 19,184 whole-exome-sequenced cancers reveals four novel signatures. Two of the signatures are confirmed in independent cohorts, and one of these signatures is associated with tobacco smoking. In summary, this report provides a reference tool for analysis of mutational signatures, a comprehensive benchmarking of bioinformatics tools for extracting signatures, and several novel mutational signatures, including one putatively attributed to direct tobacco smoking mutagenesis in bladder tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic ‘gatekeeper’ enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp–Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Å resolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg–Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Mutations in artificial self-replicating tiles: A step toward Darwinian evolution

Significance In nature, mutation is the first step of evolution, where it provides the genetic variation for the natural selection to act. Here we take a system of artificial self-replicating tiles, DNA origami, that exhibit templated reproduction. We can generate a small fraction of mutations by introducing a mismatch in hybridization between parent and daughter. We can modify the origami functionality to affect the growth rate of the mutated species, giving it less or more evolutionary advantage, and to become dominant in several generations. The introduction of mutations into an artificial self-replicating system provides new directions for research into self-assembly processes.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2024

The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress–budding at the inner nuclear membrane–is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2000

Measuring the spectrum of mutation induced by nitrogen ions and protons in the human-hamster hybrid cell line A(L)C

Astronauts can be exposed to charged particles, including protons, alpha particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human-hamster cell lines, A(L) and A(L)C. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human-hamster hybrid cell lines A(L)C and A(L). CD59(-) mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the A(L) hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid A(L)C, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in A(L)C cells than in A(L) cells. In the case of nitrogen ions, the mutations observed in A(L)C cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59(-) mutant fraction for A(L)C cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or "skipping" mutations. We demonstrate here that A(L)C cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.

Non-NASA Center↗
NASA NTRS1997

Chromosomal mutations and chromosome loss measured in a new human-hamster hybrid cell line, ALC: studies with colcemid, ultraviolet irradiation, and 137Cs gamma-rays

Small mutations, megabase deletions, and aneuploidy are involved in carcinogenesis and genetic defects, so it is important to be able to quantify these mutations and understand mechanisms of their creation. We have previously quantified a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in a hamster-human hybrid cell line AL. S1- mutants have lost expression of a human cell surface antigen, S1, which is encoded by the M1C1 gene at 11p13 so that mutants can be detected via a complement-mediated cytotoxicity assay in which S1+ cells are killed and S1- cells survive. But loss of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the AL hybrid, so that mutants that have lost the entire chromosome 11 die and escape detection. To circumvent this, we fused AL with Chinese hamster ovary (CHO) cells to produce a new hybrid, ALC, in which the requirement for maintaining 11p15.5 is relieved, allowing us to detect mutations events involving loss of 11p15.5. We evaluated the usefulness of this hybrid by conducting mutagenesis studies with colcemid, 137Cs gamma-radiation and UV 254 nm light. Colcemid induced 1000 more S1- mutants per unit dose in ALC than in AL; the increase for UV 254 nm light was only two-fold; and the increase for 137Cs gamma-rays was 12-fold. The increase in S1- mutant fraction in ALC cells treated with colcemid and 137Cs gamma-rays were largely due to chromosome loss and 11p deletions often containing a breakpoint within the centromeric region.

NASA Discipline Radiation Health↗
DOE OSTI2024

Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Mutations that cause familial Alzheimer’s disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid β-peptide (Aβ). Nevertheless, whether Aβ is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aβ production likewise led to synaptic loss. Collectively, these findings implicate the stalled process—not the products—of γ-secretase cleavage of substrates in FAD pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Hyperfusogenic Mutations Destabilize the Postfusion Six-Helix Bundle of the Measles Virus Fusion Glycoprotein

Fusion of the host membrane and viral envelope by class I viral fusion proteins is driven by the assembly of a postfusion six-helix bundle formed through antiparallel interactions between N-terminal (HR1) and C-terminal (HR2) heptad-repeat regions. Although mutations in these regions of the measles virus (MeV) fusion (F) glycoprotein are known to promote neuropathogenic and hyperfusogenic phenotypes, their effects on postfusion core stability have not been systematically examined. Here, we combine peptide biophysics and X-ray crystallography to interrogate how mutations within the HR2 domain, present in native neuropathogenic MeV isolates (e.g., L454W and N462K) and laboratory-generated hyperfusogenic variants (e.g., L454M and T461A), influence postfusion 6HB assembly. Circular dichroism (CD) spectroscopy reveals that, with few exceptions, these mutations decrease postfusion core stability, despite their association with enhanced fusion activity. We also report the first crystal structure of the wild-type MeV postfusion core as well as structures of six hyperfusogenic variants, enabling high-resolution comparison of the molecular basis of destabilization. Structural analysis shows that these effects arise from localized perturbations to steric packing, hydrogen bonding networks, and helix-stabilizing interactions within HR2, while the overall 6HB architecture remains conserved. Together, these results indicate that hyperfusogenic mutations are not associated with stabilization of the postfusion state and are instead consistent with models in which hyperfusogenicity arises from a reduction in the energetic barrier to fusion, potentially through effects on prefusion stability or triggering efficiency. These findings establish key sequence-structure–stability relationships governing coiled-coil assembly and provide a framework for the design of HR1- and HR2-based fusion inhibitors.

Genetics↗
DOE OSTI2022

“Multiagent” Screening Improves Directed Enzyme Evolution by Identifying Epistatic Mutations

Enzyme evolution has enabled numerous advances in biotechnology and synthetic biology, yet still requires many iterative rounds of screening to identify optimal mutant sequences. This is due to the sparsity of the fitness landscape, which is caused by epistatic mutations that only offer improvements when combined with other mutations. We report an approach that incorporates diverse substrate analogues in the screening process, where multiple substrates act like multiple agents navigating the fitness landscape, identifying epistatic mutant residues without a need for testing the entire combinatorial search space. We initially validate this approach by engineering a malonyl-CoA synthetase and identify numerous epistatic mutations improving activity for several diverse substrates. The majority of these mutations would have been missed upon screening for a single substrate alone. We expect that this approach can accelerate a wide array of enzyme engineering programs.

60 APPLIED LIFE SCIENCES↗