A microfluidic enzymatic sensor based on cross-linked crystals of an ancestral β-lactamase (βLa-CLECs) was developed for the detection of β-lactam antibiotics under continuous flow conditions. The sensor is presented in a modular configuration, consisting of a separated microprobe for enzymatic catalysis, allowing for precise enzyme loading, and a photonic lab-on-a-chip (PhLoC) platform for detection by in-line spectrophotometric measurements. The exceptional thermal stability of the reconstructed Precambrian β-lactamase made it suitable for the steps required in device fabrication and carrier-free immobilization treatment, while its inherent promiscuity, i.e., its capability to degrade a variety of lactam antibiotics, including man-made third-generation antibiotics, broadens its potential application. Full catalytic activity of the ancestral enzyme was retained after immobilization, and inhibition by antibiotics such as ampicillin and sulbactam was detected at concentrations as low as parts per billion. These results support the use of ancient enzymes as stable and responsive biorecognition elements in cost-effective, high-throughput analytical systems targeting environmental pollutants and pharmaceutical compounds.

ABSTRACT Natural selection acts on diversity generated by errors in the biosynthesis of the genetic material. Previous work has shown, however, that such errors are not necessarily fully random. We have used a model influenza strain and a unique-molecular-identifier-based high-throughput sequencing approach to assess the error landscape for hemagglutinin-encoding RNA. Single-site errors occur at highly variable frequencies, with differences that span several orders of magnitude, plausibly reflecting specific RNA sequence/structure patterns. Remarkably, influenza evolution/adaptation preferentially selects mutations encoded by the higher frequency errors, as shown by analyses of mutations fixed in natural strains over many decades and by analyses of antibody-escape mutations found in laboratory experiments on strains of the 2009 pandemics. Our results support that RNA error landscapes may provide information useful for predicting influenza evolution and point to high-frequency errors encoding antibody-evading mutations as potential contributors to the rapid evolution of influenza viruses.

Experimental studies support that protein engineering based on ancestral sequence reconstruction often leads to variants with biotechnologically useful biomolecular properties. These may include high stability, enhanced conformational flexibility and a modified catalysis range. Carbohydrate-active enzymes have numerous applications related to the degradation and synthesis of carbohydrates and glycoconjugates. Herein, we explore how ancestral reconstruction impacts the substrate scope of glycosidases, highly diverse enzymes that catalyze the hydrolysis of glycosidic bonds in all living cells and find applications as catalysts of the synthetic reaction. To this end, we screened 291 potential glycosidase substrates for degradation by both a modern family-1 glycosidase from the halothermophilic bacterium Halothermothrix orenii and a putative ancestral family-1 glycosidase derived from sequence reconstruction of a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for many substrates, but the ancestral glycosidase is more efficient with flavonoid glycosides bearing large-aglycone moieties. Analysis of the catalytic parameters for a selected set of substrates, alongside analysis of the library data using a supervised learning algorithm, supports the hypothesis that the modern enzyme tends to become less catalytically efficient with increasing substrate size, while this trend is not observed for the ancestral glycosidase. Molecular modeling supports that the ancestral catalysis pattern is linked to the existence of a highly flexible region of the protein and a cavity capable of accommodating large aglycones. Our results and analyses provide guidelines for the engineering of enzymes for the synthesis and hydrolysis of large glycoconjugates.

ABSTRACT Mistakes during protein synthesis, such as transcription errors, occur often and lead to non-inheritable amino acid replacements generally known as phenotypic mutations. We recently used a consensus approach in high-throughput sequencing to determine the error landscape for influenza-hemagglutinin mRNA. We found single-site errors to occur with widely different frequencies. Here we show that the most prevalent transcription error encodes a phenotypic mutation that impairs binding of broadly neutralizing antibodies. The error occurs in 0.2-0.5% of mRNA molecules and, consequently, many virions will expose hemagglutinin variants bearing the encoded amino acid replacement. Our results point to a mechanism of antibody evasion, akin to programmed recoding, in which evading mutations are encoded by transcription errors promoted by inheritable RNA sequence/structure patterns.

Enzymes are the quintessential green catalysts, but realizing their full potential for biotechnology typically requires improvement of their biomolecular properties. Catalysis enhancement, however, is often accompanied by impaired stability. Here, we show how the interplay between activity and stability in enzyme optimization can be efficiently addressed by coupling two recently proposed methodologies for guiding directed evolution. We first identify catalytic hotspots from chemical shift perturbations induced by transition-state-analogue binding and then use computational/phylogenetic design (FuncLib) to predict stabilizing combinations of mutations at sets of such hotspots. We test this approach on a previously designed de novo Kemp eliminase, which is already highly optimized in terms of both activity and stability. Most tested variants displayed substantially increased denaturation temperatures and purification yields. Notably, our most efficient engineered variant shows a ∼3-fold enhancement in activity (kcat ∼ 1700 s-1, kcat/KM ∼ 4.3 × 105 M-1 s-1) from an already heavily optimized starting variant, resulting in the most proficient proton-abstraction Kemp eliminase designed to date, with a catalytic efficiency on a par with naturally occurring enzymes. Molecular simulations pinpoint the origin of this catalytic enhancement as being due to the progressive elimination of a catalytically inefficient substrate conformation that is present in the original design. Remarkably, interaction network analysis identifies a significant fraction of catalytic hotspots, thus providing a computational tool which we show to be useful even for natural-enzyme engineering. Overall, our work showcases the power of dynamically guided enzyme engineering as a design principle for obtaining novel biocatalysts with tailored physicochemical properties, toward even anthropogenic reactions.

The protein sequence space is vast. This fact, together with the prevalence of epistasis, hampers the engineering of novel enzymes through library screening and is a major obstacle to any attempt to predict natural protein evolution. Recently, specialized methodologies have been used to determine fitness data on ~260,000 sequences for the gene of the enzyme dihydrofolate reductase and antibody affinity data for all combinations of the mutations present in the receptor-binding domain (RBD) of the Omicron strain of SARS-CoV-2 (~30,000 variants). We show that upon iterative training on a total of just a few hundred variants, various state-of-the-art AI tools (multi-layer perceptron, random forest, and XGBoost algorithms) find very high fitness variants of the enzyme and predict the antibody evasion patterns of the RBD. This work provides a basis for efficient, widely applicable, low-throughput experimental approaches to assess viral protein evolution and to engineer enzymes for biotechnological applications.

Many viruses use programmed frameshifting and stop-codon misreading to synthesize functional proteins at high levels. The underlying mechanisms involve complex RNA sequence/structure motifs and likely reflect optimization driven by natural selection of inefficient, nonprogrammed processes. Then, it follows from basic evolutionary theory that low levels of proteins generated through gene expression errors could provide viruses with some survival advantage. Here, we devise an experimental demonstration of this possibility. Phage T7 recruits the host thioredoxin as an essential processivity factor for the viral DNA polymerase. We inserted early stop codons in the thioredoxin gene and appended to its end the sequence encoding for a photoconvertible fluorescent protein. Virus replication was not abolished. Single-molecule localization microscopy showed that the phage replicates even when there are only about 10 thioredoxin molecules per host cell on average, a number orders of magnitude below typical cellular protein levels. We show that this seemingly shocking result can be understood in molecular and evolutionary terms as a consequence of the polymerase-thioredoxin complex displaying high kinetic stability and a long residence time, as these are required to ensure high polymerase processivity. More generally, our demonstration that virus replication may be enabled by proteins at exceedingly low copy number suggests that viruses have access to the wide diversity of protein variants harboring phenotypic mutations as a result of gene expression errors. This mechanism could play a role, for instance, in cross-species transmission by enabling virus survival in the new host before adaptations appear at the genetic level.

Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid-base catalysis upon loop closure. These enzymes also have a conserved phosphate binding loop that is typically highly rigid and forms a well-defined anion binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTP's high sequence similarity to its natural counterparts, ShufPTP presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active site cysteine, mechanistic promiscuity, and most notably, hyperthermostability, with a denaturation temperature likely >130 °C (>8°C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme, and showcase the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions.

Viruses are obligate parasites that establish extensive interactions with proteins and other biomolecules of their hosts. About 20% of protein molecules bear phenotypic mutations due to errors during gene expression. Phenotypic mutations are not inherited and are not purged/amplified by natural selection. Therefore, protein variants harboring phenotypic mutations remain at very low levels. Here, we show that proteins at exceedingly low levels may enable virus propagation. Bacteriophage T7 recruits the host thioredoxin as an essential processivity factor for its replisome. Thioredoxin constitutive expression yields 10000-20000 molecules per E. coli cell. We inserted early stop codons in the thioredoxin gene and appended to its end the sequence encoding for a photoconvertible fluorescent protein. Virus propagation was not abolished, indicating that some thioredoxin molecules were produced through mistranscription or mistranslation. Single-molecule localization microscopy detected 12±5 molecules per cell when an ochre codon was inserted. This work demonstrates that crucial virus-host biomolecular interactions may need occur only a few times to trigger virus propagation and supports that viruses may exploit the wide diversity of host and viral protein variants arising from gene-expression errors to establish such interactions. Immediate implications of this notion for the mechanisms of cross-species transmission and antibody evasion are discussed. ### Competing Interest Statement The authors have declared no competing interest.

Galactose oxidase (GOase) is a versatile biocatalyst with a wide range of potential applications, ranging from synthetic chemistry to bioelectrochemical devices. Previous GOase engineering by directed evolution generated the M-RQW mutant, with unprecedented new-to-nature oxidation activity at the C6-OH group of glucose, and a mutational backbone that helped to unlock its promiscuity toward other molecules, including secondary alcohols. In the current study, we have used the M-RQW mutant as a starting point to engineer a set of GOases that are very thermostable and that are easily produced at high titers in yeast, enzymes with latent activities applicable to sustainable chemistry. To boost the generation of sequence and functional diversity, the directed evolution workflow incorporated one-shot computational mutagenesis by the PROSS algorithm and ancestral sequence reconstruction. This synergetic approach helped produce a rapid rise in functional expression by Pichia pastoris, achieving g/L production in a fed-batch bioreactor while the different GOases designed were resistant to pH and high temperature, with T50 enhancements up to 27 °C over the parental M-RQW. These designs displayed latent activity against glucose and an array of secondary aromatic alcohols with different degrees of bulkiness, becoming a suitable point of departure for the future engineering of industrial GOases.

Promiscuity, the capability of catalyzing a diversity of chemical reactions, is a desirable feature in many enzymes intended for biotechnological applications. Promiscuous activities, however, are typically depressed in specialized modern enzymes. On the other hand, several ancestral reconstruction studies have reported enzymes with enhanced promiscuity. Glycosidases catalyze the hydrolysis of glycosidic bonds in all living cells but find practical applications in the synthesis of glycoconjugates as catalysts of the reverse reaction. Here, we use a library of ∼ 500 possible substrates to compare the catalytic scope of the modern family-1 glycosidase from Halothermothrix orenii with that of a putative ancestral family-1 glycosidase derived from sequence reconstruction at a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for the hydrolysis of small substrates typically used to assess the activity of family-1 glycosidases. We identify, however, a trend for the modern enzyme to become less catalytically efficient with increasing substrate size. Such trend is not apparent in the conformationally-flexible ancestral glycosidase, which is in fact the better catalyst for the hydrolysis of flavonoid glycosides. Our results support that ancestral sequence reconstruction may provide a basis for enzyme engineering for the synthesis of glycoconjugates with large glycosyl acceptors. ### Competing Interest Statement Leonardo De Maria, Martin Hayes and Francesco Falcioni are current employees and/or shareholders of AstraZeneca. The authors declare no competing interest.

Several mass spectrometry and spectroscopic techniques have been used in the search for molecular biomarkers on Mars. A major constraint is their capability to detect and identify large and complex compounds such as peptides or other biopolymers. Multiplex immunoassays can detect these compounds, but antibodies must be produced for a large number of sequence-dependent molecular targets. Ancestral Sequence Reconstruction (ASR) followed by protein "resurrection" in the lab can help to narrow the selection of targets. Herein, we propose an immunoanalytical method to identify ancient and universally conserved protein/peptide sequences as targets for identifying ancestral biomarkers in nature. We have developed, tested, and validated this approach by producing antibodies to eight previously described ancestral resurrected proteins (three β-lactamases, three thioredoxins, one Elongation Factor Tu, and one RuBisCO, all of them theoretically dated as Precambrian), and used them as a proxy to search for any potential feature of them that could be present in current natural environments. By fluorescent sandwich microarray immunoassays (FSMI), we have detected positive immunoreactions with antibodies to the oldest β-lactamase and thioredoxin proteins (ca. 4 Ga) in samples from a hydrothermal environment. Fine epitope mapping and inhibitory immunoassays allowed the identification of well-conserved epitope peptide sequences that resulted from ASR and were present in the sample. We corroborated these results by metagenomic sequencing and found several genes encoding analogue proteins with significant matches to the peptide epitopes identified with the antibodies. The results demonstrated that peptides inferred from ASR studies have true counterpart analogues in Nature, which validates and strengthens the well-known ASR/protein resurrection technique and our immunoanalytical approach for investigating ancient environments and metabolisms on Earth and elsewhere.

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

Over one hundred Mastadenovirus types infect seven orders of mammals. Virus-host coevolution may involve cospeciation, duplication, host switch and partial extinction events. We reconstruct Mastadenovirus diversification, finding that while cospeciation is dominant, the other three events are also common in Mastadenovirus evolution. Linear motifs are fast-evolving protein functional elements and key mediators of virus-host interactions, thus likely to partake in adaptive viral evolution. We study the evolution of eleven linear motifs in the Mastadenovirus E1A protein, a hub of virus-host protein–protein interactions, in the context of host diversification. The reconstruction of linear motif gain and loss events shows fast linear motif turnover, corresponding a virus-host protein–protein interaction turnover orders of magnitude faster than in model host proteomes. Evolution of E1A linear motifs is coupled, indicating functional coordination at the protein scale, yet presents motif-specific patterns suggestive of convergent evolution. We report a pervasive association between Mastadenovirus host diversification events and the evolution of E1A linear motifs. Eight of 17 host switches associate with the gain of one linear motif and the loss of four different linear motifs, while five of nine partial extinctions associate with the loss of one linear motif. The specific changes in E1A linear motifs during a host switch or a partial extinction suggest that changes in the host molecular environment lead to modulation of the interactions with the retinoblastoma protein and host transcriptional regulators. Altogether, changes in the linear motif repertoire of a viral hub protein are associated with adaptive evolution events during Mastadenovirus evolution.

The routine generation of enzymes with completely new active sites is a major unsolved problem in protein engineering. Advances in this field have thus far been modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral β-lactamase with a new active site that is capable of efficiently catalyzing Kemp elimination. The engineering of our Kemp eliminase involved minimalist design based on a single function-generating mutation, inclusion of an extra polypeptide segment at a position close to the de novo active site, and sharply focused, low-throughput library screening. Nevertheless, its catalytic parameters (kcat/KM~2·105 M−1 s−1, kcat~635 s−1) compare favorably with the average modern natural enzyme and match the best proton-abstraction de novo Kemp eliminases that are reported in the literature. The general implications of our results for de novo enzyme engineering are discussed.

A general protocol for the experimental assessment of bacteriophage adaptation to new hosts is described. We use as a model system the lytic phage T7 and an engineered E. coli strain modified to hamper the recruitment of a known proviral factor. Our protocol includes steps of phage amplification, plaque and liquid lysis assays, and DNA extraction for next-generation sequencing of the viral genome over several rounds of laboratory evolution thus allowing the investigation of the sequence determinants of viral adaptation. For complete information on the generation and use of this protocol, please refer to Luzon-Hidalgo et al. (2021).

TEM-1 β-lactamase degrades β-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian β-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. In this work, we report an ancestral sequence reconstruction exercise targeting ancient nodes during the evolution of family-1 glycosidases and present the exceptional properties of a putative resurrected ancestor of bacterial and eukaryotic enzymes. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures for ∼1400 modern glycosidases show a porphyrin ring in the enzyme. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein resurrection to reveal valuable but unusual protein features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.