Biology subjects

Liu, C. C.

Publications and source records attributed to Liu, C. C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Rapidly inducible yeast surface display for antibody evolution with OrthoRep

We recently developed autonomous hypermutation yeast surface display (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence activated cell sorting (FACS). AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 hours of induction. Here, we report an updated version of the AHEAD platform that utilizes a synthetic {beta}-estradiol induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system as well as traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.

synthetic biology↗

bioRxiv2024

Psychobiological regulation of plasma and saliva GDF15 dynamics in health and mitochondrial diseases

Growth Differentiation Factor 15 (GDF15) is a protein that reflects mitochondrial energetic stress and is linked to physical and mental health symptoms, aging, and mortality. Here, we tested the hypothesis that GDF15 is a stress-responsive biomarker through a series of observational and experimental studies. We report four main findings. First, in the UK Biobank (n=53,026) and Framingham Heart Study (FHS) Offspring (n=3,460) cohorts, plasma GDF15 levels were elevated in individuals with symptoms of depression and anxiety. In the FHS cohort, GDF15 was also higher in participants exposed to chronic psychosocial stressors, including lower educational attainment, lower family income, and higher job strain. Second, plasma GDF15 levels in the FHS cohort correlated positively with epigenetic clocks measuring biological aging and effect sizes of GDF15 associations with psychosocial stressors were comparable to those observed for the clocks. Third, in a two-participant intensive-sampling study (n=112 days), saliva GDF15 showed a robust awakening response similar to established stress-related hormones. However, it exhibited a distinct negative pattern, peaking at waking and declining by 42-92% within 30-45 minutes. Finally, in two laboratory experiments (n=148), acute social-evaluative stress significant increased GDF15 levels in plasma and saliva within minutes. Together, these findings suggest that psychosocial stress may contribute to mitochondrial energetic stress indexed by GDF15, with implications for aging and health. This work opens new avenues for using GDF15 as a non-invasive biomarker to study the biological embedding of stress and its impact on aging trajectories. Significance statementGrowth Differentiation Factor 15 (GDF15) is a circulating protein elevated with mitochondrial energetic stress, aging, and diseases. Our findings show that GDF15 is elevated with depressive and anxiety symptoms and in those exposed to chronic psychosocial stress. Elevated plasma GDF15 also correlates with accelerated biological aging, as measured by epigenetic clocks. The effect sizes linking GDF15 to psychosocial stressors were comparable to those observed for the epigenetic clocks. Saliva GDF15 shows a robust negative awakening response characterized by elevated levels at awakening before declining within 30-45 minutes. Acute social-evaluative stress induced increase in plasma and saliva GDF15. Together, these findings suggest GDF15 can be used to study the energetic mechanisms for the biological embedding of stress across the lifespan.

physiology↗

bioRxiv2023

Continuous evolution of user-defined genes at 1-million-times the genomic mutation rate

When nature maintains or evolves a genes function over millions of years at scale, it produces a diversity of homologous sequences whose patterns of conservation and change contain rich structural, functional, and historical information about the gene. However, natural gene diversity likely excludes vast regions of functional sequence space and includes phylogenetic and evolutionary eccentricities, limiting what information we can extract. We introduce an accessible experimental approach for compressing long-term gene evolution to laboratory timescales, allowing for the direct observation of extensive adaptation and divergence followed by inference of structural, functional, and environmental constraints for any selectable gene. To enable this approach, we developed a new orthogonal DNA replication (OrthoRep) system that durably hypermutates chosen genes at a rate of >10-4 substitutions per base in vivo. When OrthoRep was used to evolve a conditionally essential maladapted enzyme, we obtained thousands of unique multi-mutation sequences with many pairs >60 amino acids apart (>15% divergence), revealing known and new factors influencing enzyme adaptation. The fitness of evolved sequences was not predictable by advanced machine learning models trained on natural variation. We suggest that OrthoRep supports the prospective and systematic discovery of constraints shaping gene evolution, uncovering of new regions in fitness landscapes, and general applications in biomolecular engineering.

synthetic biology↗

bioRxiv2022

Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted by metastasis

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment (TME), recent studies in mice identified responses in lymph nodes (LN) as essential; however, the role of LN in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LN, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the TME. After anti-PD-L1 immunotherapy, Tpex in uninvolved LN reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LN, these response hallmarks were impaired by immunosuppressive cellular niches. Our results identify important roles for LN in anti-tumor immune responses in humans.

immunology↗

bioRxiv2022

Robust phenotyping of highly multiplexed tissue imaging data using pixel-level clustering

While technologies for multiplexed imaging have provided an unprecedented understanding of tissue composition in health and disease, interpreting this data remains a significant computational challenge. To understand the spatial organization of tissue and how it relates to disease processes, imaging studies typically focus on cell-level phenotypes. However, images can capture biologically important objects that are outside of cells, such as the extracellular matrix. Here, we developed a pipeline, Pixie, that achieves robust and quantitative annotation of pixel-level features using unsupervised clustering and show its application across a variety of biological contexts and multiplexed imaging platforms. Furthermore, current cell phenotyping strategies that rely on unsupervised clustering can be labor intensive and require large amounts of manual cluster adjustments. We demonstrate how pixel clusters that lie within cells can be used to improve cell annotations. We comprehensively evaluate pre-processing steps and parameter choices to optimize clustering performance and quantify the reproducibility of our method. Importantly, Pixie is open source and easily customizable through a user-friendly interface.

bioinformatics↗

bioRxiv2022

An in silico method to assess antibody fragment polyreactivity

Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse naive synthetic camelid antibody fragment ( nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our models performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

pharmacology and toxicology↗

bioRxiv2021

Molecular recording of sequential cellular events into DNA

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cells genome, allowing for the later reconstruction of cellular experiences using high-throughput DNA sequencing1. Existing DNA recorders have achieved high-information recording2-15, durable recording3,5-10,13,15-19, multiplexed recording of several cellular signals5-8,19,20, and temporally resolved signal recording5-8,19,20, but not all at the same time in mammalian cells. We present a DNA recorder called peCHYRON (prime editing21 Cell HistorY Recording by Ordered iNsertion) that does. In peCHYRON, mammalian cells are engineered to express prime editor and a collection of prime editing guide RNAs21 (pegRNAs) that facilitate iterative rounds of prime editing. In each round of editing, prime editor inserts a variable triplet DNA sequence alongside a constant propagator sequence that deactivates the previous and activates the next step of insertion. Editing can continue indefinitely because each insertion adds the complete sequence needed to initiate the next step. Because only one active target site is present at any given time, insertions accumulate sequentially, in a unidirectional order. Thus, temporal information is preserved in the order of insertions. Durability is achieved through the use of a prime editor that only nicks a single DNA strand, effectively avoiding deletion mutations that could otherwise corrupt the information stored at the recording locus. High-information content is established by co-expressing a variety of pegRNAs, each harboring unique triplet DNA sequences. We demonstrate that constitutive expression of such a library of pegRNAs generates insertion patterns that support straightforward reconstruction of cell lineage relationships. In an alternative pegRNA expression scheme, we also achieve multiplexed recording by manually pulsing expression of different pegRNAs, then reconstructing pulse sequences from the peCHYRON records. Additionally, we coupled the expression of specific pegRNAs to specific biological stimuli, which allowed temporally resolved, multiplexed recording of chemical exposures in populations of mammalian cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/467507v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@3dcbb2org.highwire.dtl.DTLVardef@1081b72org.highwire.dtl.DTLVardef@141fed3org.highwire.dtl.DTLVardef@1c0e8ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2021

Reproducible, high-dimensional imaging in archival human tissue by Multiplexed Ion Beam Imaging by Time-of-Flight (MIBI-TOF)

Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institutes (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R2 = 0.94 {+/-} 0.04) and frequency (R2 = 0.95 {+/-} 0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R2 = 0.85 {+/-} 0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between replicates yielded an average correlation of R2 = 0.92 {+/-} 0.06. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.

pathology↗

bioRxiv2021

Large-scale comparative genomics of Salmonella enterica to refine the organization of the global Salmonella population structure

1.Since the introduction of the White-Kauffmann-Le Minor (WKL) scheme for Salmonella serotyping, the nomenclature remains the most widely used for reporting the disease prevalence of Salmonella enterica across the globe. With the advent of whole genome sequencing (WGS), traditional serotyping has been increasingly replaced by in-silico methods that couple the detection of genetic variations in antigenic determinants with sequence-based typing. However, despite the integration of genomic-based typing by in-silico serotyping tools such as SeqSero2 and SISTR, in-silico serotyping in certain contexts remains ambiguous and insufficiently informative due to polyphyletic serovars. Furthermore, in spite of the widespread acknowledgement of polyphyly from genomic studies, the serotyping nomenclature remains unaltered. To prompt refinements to the Salmonella typing nomenclature for disease reporting, we herein performed a systematic characterization of putative polyphyletic serovars and the global Salmonella population structure by comparing 180,098 Salmonella genomes (representing 723 predicted serovars) from GenomeTrakr and PubMLST databases. We identified a range of core genome MLST typing thresholds that result in stable population structure, potentially suitable as the foundation of a genomic-based typing nomenclature for longitudinal surveillance. From the genomic comparisons of hundreds of predicted serovars, we demonstrated that in-silico serotyping classifications do not consistently reflect the population divergence observed at the genomic level. The organization of Salmonella subpopulations based on antigenic determinants can be confounded by homologous recombination and niche adaptation, resulting in shared classification of highly divergent genomes and misleading distinction between highly similar genomes. In consideration of the pivotal role of Salmonella serotyping, a compendium of putative polyphyletic serovars was compiled and made publicly available to provide additional context for future interpretations of in-silico serotyping results in disease surveillance settings. To refine the typing nomenclatures used in Salmonella surveillance reports, we foresee an improved typing scheme to be a hybrid that integrates both genomic and antigenic information such that the resolution from WGS is leveraged to improve the precision of subpopulation classifications while preserving the common names defined by the WKL scheme. Lastly, we stress the importance of controlled vocabulary integration for typing information in open data settings in order for the global Salmonella population dynamics to be fully trackable. 2. Impact StatementSalmonella enterica (S. enterica) is a major foodborne pathogen responsible for an annual incidence rate of more than 90 million cases of foodborne illnesses worldwide. To surveil the high order Salmonella lineages, compare disease prevalence across jurisdictions worldwide, and inform risk assessments, in-silico serotyping has been established as the gold standard for typing the bacteria. However, despite previous Salmonella genomic studies reporting discordance between phylogenomic clades and serovars, refinements have yet been made to the serotyping scheme. Here, we analyzed over 180,000 Salmonella genomes representing 723 predicted serovars to subdivide the population into evolutionarily stable clusters in order to propose a stable organization of the Salmonella population structure that can form the basis of a genomic-based typing scheme for the pathogen. We described numerous instances in which genomes between serotypes are more similar than genomes within a serotype to reflect the inconsistencies of subpopulation classifications based on antigenic determinants. Moreover, we found inconsistencies between predicted serovars and reported serovars which highlighted potential errors in existing in-silico serotyping tools and the need to implement controlled vocabularies for reporting Salmonella subtypes in public databases. The findings of our study aim to motivate the future development of a standardized genomic-based typing nomenclature that more accurately captures the natural populations of S. enterica. 3. Data SummaryThe assembly accession numbers of the genomes analyzed in this study (n = 204,952) and the associated metadata (e.g. sampling location, collection date, FTP address for retrieval) are documented in Table S1. The GenomeTrakr genomes were retrieved from the National Center for Biological Information GenBank database. The PubMLST genomes were retrieved using the BIGSdb API.

genomics↗

bioRxiv2021

Using Continuous Directed Evolution to Improve Enzymes for Plant Applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzymes activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful new way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the Saccharomyces cerevisiae OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt ( plantize) enzymes from prokaryotes - especially exotic prokaryotes - to function well in mild, plant-like conditions. One-sentence summaryContinuous directed evolution using the yeast OrthoRep system is a powerful new way to improve enzymes for use in plant engineering as illustrated by plantizing a bacterial thiamin synthesis enzyme..

synthetic biology↗

bioRxiv2021

Single-synapse analyses of Alzheimers disease implicate pathologic tau, DJ1, CD47, and ApoE

Synaptic molecular characterization is limited for Alzheimers disease (AD). We used mass cytometry to quantify 38 probes in approximately 17 million single synaptic events from human brains without pathologic change or with pure AD or Lewy body disease (LBD), non-human primates (NHP), and PS/APP mice. Synaptic molecular integrity in humans and NHP was similar. Although not detected in human synapses, A{beta} was in PS/APP mice synaptic events. Clustering and pattern identification of human synapses showed expected disease-specific differences, like increased hippocampal pathologic tau in AD and reduced caudate dopamine transporter in LBD, and revealed novel findings including increased hippocampal CD47 and lowered DJ1 in AD and higher ApoE in AD dementia. Our results were independently supported by multiplex ion beam imaging of intact tissue.

neuroscience↗

bioRxiv2021

Evolving small-molecule biosensors with improved performance and reprogrammed ligand specificity using OrthoRep

Genetically-encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand specificities remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with high dynamic range, reprogrammed activity towards desired non-cognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that propagation of host yeast cells resulted in BenMs rapid and continuous diversification. When these cells were subjected to cycles of culturing and sorting on BenM activity in the presence and absence of its cognate ligand, muconic acid, or the non-cognate ligand, adipic acid, we obtained multiple BenM variants that respond to their corresponding ligands. These biosensors outperform previously-engineered BenM-based biosensors by achieving substantially greater dynamic range (up to ~180-fold-induction) and broadened operational range. Expression of select BenM variants in the presence of a muconic acid biosynthetic pathway demonstrated sensitive biosensor activation without saturating response, which should enable pathway and host engineering for higher production of muconic and adipic acids. Given the streamlined manner in which high-performance and versatile biosensors were evolved using OrthoRep, this study provides a template for generating custom biosensors for metabolic pathway engineering and other biotechnology goals.

synthetic biology↗

bioRxiv2021

Experiment level curation identifies high confidence transcriptional regulatory interactions in neurodevelopment

To facilitate the development of large-scale transcriptional regulatory networks (TRNs) that may enable in-silico analyses of disease mechanisms, a reliable catalogue of experimentally verified direct transcriptional regulatory interactions (DTRIs) is needed for training and validation. There has been a long history of using low-throughput experiments to validate single DTRIs. Therefore, we hypothesize that a reliable set of DTRIs could be produced by curating the published literature for such evidence. In our survey of previous curation efforts, we identified the lack of details about the quantity and the types of experimental evidence to be a major gap, despite the importance of such details for the identification of bona fide DTRIs. We developed a curation protocol to inspect the published literature for support of DTRIs at the experiment level, focusing on genes important to the development of the mammalian nervous system. We sought to record three types of low-throughput experiments: Transcription factor (TF) perturbation, TF-DNA binding, and TF-reporter assays. Using this protocol, we examined a total of 1,310 papers to assemble a collection of 1,499 unique DTRIs, involving 251 TFs and 825 target genes, many of which were not reported in any other DTRI resource. The majority of DTRIs (965, 64%) were supported by two or more types of experimental evidence and 27% were supported by all three. Of the DTRIs with all three types of evidence, 170 had been tested using primary tissues or cells and 44 had been tested directly in the central nervous system. We used our resource to document research biases among reports towards a small number of well-studied TFs. To demonstrate a use case for this resource, we compared our curation to a previously published high-throughput perturbation screen and found significant enrichment of the curated targets among genes differentially expressed in the developing brain in response to Pax6 deletion. This study demonstrates a proof-of-concept for the assembly of a high confidence DTRI resource in order to support the development of large-scale TRNs. Author SummaryThe capacity to computationally reconstruct gene regulatory networks using large-scale biological data is currently limited by the absence of a high confidence set of one-to-one regulatory interactions. Given the lengthy history of using small scale experimental assays to investigate individual interactions, we hypothesize that a reliable collection of gene regulatory interactions could be compiled by systematically inspecting the published literature. To this end, we developed a curation protocol to examine and record evidence of regulatory interactions at the individual experiment level. Focusing on the area of brain development, we applied our pipeline to 1,310 publications. We identified 3,601 individual experiments, providing detailed information about 1,499 regulatory interactions. Many of these interactions have verified activity specifically in the embryonic brain. By capturing reports of regulatory interactions at this level of granularity, we present a resource that is more interpretable than other similar resources.

genomics↗

bioRxiv2021

Transition to invasive breast cancer is associated with progressive changes in the structure and composition of tumor stroma

Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand how the tumor microenvironment (TME) changes with transition to IBC, we used Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to analyze 140 clinically annotated surgical resections covering the full spectrum of breast cancer progression. We compared normal, DCIS, and IBC tissues using machine learning tools for multiplexed cell segmentation, pixel-based clustering, and object morphometrics. Transition from DCIS to IBC was found to occur along a trajectory marked by coordinated shifts in location and function of myoepithelium, fibroblasts, and infiltrating immune cells in the surrounding stroma. Taken together, this comprehensive study within the HTAN Breast PreCancer Atlas offers insight into the etiologies of DCIS, its transition to IBC, and emphasizes the importance of the TME stroma in promoting these processes.

cancer biology↗

bioRxiv2020

Rapid generation of potent antibodies by autonomous hypermutation in yeast

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens (e.g., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe Autonomous Hypermutation yEast surfAce Display (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naive nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw [~]580-fold and [~]925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.

bioengineering↗

bioRxiv2020

Potential For Applying Continuous Directed Evolution To Plant Enzymes

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in todays agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cells growth rate to the target genes function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

synthetic biology↗

bioRxiv2020

Scalable, continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolved the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only onTmTrpB’s primary activity (synthesizing L-tryptophan from indole and L-serine). We find that the resulting sequence-diverseTmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of new biomolecular functions.Competing Interest StatementC.E.B and F.H.A. are co-founders of Aralez Bio, focusing on the enzymatic synthesis of unnatural amino acids.View Full Text

synthetic biology↗

bioRxiv2020

Automated continuous evolution of proteins in vivo

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multi-parameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multi-mutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.

synthetic biology↗