The EccDNA Replicon: A Heritable, Extranuclear Vehicle That Enables Gene Amplification and Glyphosate Resistance in Amaranthus palmeri.

Gene copy number variation is a predominant mechanism used by organisms to respond to selective pressures from the environment. This often results in unbalanced structural variations that perpetuate as adaptations to sustain life. However, the underlying mechanisms that give rise to gene proliferation are poorly understood. Here, we show a unique result of genomic plasticity in Amaranthus palmeri: a massive, ∼400-kb extrachromosomal circular DNA (eccDNA) that harbors the 5-ENOYLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) gene and 58 other genes whose encoded functions traverse detoxification, replication, recombination, transposition, tethering, and transport. Gene expression analysis under glyphosate stress showed transcription of 41 of these 59 genes, with high expression of EPSPS, as well as genes coding for aminotransferases, zinc finger proteins, and several uncharacterized proteins. The genomic architecture of the eccDNA replicon is composed of a complex arrangement of repeat sequences and mobile genetic elements interspersed among arrays of clustered palindromes that may be crucial for stability, DNA duplication and tethering, and/or a means of nuclear integration of the adjacent and intervening sequences. Comparative analysis of orthologous genes in grain amaranth (Amaranthus hypochondriacus) and waterhemp (Amaranthus tuberculatus) suggests that higher order chromatin interactions contribute to the genomic origins of the A. palmeri eccDNA replicon structure.

Flowthrough of239PU and55FE during RNA extraction.

Analysis of gene expression has become an important tool in understanding low-dose effect mechanisms of ionizing radiation at the cellular level. Metal binding to nucleic acids needs to be considered when interpreting these results, as some radioactive metals, particularly actinides, may produce free radicals and cause oxidative stress damage via chemical means at rates much higher than free radical formation related to their radiological properties. Bacteria exposedin situto low dose rates of plutonium-239 (239Pu) and iron-55 (55Fe) were previously analysed for gene expression. The work herein was motivated by an interest in more precisely identifying the distribution of radionuclides in these bacteria as well as the practical need to ensure appropriate transport and handling of the associated ribonucleic acid (RNA) extractions. RNA extractions were performed on bacteria growth media with and without bacteria cells (i.e. with and without RNA) at several different concentrations of239Pu and55Fe to inform the level of specificity of the extraction membrane as well as provide insight into internal (uptake) vs external (sorption) accumulation of these radionuclides in bacteria cells. Results of the study suggest that239Pu and55Fe detected in RNA extraction samples during long term cell studies is the result of binding to RNA prior to the time of extraction, as opposed to flow through or binding after cell lysis, and it highlights the practical importance of nucleic acid sample characterization to radiation protection more generally.

Engineering heterologous enzyme secretion in Yarrowia lipolytica.

BACKGROUND: Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. RESULTS: By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and [Formula: see text]-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. CONCLUSIONS: Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.

Survey of nonconventional yeasts for lipid and hydrocarbon biotechnology.

Nonconventional yeasts have an untapped potential to expand biotechnology and enable process development necessary for a circular economy. They are especially convenient for the field of lipid and hydrocarbon biotechnology because they offer faster growth than plants and easier scalability than microalgae and exhibit increased tolerance relative to some bacteria. The ability of industrial organisms to import and metabolically transform lipids and hydrocarbons is crucial in such applications. Here, we assessed the ability of 14 yeasts to utilize 18 model lipids and hydrocarbons from six functional groups and three carbon chain lengths. The studied strains covered 12 genera from nine families. Nine nonconventional yeasts performed better than Saccharomyces cerevisiae, the most common industrial yeast. Rhodotorula toruloides, Candida maltosa, Scheffersomyces stipitis, and Yarrowia lipolytica were observed to grow significantly better and on more types of lipids and lipid molecules than other strains. They were all able to utilize mid- to long-chain fatty acids, fatty alcohols, alkanes, alkenes, and dicarboxylic acids, including 28 previously unreported substrates across the four yeasts. Interestingly, a phylogenetic analysis showed a short evolutionary distance between the R. toruloides, C. maltosa, and S. stipitis, even though R. toruloides is classified under a different phylum. This work provides valuable insight into the lipid substrate range of nonconventional yeasts that can inform species selection decisions and viability of lipid feedstocks.

Accumulation of radio-iron and plutonium, alone and in combination, inPseudomonas putidagrown in liquid cultures.

The impact of low doses of ionising radiation on biological and environmental systems have been historically difficult to study. Modern biological tools have provided new methods for studying these mechanisms but applying these tools to a dose-response relationship may require refinement of dosimetric techniques that incorporate a detailed understand of radionuclide accumulation in biological cells, particularly when assessing the impact of low doses of ionising radiation. In this workPseudomonas putida (KT2440) grown in liquid culture was exposed to low dose rates (10-20 mGy d-1) of239Pu and55Fe, both alone and in combination, for a period of 20 days, and the accumulation of239Pu and55Fe in cell pellets was analysed via liquid scintillation counting. The study also considered of cells grown with239Pu and stable Fe (primarily56Fe). In addition to the analysis of cell pellet and media samples, this work includes analysis of the radiological content of ribonucleic acid extraction samples to examine uptake of radionuclides. Results indicate that239Pu inhibited the uptake of55Fe, and that the presence of stable and radioactive isotopes of Fe in cultures may promote pathways for Fe accumulation that are used by239Pu. The work herein provides foundational insight into future dosimetric models for our work with environmental bacteria.

Polymersomes for Therapeutic Delivery of Protein and Nucleic Acid Macromolecules: From Design to Therapeutic Applications.

Macromolecule-based therapeutic agents, particularly proteins, antigens, monoclonal antibodies, transcription factors, nucleic acids, and gene editing enzymes, have the potential to offer cures for previously untreatable diseases. However, they present an enormous delivery challenge due to poor absorption and rapid metabolism in the body. Polymersomes have tremendous potential in delivering these agents to their desired intracellular location due to increased circulation times, decreased macromolecule degradation, and decreased immune responses. In this Review, we highlight the key factors in design, development, and improved performance of these vesicles for macromolecular delivery. The recent progress made toward preclinical application of these vesicles for protein and gene delivery is also covered.

Identification of oleaginous yeasts that metabolize aromatic compounds.

The valorization of lignin is critical for the economic viability of the bioeconomy. Microbial metabolism is advantageous for handling the myriad of aromatic compounds resulting from lignin chemical or enzymatic depolymerization. Coupling aromatic metabolism to fatty acid biosynthesis makes possible the production of biofuels, oleochemicals, and other fine/bulk chemicals derived from lignin. Our previous work identified Cutaneotrichosporon oleaginosus as a yeast that could accumulate nearly 70% of its dry cell weight as lipids using aromatics as a sole carbon source. Expanding on this, other oleaginous yeast species were investigated for the metabolism of lignin-relevant monoaromatics. Thirty-six oleaginous yeast species from the Phaff yeast collection were screened for growth on several aromatic compounds representing S-, G-, and H- type lignin. The analysis reported in this study suggests that aromatic metabolism is largely segregated to the Cutaenotrichosporon, Trichosporon, and Rhodotorula clades. Each species tested within each clade has different properties with respect to the aromatics metabolized and the concentrations of aromatics tolerated. The combined analysis suggests that Cutaneotrichosporon yeast are the best suited to broad spectrum aromatic metabolism and support its development as a model system for aromatic metabolism in yeast.

Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica.

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.

Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica.

Yarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR-Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

Urea and urine are a viable and cost-effective nitrogen source for Yarrowia lipolytica biomass and lipid accumulation.

Yarrowia lipolytica is an industrial yeast that has been used in the sustainable production of fatty acid-derived and lipid compounds due to its high growth capacity, genetic tractability, and oleaginous properties. This investigation examines the possibility of utilizing urea or urine as an alternative to ammonium sulfate as a nitrogen source to culture Y. lipolytica. The use of a stoichiometrically equivalent concentration of urea in lieu of ammonium sulfate significantly increased cell growth when glucose was used as the carbon source. Furthermore, Y. lipolytica growth was equally improved when grown with synthetic urine and real human urine. Equivalent or better lipid production was achieved when cells are grown on urea or urine. The successful use of urea and urine as nitrogen sources for Y. lipolytica growth highlights the potential of using cheaper media components as well as exploiting and recycling non-treated human waste streams for biotechnology processes.

Metabolism of aromatics by Trichosporon oleaginosus while remaining oleaginous.

BACKGROUND: The oleaginous yeast, Trichosporon oleaginosus, has been extensively studied for its ability to metabolize non-conventional feedstocks. These include phenol-containing waste streams, such as distillery wastewater, or streams consisting of non-conventional sugars, such as hydrolyzed biomass and various bagasse. An initial BLAST search suggests this yeast has putative aromatic metabolizing genes. Given the desirability to valorize underutilized feedstocks such as lignin, we investigated the ability of T. oleaginosus to tolerate and metabolize lignin-derived aromatic compounds. RESULTS: Trichosporon oleaginosus can tolerate and metabolize model lignin monoaromatics and associated intermediates within funneling pathways. Growth rates and biomass yield were similar to glucose when grown in 4-hydroxybenzoic acid (pHBA) and resorcinol, but had an increased lag phase when grown in phenol. Oleaginous behavior was observed using resorcinol as a sole carbon source. Fed-batch feeding resulted in lipid accumulation of 69.5% on a dry weight basis. CONCLUSIONS: Though the exact pathway of aromatic metabolism remains to be determined for T. oleaginosus, the results presented in this work motivate use of this organism for lignin valorization and phenolic wastewater bioremediation. Trichosporon oleaginosus is the first yeast shown to be oleaginous while growing on aromatic substrates, and shows great promise as a model industrial microbe for biochemical and biofuel production from depolymerized lignin.

Structural basis of regulation of von Willebrand factor binding to glycoprotein Ib.

Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα ß-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.

Structural and energetic determinants of tyrosylprotein sulfotransferase sulfation specificity.

MOTIVATION: Tyrosine sulfation is a type of post-translational modification (PTM) catalyzed by tyrosylprotein sulfotransferases (TPST). The modification plays a crucial role in mediating protein-protein interactions in many biologically important processes. There is no well-defined sequence motif for TPST sulfation, and the underlying determinants of TPST sulfation specificity remains elusive. Here, we perform molecular modeling to uncover the structural and energetic determinants of TPST sulfation specificity. RESULTS: We estimate the binding affinities between TPST and peptides around tyrosines of both sulfated and non-sulfated proteins to differentiate them. We find that better differentiation is achieved after including energy costs associated with local unfolding of the tyrosine-containing peptide in a host protein, which depends on both the peptide's secondary structures and solvent accessibility. Local unfolding renders buried peptide-with ordered structures-thermodynamically available for TPST binding. Our results suggest that both thermodynamic availability of the peptide and its binding affinity to the enzyme are important for TPST sulfation specificity, and their interplay results into great variations in sequences and structures of sulfated peptides. We expect our method to be useful in predicting potential sulfation sites and transferable to other TPST variants. Our study may also shed light on other PTM systems without well-defined sequence and structural specificities. AVAILABILITY AND IMPLEMENTATION: All the data and scripts used in the work are available at http://dlab.clemson.edu/research/Sulfation.

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6ß and WFS1 knockdown on fed-batch production of IgG1.

Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6ß and WFS1, are rational engineering targets. Although both ATF6ß and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6ß decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6ß and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6ß improved the UPR specifically later in fed-batch leading to increased overall productivity.

Biological Upcycling of Plastics Waste.

Plastic wastes accumulate in the environment, impacting wildlife and human health and representing a significant pool of inexpensive waste carbon that could form feedstock for the sustainable production of commodity chemicals, monomers, and specialty chemicals. Current mechanical recycling technologies are not economically attractive due to the lower-quality plastics that are produced in each iteration. Thus, the development of a plastics economy requires a solution that can deconstruct plastics and generate value from the deconstruction products. Biological systems can provide such value by allowing for the processing of mixed plastics waste streams via enzymatic specificity and using engineered metabolic pathways to produce upcycling targets. We focus on the use of biological systems for waste plastics deconstruction and upcycling. We highlight documented and predicted mechanisms through which plastics are biologically deconstructed and assimilated and provide examples of upcycled products from biological systems. Additionally, we detail current challenges in the field, including the discovery and development of microorganisms and enzymes for deconstructing non-polyethylene terephthalate plastics, the selection of appropriate target molecules to incentivize development of a plastic bioeconomy, and the selection of microbial chassis for the valorization of deconstruction products.

Engineering Yarrowia lipolytica for the biosynthesis of geraniol.

Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast.

Global Transcriptional Response of Escherichia coli Exposed In Situ to Different Low-Dose Ionizing Radiation Sources.

Characterization of biological and chemical responses to ionizing radiation by various organisms is essential for potential applications in bioremediation, alternative modes of detecting nuclear material, and national security. Escherichia coli DH10ß is an optimal system to study the microbial response to low-dose ionizing radiation at the transcriptional level because it is a well-characterized model bacterium and its responses to other environmental stressors, including those to higher radiation doses, have been elucidated in prior studies. In this study, RNA sequencing with downstream transcriptomic analysis (RNA-seq) was employed to characterize the global transcriptional response of stationary-phase E. coli subjected to 239Pu, 3H (tritium), and 55Fe, at an approximate absorbed dose rate of 10 mGy day-1 for 1 day and 15 days. Differential expression analysis identified significant changes in gene expression of E. coli for both short- and long-term exposures. Radionuclide source exposure induced differential expression in E. coli of genes involved in biosynthesis pathways of nuclear envelope components, amino acids, and siderophores, transport systems such as ABC transporters and type II secretion proteins, and initiation of stress response and regulatory systems of temperature stress, the RpoS regulon, and oxidative stress. These findings provide a basic understanding of the relationship between low-dose exposure and biological effect of a model bacterium that is critical for applications in alternative nuclear material detection and bioremediation. IMPORTANCE Escherichia coli strain DH10ß, a well-characterized model bacterium, was subjected to short-term (1-day) and long-term (15-day) exposures to three different in situ radiation sources comprised of radionuclides relevant to nuclear activities to induce a measurable and identifiable genetic response. We found E. coli had both common and unique responses to the three exposures studied, suggesting both dose rate- and radionuclide-specific effects. This study is the first to provide insights into the transcriptional response of a microorganism in short- and long-term exposure to continuous low-dose ionizing radiation with multiple in situ radionuclide sources and the first to examine microbial transcriptional response in stationary phase. Moreover, this work provides a basis for the development of biosensors and informing more robust dose-response relationships to support ecological risk assessment.

High-Efficiency Multiplexed Cytosine Base Editors for Natural Product Synthesis in Yarrowia lipolytica.

Yarrowia lipolytica is an industrial host with a high fatty acid flux. Even though CRISPR-based tools have accelerated its metabolic engineering, there remains a need to develop tools for rapid multiplexed strain engineering to accelerate the design-build-test-learn cycle. Base editors have the potential to perform high-efficiency multiplexed gene editing because they do not depend upon double-stranded DNA breaks. Here, we identified that base editors are less toxic than CRISPR-Cas9 for multiplexed gene editing. We increased the editing efficiency by removing the extra nucleotides between tRNA and gRNA and increasing the base editor and gRNA copy number in a Ku70 deficient strain. We achieved five multiplexed gene editing in the ΔKu70 strain at 42% efficiency. Initially, we were unsuccessful at performing multiplexed base editing in NHEJ competent strain; however, we increased the editing efficiency by using a co-selection approach to enrich base editing events. Base editor-mediated canavanine gene (CAN1) knockout provided resistance to the import of canavanine, which enriched the base editing in other unrelated genetic loci. We performed multiplexed editing of up to three genes at 40% efficiency in the Po1f strain through the CAN1 co-selection approach. Finally, we demonstrated the application of multiplexed cytosine base editor for rapid multigene knockout to increase naringenin production by 2-fold from glucose or glycerol as a carbon source.

Theory for High-Throughput Genetic Interaction Screening.

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most ∼100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose ∼105 spectrally unique, genetically encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was < 9 h for 95% of samples and < 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.