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.

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.

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.

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.

Synthetic Biology towards Engineering Microbial Lignin Biotransformation.

Lignin is the second most abundant biopolymer on earth and is a major source of aromatic compounds; however, it is vastly underutilized owing to its heterogeneous and recalcitrant nature. Microorganisms have evolved efficient mechanisms that overcome these challenges to depolymerize lignin and funnel complex mixtures of lignin-derived monomers to central metabolites. This review summarizes recent synthetic biology efforts to enhance lignin depolymerization and aromatic catabolism in bacterial and fungal hosts for the production of both natural and novel bioproducts. We also highlight difficulties in engineering complex phenotypes and discuss the outlook for the future of lignin biological valorization.

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.

A Strong Hybrid Fatty Acid Inducible Transcriptional Sensor Built From Yarrowia lipolytica Upstream Activating and Regulatory Sequences.

The engineering of Yarrowia lipolytica to accumulate lipids with high titers and productivities has been enabled with a handful of constitutive promoters for pathway engineering. However, the development of promoters that are both strong and lipid responsive could greatly benefit the bioproduction efficiency of lipid-derived oleochemicals in oleaginous yeast. In this study, a fatty acid regulated hybrid promoter for use in Y. lipolytica is engineered. A 200 bp upstream regulatory sequence in the peroxisomal acyl CoA oxidase 2 (POX2) promoter is identified. Further analysis of the promoter sequence reveal a regulatory sequence, that when used in tandem repeats, lead to a 48-fold induction of gene expression relative to glucose and fourfold higher than the native POX2 promoter. To date, this is the strongest inducible promoter reported in Y. lipolytica. Taken together, the results show that it is possible to engineer strong promoters that retain strong inducibility. These types of promoters will be useful in controlling metabolism and as fatty acid sensors.

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica.

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.

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.

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.

Calcium-induced folding of a beta roll motif requires C-terminal entropic stabilization.

Beta roll motifs are associated with several proteins secreted by the type 1 secretion system (T1SS). Located just upstream of the C-terminal T1SS secretion signal, they are believed to act as calcium-induced switches that prevent folding before secretion. Bordetella pertussis adenylate cyclase (CyaA) toxin has five blocks of beta roll motifs (or repeats-in-toxin motifs) separated by linkers. The block V motif on its own has been reported to be non-responsive to calcium. Only when the N- and C-terminal linkers, or flanking groups, were fused did the motif bind calcium and fold. In an effort to understand the requirements for beta roll folding, we have truncated the N- and C-terminal flanks at several locations to determine the minimal essential sequences. Calcium-responsive beta roll folding occurred even in the absence of the natural N-terminal flank. The natural C-terminal flank could not be truncated without decreased calcium affinity and only partially truncated before losing calcium-responsiveness. Globular protein fusion at the C-terminus likewise enabled calcium-induced folding but fusions solely at the N-terminus failed. This demonstrates that calcium-induced folding is an inherent property of the beta roll motif rather than the flanking groups. Given the disparate nature of the observed functional flanking groups, C-terminal fusions appear to confer calcium-responsiveness to the beta roll motif via a non-specific mechanism, suggesting that entropic stabilization of the unstructured C-terminus can enable beta roll folding. Increased calcium affinity was observed when the natural C-terminal flank was used to enable calcium-induced folding, pointing to its cooperative participation in beta roll formation. This work indicates that a general principle of C-terminal entropic stabilization can enable stimulus-responsive repeat protein folding, while the C-terminal flank has a specific role in tuning calcium-responsive beta roll formation. These observations are in stark contrast to what has been reported for other repeat proteins.

A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase.

A better understanding of the conformational changes exhibited by intrinsically disordered proteins is necessary as we continue to unravel their myriad biological functions. In repeats in toxin (RTX) domains, calcium binding triggers the natively unstructured domain to adopt a beta roll structure. Here we present an in vitro Forster resonance energy transfer (FRET)-based method for the investigation of the conformational behavior of an RTX domain from the Bordetella pertussis adenylate cyclase consisting of nine repeat units. Equilibrium and stopped-flow FRET between fluorescent proteins, attached to the termini of the domain, were measured in an analysis of the end-to-end distance changes in the RTX domain. The method was complemented with circular dichroism spectroscopy, tryptophan fluorescence, and bis-ANS dye binding. High ionic strength was observed to decrease the calcium affinity of the RTX domain. A truncation and single amino acid mutations yielded insights into the structural determinants of beta roll formation. Mutating the conserved Asp residue in one of the nine repeats significantly reduced the affinity of the domains for calcium ions. Removal of the sequences flanking the repeat domain prevented folding, but replacing them with fluorescent proteins restored the conformational behavior, suggesting an entropic stabilization. The FRET-based method is a useful technique that complements other low-resolution techniques for investigating the dynamic conformational behavior of the RTX domain and other intrinsically disordered protein domains.

Characterization of the 4D5Flu single-chain antibody with a stimulus-responsive elastin-like peptide linker: a potential reporter of peptide linker conformation.

Single-chain antibodies (scFvs) are comprised of IgG variable light and variable heavy domains tethered together by a peptide linker whose length and sequence can affect antigen binding properties. The ability to modulate antigen binding affinity through the use of environmental triggers would be of great interest for many biotechnological applications. We have characterized the antigen binding properties of an anti-fluorescein scFv, 4D5Flu, containing stimulus-responsive short elastin-like peptide linkers and nonresponsive flexible linkers. Comparison of length-matched flexible and short elastin-like peptide linkers indicates that a stimulus-responsive linker can confer stimulus-responsive control of fluorescein binding. A linker length of either six or 10 amino acids proved to have the largest thermally induced response. Similar differences in binding free energy changes indicate a common underlying mechanism of thermal responsiveness. Contrary to the thermal behavior, the effect of salt, another elastin beta-turn-inducing stimulus, stabilized antigen binding in the six- and 10-amino-acid linkers such that elastin-like linkers became less stimulus-responsive as compared with flexible linkers. Again, the thermodynamic analysis indicates a common mechanism of salt responsiveness. Characterization of the room-temperature binding affinities and evidence indicating a dimeric state of the scFvs concomitantly suggest the major contribution to the stimulus-responsive behavior derives from the perturbation of interdomain associations, rather than the linker-constrained disruption of the intramolecular association. The ability to use stimulus-responsive peptide modules to exert a novel control over protein function will likely find application in the creation of allosteric antibodies and scFv-based biosensors, and as a platform to enable the evolution of new stimulus-responsive peptides.