Thraustochytrid hosts for expression of proteins relevant to SARS-CoV-2 intervention.

The emergence of COVID-19 as a global pandemic had sharply illustrated the limitations of research and development pipelines and scaled manufacturing. Although existing vaccines were created in record time, global deployment remains limited by regional production scales. Similarly, the most effective treatments for infected COVID-19 patients are also constrained by production scales as well as by the cost of production and thus expense per treatment. The need to produce these interventions more cost-effectively, at larger scales, in less time while retaining high quality is paramount. The ConamaxTM platform is based on a Thraustochytrid-an order of microorganisms well established in industry for world-scale production of omega-3 fatty acids by fermentation. Thraustochytrids, and the species Aurantiochytrium acetophilum in particular, possess a number of innate qualities which make it ideal for production of monoclonal antibodies and other biotherapeutic proteins. In this study, the Conamax system was used to produce several targets which may be relevant as interventions in the fight against COVID-19; an anti-SARS-CoV-2 antibody (CR3022), tocilizumab, and the ACE2 receptor. Our system was capable of producing all of these targets and each was assayed in vitro for an activity which confirmed proper structural folding. Purified CR3022 antibody produced from Conamax was capable of reducing the cytopathic effect of SARS-CoV-2. Conamax-derived tocilizumab was shown to bind to its target IL6R. Both the full-length and soluble versions of ACE2 protein produced in the Conamax platform exhibited ACE2-specific proteolytic activity. These data indicate that the Conamax platform has great potential in the production of therapeutic agents.

Interfacing whispering gallery mode optical microresonator biosensors with the plant defense elicitor chitin.

The biomaterial class of chitooligosaccharides (chitin), commonly found in insects and fungi, is one of the most abundant on earth. Substantial evidence implicates chitin in mediating a diverse array of plant cellular signaling events, including the induction of plant defense mechanisms against invading pests. However, these recognition and mediation mechanisms, including the binding kinetics between chitin and their plant recognition receptors, are not fully understood. Therefore, the creation of a platform capable of both interfacing with chitin and plant cell receptors, and monitoring their interactions, would significantly advance our understanding of this plant defense elicitor. Recently, a label-free, highly sensitive biosensor platform, based on Whispering Gallery Mode optical microresonators, has been developed to study such biomolecular interactions. Here, we demonstrate how this unique platform can be interfaced with chitin using simple carbohydrate chemistry. The surface chemistry is demonstrated using X-ray photoelectron spectroscopy, fluorescence microscopy, optical profilometry, ellipsometry, and contact angle measurements. The resulting surface is uniform, with an average surface roughness of 1.25nm, and is active toward chitin recognition elements. Optical loss measurements using standard quantitative cavity analysis techniques demonstrate that the bioconjugated platforms maintain the high performance (Q>10(6)) required to track binding interactions in this system. The platform is able to detect lectin, which binds COs, at 10µg/mL concentration. This biosensor platform's unique capabilities for label-free, high sensitivity biodetection, when properly interfaced with the biomaterials of interest, could provide the basis for a robust analytical technique to probe the binding dynamics of chitin-plant cell receptors.

A soybean acyl carrier protein, GmACP, is important for root nodule symbiosis.

Legumes (members of family Fabaceae) establish a symbiotic relationship with nitrogen-fixing soil bacteria (rhizobia) to overcome nitrogen source limitation. Single root hair epidermal cells serve as the entry point for bacteria to infect the host root, leading to development of a new organ, the nodule, which the bacteria colonize. In the present study, the putative role of a soybean acyl carrier protein (ACP), GmACP (Glyma18g47950), was examined in nodulation. ACP represent an essential cofactor protein in fatty acid biosynthesis. Phylogenetic analysis of plant ACP protein sequences showed that GmACP was classified in a legume-specific clade. Quantitative reverse-transcription polymerase chain reaction analysis demonstrated that GmACP was expressed in all soybean tissues but showed higher transcript accumulation in nodule tissue. RNA interference-mediated gene silencing of GmACP resulted in a significant reduction in nodule numbers on soybean transgenic roots. Fluorescent protein-labeled GmACP was localized to plastids in planta, the site of de novo fatty acid biosynthesis in plants. Analysis of the fatty acid content of root tissue silenced for GmACP expression, as determined by gas chromatography-mass spectrometry, showed an approximately 22% reduction, specifically in palmitic and stearic acid. Taken together, our data provide evidence that GmACP plays an important role in nodulation.

Predicting gene regulatory networks of soybean nodulation from RNA-Seq transcriptome data.

BACKGROUND: High-throughput RNA sequencing (RNA-Seq) is a revolutionary technique to study the transcriptome of a cell under various conditions at a systems level. Despite the wide application of RNA-Seq techniques to generate experimental data in the last few years, few computational methods are available to analyze this huge amount of transcription data. The computational methods for constructing gene regulatory networks from RNA-Seq expression data of hundreds or even thousands of genes are particularly lacking and urgently needed. RESULTS: We developed an automated bioinformatics method to predict gene regulatory networks from the quantitative expression values of differentially expressed genes based on RNA-Seq transcriptome data of a cell in different stages and conditions, integrating transcriptional, genomic and gene function data. We applied the method to the RNA-Seq transcriptome data generated for soybean root hair cells in three different development stages of nodulation after rhizobium infection. The method predicted a soybean nodulation-related gene regulatory network consisting of 10 regulatory modules common for all three stages, and 24, 49 and 70 modules separately for the first, second and third stage, each containing both a group of co-expressed genes and several transcription factors collaboratively controlling their expression under different conditions. 8 of 10 common regulatory modules were validated by at least two kinds of validations, such as independent DNA binding motif analysis, gene function enrichment test, and previous experimental data in the literature. CONCLUSIONS: We developed a computational method to reliably reconstruct gene regulatory networks from RNA-Seq transcriptome data. The method can generate valuable hypotheses for interpreting biological data and designing biological experiments such as ChIP-Seq, RNA interference, and yeast two hybrid experiments.

Cytochrome b5 coexpression increases Tetrahymena thermophila Δ6 fatty acid desaturase activity in Saccharomyces cerevisiae.

Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2(Δ9,12) substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b5 protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b5 domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b5s were coexpressed with the desaturase. Coexpression of a cytochrome b5 from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b5 proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.

Structural analysis and cellular localization of polyunsaturated C27 hydrocarbons in the marine dinoflagellate, Pyrocystis lunula (Dinophyceae).

A neutral lipid fraction obtained from two strains of the permanently sheathed dinoflagellate, Pyrocystis lunula, was found to contain three polyunsaturated C27 hydrocarbons as abundant lipid components. A combination of mass spectrometry techniques was used to identify these compounds as n-heptacosa-3,6,9,12,15,18-hexaene (C27:6), approx. 0.7 ng/sheathed cell), n-heptacosa-3,6,9,12,15,18,21-heptaene (C27:7), approx. 2 ng/sheathed cell), and n-heptacosa-3,6,9,12,15,18,21,24-octaene (C27:8), approx. 2 ng/sheathed cell). Polyunsaturated C21, C23, and C25 hydrocarbons were also found at lesser amounts of approximately 0.2-0.5 ng/sheathed cell. Fluorescent microscopy revealed Nile red staining in both the vegetative cell and structures within the outer sheath surrounding the cell. These hydrocarbons were not present in two other species of Pyrocystis, P. fusiformis and P. noctiluca. Although their function(s) is not known, previous studies have shown and hypothesized that similar hydrocarbons function in carbon storage, buoyancy regulation, or signaling.

Free sterol composition of species in the dinoflagellate genus Pyrocystis: a spectrum of sterol diversity.

The dinoflagellate genus Pyrocystis includes a small number of marine species, which spend the majority of their life cycles as nonmotile cells within a carbohydrate sheath, and which are found ubiquitously throughout the world's oceans. The biochemistry of this model dinoflagellate genus has been widely studied due to its ability to bioluminesce. However, Pyrocystis has been comparatively understudied with respect to its lipid biochemistry, in particular that of sterols. To date, examination of the sterols of Pyrocystis has focused primarily upon Pyrocystis lunula, which produces cholesterol and 4,24-dimethyl-5α-cholestan-3ß-ol as its predominant sterols, while it lacks the common dinoflagellate sterol, dinosterol. We have examined the sterol composition of the two other commercially available species of Pyrocystis, Pyrocystis fusiformis and Pyrocystis noctiluca. Pyrocystis noctiluca possesses dinosterol as its most abundant sterol, while P. fusiformis possesses dinosterol and 4,24-dimethyl-5α-cholestan-3ß-ol as the predominant sterols, placing it at an intermediate position between P. lunula and P. noctiluca, as based on sterol composition. The potential limitations of the dinoflagellate sterol biomarker dinosterol are also explored in this study due to its notable absence in P. lunula.

Sterols of the syndinian dinoflagellate Amoebophrya sp., a parasite of the dinoflagellate Alexandrium tamarense (Dinophyceae).

Several harmful photosynthetic dinoflagellates have been examined over past decades for unique chemical biomarker sterols. Little emphasis has been placed on important heterotrophic genera, such as Amoebophrya, an obligate, intracellular parasite of other, often harmful, dinoflagellates with the ability to control host populations naturally. Therefore, the sterol composition of Amoebophrya was examined throughout the course of an infective cycle within its host dinoflagellate, Alexandrium tamarense, with the primary intent of identifying potential sterol biomarkers. Amoebophrya possessed two primary C(27) sterols, cholesterol and cholesta-5,22Z-dien-3beta-ol (cis-22-dehydrocholesterol), which are not unique to this genus, but were found in high relative percentages that are uncommon to other genera of dinoflagellates. Because the host also possesses cholesterol as one of its major sterols, carbon-stable isotope ratio characterization of cholesterol was performed in order to determine whether it was produced by Amoebophrya or derived intact from the host. Results indicated that cholesterol was not derived intact from the host. A comparison of the sterol profile of Amoebophrya to published sterol profiles of phylogenetic relatives revealed that its sterol profile most closely resembles that of the (proto)dinoflagellate Oxyrrhis marina rather than other extant genera.