ASU Scholarship Showcase

Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H₂ under certain physiological conditions. However, most produce only little H₂, revert readily to H₂ consumption, and suffer from hydrogenase sensitivity to O₂. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H₂ production potential. Because of their ecological origin in environments that become quickly anoxic in the dark, we hypothesized that this differential ability may have evolved to serve a role in the fermentation of the photosynthate. Here we show that, when forced to ferment internal substrate, these cyanobacteria display desirable characteristics of physiological H₂ production. Among them, the strain L. aestuarii BL J had the fastest specific rates and attained the highest H₂ concentrations during fermentation of photosynthate, which proceeded via a mixed acid fermentation pathway to yield acetate, ethanol, lactate, H₂, CO₂, and pyruvate. Contrary to expectations, the H₂ yield per mole of glucose was only average compared to that of other cyanobacteria. Thermodynamic analyses point to the use of electron donors more electronegative than NAD(P)H in Lyngbya hydrogenases as the basis for its strong H₂ production ability. In any event, the high specific rates and H₂ concentrations coupled with the lack of reversibility of the enzyme, at the expense of internal, photosynthetically generated reductants, makes L. aestuarii BL J and/or its enzymes, a potentially feasible platform for large-scale H₂ production.

Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs that produce filaments that bind the soil together, and support an array of heterotrophic microorganisms. These microorganisms in turn contribute to soil stability and biogeochemistry of BSCs. Non-cyanobacterial populations of BSCs are less well known than cyanobacterial populations. Therefore, we attempted to isolate a broad range of numerically significant and phylogenetically representative BSC aerobic heterotrophs. Combining simple pre-treatments (hydration of BSCs under dark and light) and isolation strategies (media with varying nutrient availability and protection from oxidative stress) we recovered 402 bacterial and one fungal isolate in axenic culture, which comprised 116 phylotypes (at 97% 16S rRNA gene sequence homology), 115 bacterial and one fungal. Each medium enriched a mostly distinct subset of phylotypes, and cultivated phylotypes varied due to the BSC pre-treatment. The fraction of the total phylotype diversity isolated, weighted by relative abundance in the community, was determined by the overlap between isolate sequences and OTUs reconstructed from metagenome or metatranscriptome reads. Together, more than 8% of relative abundance of OTUs in the metagenome was represented by our isolates, a cultivation efficiency much larger than typically expected from most soils. We conclude that simple cultivation procedures combined with specific pre-treatment of samples afford a significant reduction in the culturability gap, enabling physiological and metabolic assays that rely on ecologically relevant axenic cultures.

Previously, our group engineered a plant-derived monoclonal antibody (MAb) (pHu-E16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed several pHu-E16 variants to improve its efficacy. These variants included a single-chain variable fragment (scFv) of pHu-E16 fused to the heavy chain (HC) constant domains (CH1-3) of human IgG (pHu-E16scFv-CH1-3) and a tetravalent molecule (Tetra pHu-E16) assembled from pHu-E16scFv-CH1-3 with a second pHu-E16scFv fused to the light chain (LC) constant region. pHu-E16scFv-CH1-3 and Tetra pHu-E16 were efficiently expressed and assembled in plants. To assess the impact of differences in N-linked glycosylation on pHu-E16 variant assembly and function, we expressed additional pHu-E16 variants with various combinations of HC and LC components.

Our study revealed that proper pairing of HC and LC was essential for the complete N-glycan processing of antibodies in both plant and animal cells. Associated with their distinct N-glycoforms, pHu-E16, pHu-E16scFv-CH1-3 and Tetra pHu-E16 exhibited differential binding to C1q and specific Fcγ receptors (FcγR). Notably, none of the plant-derived Hu-E16 variants showed antibody-dependent enhancement (ADE) activity in CD32A+ human cells, suggesting the potential of plant-produced antibodies to minimize the adverse effect of ADE. Importantly, all plant-derived MAb variants exhibited at least equivalent in vitro neutralization and in vivo protection in mice compared to mammalian cell-produced Hu-E16. This study demonstrates the capacity of plants to express and assemble a large, complex and functional IgG-like tetravalent mAb variant and also provides insight into the relationship between MAb N-glycosylation, FcγR and C1q binding, and ADE. These new insights may allow the development of safer and cost effective MAb-based therapeutics for flaviviruses, and possibly other pathogens.

Background: The extracellular sunscreen scytonemin is the most common and widespread indole-alkaloid among cyanobacteria. Previous research using the cyanobacterium Nostoc punctiforme ATCC 29133 revealed a unique 18-gene cluster (NpR1276 to NpR1259 in the N. punctiforme genome) involved in the biosynthesis of scytonemin. We provide further genomic characterization of these genes in N. punctiforme and extend it to homologous regions in other cyanobacteria.

Results: Six putative genes in the scytonemin gene cluster (NpR1276 to NpR1271 in the N. punctiforme genome), with no previously known protein function and annotated in this study as scyA to scyF, are likely involved in the assembly of scytonemin from central metabolites, based on genetic, biochemical, and sequence similarity evidence. Also in this cluster are redundant copies of genes encoding for aromatic amino acid biosynthetic enzymes. These can theoretically lead to tryptophan and the tyrosine precursor, p-hydroxyphenylpyruvate, (expected biosynthetic precursors of scytonemin) from end products of the shikimic acid pathway. Redundant copies of the genes coding for the key regulatory and rate-limiting enzymes of the shikimic acid pathway are found there as well. We identified four other cyanobacterial strains containing orthologues of all of these genes, three of them by database searches (Lyngbya PCC 8106, Anabaena PCC 7120, and Nodularia CCY 9414) and one by targeted sequencing (Chlorogloeopsis sp. strain Cgs-089; CCMEE 5094). Genomic comparisons revealed that most scytonemin-related genes were highly conserved among strains and that two additional conserved clusters, NpF5232 to NpF5236 and a putative two-component regulatory system (NpF1278 and NpF1277), are likely involved in scytonemin biosynthesis and regulation, respectively, on the basis of conservation and location. Since many of the protein product sequences for the newly described genes, including ScyD, ScyE, and ScyF, have export signal domains, while others have putative transmembrane domains, it can be inferred that scytonemin biosynthesis is compartmentalized within the cell. Basic structural monomer synthesis and initial condensation are most likely cytoplasmic, while later reactions are predicted to be periplasmic.

Conclusion: We show that scytonemin biosynthetic genes are highly conserved among evolutionarily diverse strains, likely include more genes than previously determined, and are predicted to involve compartmentalization of the biosynthetic pathway in the cell, an unusual trait for prokaryotes.

Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

The threat of West Nile virus (WNV) epidemics with increasingly severe neuroinvasive infections demands the development and licensing of effective vaccines. To date, vaccine candidates based on inactivated, live-attenuated, or chimeric virus, and viral DNA and WNV protein subunits have been developed. Some have been approved for veterinary use or are under clinical investigation, yet no vaccine has been licensed for human use. Reaching the milestone of a commercialized human vaccine, however, may largely depend on the economics of vaccine production. Analysis suggests that currently only novel low-cost production technologies would allow vaccination to outcompete the cost of surveillance and clinical treatment. Here, we review progress using plants to address the economic challenges of WNV vaccine production. The advantages of plants as hosts for vaccine production in cost, speed and scalability, especially those of viral vector-based transient expression systems, are discussed. The progress in developing WNV subunit vaccines in plants is reviewed within the context of their expression, characterization, downstream processing, and immunogenicity in animal models. The development of vaccines based on enveloped and non-enveloped virus-like particles is also discussed. These advancements suggest that plants may provide a production platform that offers potent, safe and affordable human vaccines against WNV.

N₂ fixation and ammonia oxidation (AO) are the two most important processes in the nitrogen (N) cycle of biological soil crusts (BSCs). We studied the short-term response of acetylene reduction assay (ARA) rates, an indicator of potential N₂ fixation, and AO rates to temperature (T, -5°C to 35°C) in BSC of different successional stages along the BSC ecological succession and geographic origin (hot Chihuahuan and cooler Great Basin deserts). ARA in all BSCs increased with T until saturation occurred between 15 and 20°C, and declined at 30–35°C. Culture studies using cyanobacteria isolated from these crusts indicated that the saturating effect was traceable to their inability to grow well diazotrophically within the high temperature range. Below saturation, temperature response was exponential, with Q₁₀ significantly different in the two areas (~ 5 for Great Basin BSCs; 2–3 for Chihuahuan BSCs), but similar between the two successional stages. However, in contrast to ARA, AO showed a steady increase to 30–35°C in Great Basin, and Chihuhuan BSCs showed no inhibition at any tested temperature. The T response of AO also differed significantly between Great Basin (Q₁₀ of 4.5–4.8) and Chihuahuan (Q₁₀ of 2.4–2.6) BSCs, but not between successional stages. Response of ARA rates to T did not differ from that of AO in either desert. Thus, while both processes scaled to T in unison until 20°C, they separated to an increasing degree at higher temperature. As future warming is likely to occur in the regions where BSCs are often the dominant living cover, this predicted decoupling is expected to result in higher proportion of nitrates in soil relative to ammonium. As nitrate is more easily lost as leachate or to be reduced to gaseous forms, this could mean a depletion of soil N over large landscapes globally.

Endolithic microbial communities are prominent features of intertidal marine habitats, where they colonize a variety of substrates, contributing to their erosion. Almost 2 centuries worth of naturalistic studies focused on a few true-boring (euendolithic) phototrophs, but substrate preference has received little attention. The Isla de Mona (Puerto Rico) intertidal zone offers a unique setting to investigate substrate specificity of endolithic communities since various phosphate rock, limestone and dolostone outcrops occur there. High-throughput 16S rDNA genetic sampling, enhanced by targeted cultivation, revealed that, while euendolithic cyanobacteria were dominant operational taxonomic units (OTUs), the communities were invariably of high diversity, well beyond that reported in traditional studies and implying an unexpected metabolic complexity potentially contributed by secondary colonizers. While the overall community composition did not show differences traceable to the nature of the mineral substrate, we detected specialization among particular euendolithic cyanobacterial clades towards the type of substrate they excavate but only at the OTU phylogenetic level, implying that close relatives have specialized recurrently into particular substrates. The cationic mineral component was determinant in this preference, suggesting the existence in nature of alternatives to the boring mechanism described in culture that is based exclusively on transcellular calcium transport.

The filamentous, non-heterocystous cyanobacterium Lyngbya aestuarii is an important contributor to marine intertidal microbial mats system worldwide. The recent isolate L. aestuarii BL J, is an unusually powerful hydrogen producer. Here we report a morphological, ultrastructural, and genomic characterization of this strain to set the basis for future systems studies and applications of this organism. The filaments contain circa 17 μm wide trichomes, composed of stacked disk-like short cells (2 μm long), encased in a prominent, laminated exopolysaccharide sheath. Cellular division occurs by transversal centripetal growth of cross-walls, where several rounds of division proceed simultaneously. Filament division occurs by cell self-immolation of one or groups of cells (necridial cells) at the breakage point. Short, sheath-less, motile filaments (hormogonia) are also formed. Morphologically and phylogenetically L. aestuarii belongs to a clade of important cyanobacteria that include members of the marine Trichodesmiun and Hydrocoleum genera, as well as terrestrial Microcoleus vaginatus strains, and alkalyphilic strains of Arthrospira. A draft genome of strain BL J was compared to those of other cyanobacteria in order to ascertain some of its ecological constraints and biotechnological potential.

The genome had an average GC content of 41.1%. Of the 6.87 Mb sequenced, 6.44 Mb was present as large contigs (>10,000 bp). It contained 6515 putative protein-encoding genes, of which, 43% encode proteins of known functional role, 26% corresponded to proteins with domain or family assignments, 19.6% encode conserved hypothetical proteins, and 11.3% encode apparently unique hypothetical proteins. The strain's genome reveals its adaptations to a life of exposure to intense solar radiation and desiccation. It likely employs the storage compounds, glycogen, and cyanophycin but no polyhydroxyalkanoates, and can produce the osmolytes, trehalose, and glycine betaine. According to its genome, BL J strain also has the potential to produce a plethora of products of biotechnological interest such as Curacin A, Barbamide, Hemolysin-type calcium-binding toxin, the suncreens scytonemin, and mycosporines, as well as heptadecane and pentadecane alkanes. With respect to hydrogen production, initial comparisons of the genetic architecture and sequence of relevant genes and loci, and a comparative model of protein structure of the NiFe bidirectional hydrogenase, did not reveal conspicuous differences that could explain its unusual hydrogen producing capacity.

Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other diseases due to their specificity and potency. While mammalian cell-produced mAbs have shown promise as therapeutic candidates against several flaviviruses, their eventual approval for human application still faces several challenges including their potential risk of predisposing treated patients to more severe secondary infection by a heterologous flavivirus through antibody-dependent enhancement (ADE). The high cost associated with mAb production in mammalian cell cultures also poses a challenge for the feasible application of these drugs to the developing world where the majority of flavivirus infection occurs. Here, we review the current therapeutic mAb candidates against various flaviviruses including West Nile (WNV), Dengue virus (DENV), and ZIKV. The progress of using plants for developing safer and more economical mAb therapeutics against flaviviruses is discussed within the context of their expression, characterization, downstream processing, neutralization, and in vivo efficacy. The progress of using plant glycoengineering to address ADE, the major impediment of flavivirus therapeutic development, is highlighted. These advancements suggest that plant-based systems are excellent alternatives for addressing the remaining challenges of mAb therapeutic development against flavivirus and may facilitate the eventual commercialization of these drug candidates.