Sanchez-Alvarez, EL, Gonzalez-Ledezma G, Prats JAB, Stephano-Hornedo JL, Hildebrand M.
2017.
Evaluating Marinichlorella kaistiae KAS603 cell size variation, growth and TAG accumulation resulting from rapid adaptation to highly diverse trophic and salinity cultivation regimes. Algal Research-Biomass Biofuels and Bioproducts. 25:12-24.
10.1016/j.algal.2017.03.027 AbstractThe ability of a microalgal species to adapt to changes in cultivation environment is likely to be beneficial for a successful biofuel/bioproduct production system, because the species could maintain high yields under diverse seasonal or cultivation conditions. Examining factors that enable culturing flexibility in a single species could provide clearer insights than when comparing different species because it will reduce interspecies variability. Marinichlorella kaistiae KAS603 is an excellent model to evaluate mechanisms involved in adaptation to different cultivation regimes. We have studied cell growth, size, triacylglycerol (TAG) accumulation and life cycle stages in this multiple fission dividing strain under a wide variety of conditions, ranging from autotrophic growth in freshwater to mixotrophic growth in fresh and seawater, and to autotrophic growth under saline and hypersaline conditions. Such conditions influence the division mode of the strain, which is linked to biomass and TAG yield. Based on lab and pilot plant experiments, we have discovered that the fastest TAG accumulation takes place under mixotrophy in freshwater, highest yield (culture density plus TAG) condition occurs under mixotrophy in sea water, and the best outdoor culture condition to achieve growth with fewer invasive species is hypersaline natural seawater. In addition to characterizing growth and TAG accumulation characteristics under a wide variety of cultivation conditions, this work sets the stage for investigation into the mechanisms that enable diverse cultivation adaptations, and contribute to the development of this environmentally flexible microalga as a production feedstock.
Shrestha, RP, Hildebrand M.
2017.
Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica. Microbial Cell Factories. 16
10.1186/s12934-017-0760-3 AbstractBackground: An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. Results: In this study, we evaluate a number of promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Conclusions: Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.
Davis, A, Crum LT, Corbeil LB, Hildebrand M.
2017.
Expression of Histophilus somni IbpA DR2 protective antigen in the diatom Thalassiosira pseudonana. Applied Microbiology and Biotechnology. 101:5313-5324.
10.1007/s00253-017-8267-8 AbstractIncreasing demand for the low-cost production of valuable proteins has stimulated development of novel expression systems. Many challenges faced by existing technology may be overcome by using unicellular microalgae as an expression platform due to their ability to be cultivated rapidly, inexpensively, and in large scale. Diatoms are a particularly productive type of unicellular algae showing promise as production organisms. Here, we report the development of an expression system in the diatom Thalassiosira pseudonana by expressing the protective IbpA DR2 antigen from Histophilus somni for the production of a vaccine against bovine respiratory disease. The utilization of diatoms with their typically silicified cell walls permitted development of silicon-responsive transcription elements to induce protein expression. Specifically, we demonstrate that transcription elements from the silicon transporter gene SIT1 are sufficient to drive high levels of IbpA DR2 expression during silicon limitation and growth arrest. These culture conditions eliminate the flux of cellular resources into cell division processes, yet do not limit protein expression. In addition to improving protein expression levels by molecular manipulations, yield was dramatically increased through cultivation enhancement including elevated light and CO2 supplementation. We substantially increased recombinant protein production over starting levels to 1.2% of the total sodium dodecyl sulfate-extractable protein in T. pseudonana, which was sufficient to conduct preliminary immunization trials in mice. Mice exposed to 5 mu g of diatom-expressed DR2 in whole or sonicated cells (without protein purification) exhibited a modest immune response without the addition of adjuvant.
Hildebrand, M, Manandhar-Shrestha K, Abbriano R.
2017.
Effects of chrysolaminarin synthase knockdown in the diatom Thalassiosira pseudonana: Implications of reduced carbohydrate storage relative to green algae. Algal Research-Biomass Biofuels and Bioproducts. 23:66-77.
10.1016/j.algal.2017.01.010 AbstractIn all organisms, the flux of carbon through the fundamental pathways of glycolysis, gluconeogenesis and the pyruvate hub is a core process related to growth and productivity. In unicellular microalgae, the complexity and intracellular location of specific steps of these pathways can vary substantially. In addition, the location and chemical nature of storage carbohydrate can be substantially different. The role of starch storage in green algae has been investigated, but thus far, only a minimal understanding of the role of carbohydrate storage in diatoms as the beta- 1,3-glucan chrysolaminarin has been achieved. In this report, we aimed to determine the effect of specifically reducing the ability of Thalassiosira pseudonana cells to accumulate chrysolaminarin by knocking down transcript levels of the chrysolaminarin synthase gene. We monitored changes in chrysolaminarin and triacylglycerol (TAG) levels during growth and silicon starvation. Transcript- level changes in genes encoding steps in chrysolaminarin metabolism, and cytoplasmic and chloroplast glycolysis/ gluconeogenesis, were monitored during silicon limitation, highlighting the carbon flux processes involved. We demonstrate that knockdown lines accumulate less chrysolaminarin and have a transiently increased TAG level, with minimal detriment to growth. The results provide insight into the role of chrysolaminarin storage in diatoms, and further discussion highlights differences between diatoms and green algae in carbohydrate storage processes and the effect of reducing carbohydrate stores on growth and productivity. (c) 2017 The Authors. Published by Elsevier B.V.
Nazmi, A, Hauck R, Davis A, Hildebrand M, Corbeil LB, Gallardo RA.
2017.
Diatoms and diatomaceous earth as novel poultry vaccine adjuvants. Poultry Science. 96:288-294.
10.3382/ps/pew250 AbstractDiatoms are single cell eukaryotic microalgae; their surface possesses a porous nanostructured silica cell wall or frustule. Diatomaceous earth (DE) or diatomite is a natural siliceous sediment of diatoms. Since silica has been proved to have adjuvant capabilities, we propose that diatoms and DE may provide an inexpensive and abundant source of adjuvant readily available to use in livestock vaccines. In a first experiment, the safety of diatoms used as an adjuvant for in-ovo vaccination was investigated. In a second experiment, we assessed the humoral immune response after one in-ovo vaccination with inactivated Newcastle Disease Virus (NDV) and DE as adjuvant followed by 2 subcutaneous boosters on d 21 and 29 of age. In both experiments, results were compared to Freund's incomplete adjuvant and aluminum hydroxide. No detrimental effects on hatchability and chick quality were detected after in-ovo inoculation of diatoms and DE in experiments 1 and 2 respectively. In experiment 2 no humoral responses were detected after the in-ovo vaccination until 29 d of age. Seven d after the second subcutaneous booster an antibody response against NDV was detected in chickens that had received vaccines adjuvanted with Freund's incomplete adjuvant, aluminum hydroxide, and DE. These responses became significantly higher 10 d after the second booster. Finally, 15 d after the second booster, the humoral responses induced by the vaccine with Freund's incomplete adjuvant were statistically higher, followed by comparable responses induced by vaccines containing DE or aluminum hydroxide that were significantly higher than DE+PBS, PBS+INDV and PBS alone. From an applied perspective, we can propose that DE can serve as a potential adjuvant for vaccines against poultry diseases.