Emiliania huxleyi, a ubiquitous marine algae, was cultured under replete and Cu-limiting conditions to investigate Cu uptake strategies involving thiols and associated redox reactions; comparisons to a model diatom, Thalassiosira pseudonana, were also drawn. Cu-limitation increased rates of cell surface reduction of Cu(II) to Cu(I) in E. huxleyi but not in T. pseudonana. Furthermore, Cu-limited E. huxleyi cells took up more Cu when cysteine was present compared to when no ligand was added, although a dependence on cysteine concentration was not observed. In contrast, Cu uptake by replete cells was dependent upon the relative abundance of inorganic species [Cu(I)']. We also show that cysteine can increase the bioavailability of Cu to Cu-limited cells, of both species, through the reductive release of Cu(I) from fairly strong Cu(II) ligands such as EDTA. Finally, support for a mechanism involving uptake of a Cys-Cu complex in E. huxleyi is drawn from the observation that Cu-limitation significantly enhances cysteine uptake by transporters that exhibit Michaelis-Menten kinetics. These Cu uptake strategies help explain the presence and distribution of dissolved thiols in surface seawater and have implications for the biogeochemical cycling of Cu in low Cu environments.
BackgroundWe investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N-terminally fused to the coding region of cel6A, an endoglucanase from Thermobifida fusca. We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5′UTR, previously reported to enhance protein expression, to regulate the expression of the TetC-cel6A gene. We further investigated the accumulation of TetC-Cel6A under N-deplete growth conditions.ResultsBoth of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII-cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N-deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N-replete medium.ConclusionsThe DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1127-7) contains supplementary material, which is available to authorized users.
Microalgae have been increasingly recognized in the fields of environmental and biomedical engineering because of its use as base materials for biofuels or biomedical products, and also the urgent needs to control harmful algal blooms protecting water resources worldwide. Central to the theme is the growth rate of microalgae under the influences of various environmental cues including nutrients, pH, oxygen tension and light intensity. Current microalgal culture systems, e.g. raceway ponds or chemostats, are not designed for system parameter optimizations of cell growth. In this article, we present the development of an array microfluidic system for high throughput studies of microalgal growth under well defined environmental conditions. The microfluidic platform consists of an array of microhabitats flanked by two parallel side channels, all of which are patterned in a thin agarose gel membrane. The unique feature of the device is that each microhabitat is physically confined suitable for both motile and non-motile cell culture, and at the same time, the device is transparent and can be perfused through the two side channels amendable for precise environmental control of photosynthetic microorganisms. This microfluidic system is used to study the growth kinetics of a model microalgal strain, Chlamydomonas reinhardtii (C. reinhardtii), under ammonium (NH4Cl) concentration gradients. Experimental results show that C. reinhardtii follows Monod growth kinetics with a half-saturation constant of 1.2 ± 0.3 μM. This microfluidic platform provides a fast (~50 fold speed increase), cost effective (less reagents and human intervention) and quantitative technique for microalgal growth studies, in contrast to the current chemostat or batch cell culture system. It can be easily extended to investigate growth kinetics of other microorganisms under either single or co-culture setting.
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