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Functional categorization of cDNA clones that were isolated by SSH. The up-regulated genes in C. protothecoides photosynthesis– fermentation approach were identified by SSH. The clones were categorized according to their homology to known genes in both NCBI and JGI databases. The obtained clones were categorized into different functions shown in the pie chart as follows (and Table S1): metabolism (10.34 %), energy (10.34 %), cell growth/division (1.15 %), transcription (6.90 %), protein synthesis (22.99 %), protein destination and storage (1.15 %), transporters (5.75 %), signal transduction (2.30 %), disease/defense (2.30 %), secondary metabolism (2.30 %), unclassified (24.14 %), and unknown (10.34 %)
Source publication
A suppression subtractive hybridization cDNA library was used to screen the differently expressed (up-regulated) genes in the photosynthesis-fermentation approach (PFA) of Chlorella protothecoides cultivation. A total of 87 clones were obtained and sequenced, in which 78 clones were homologous to known genes in databases. Among them, the ammonium t...
Citations
... Although the relevant metabolic pathway of ammonium, glucose and CO 2 in autotrophy or heterotrophy has been reported in some studies (Garcia et al., 2011;Liu et al., 2015;Wu et al., 2016), the underlying mechanisms related to high-efficient ammonium assimilation coupled with nutrient components biosynthesis in mixotrophic green microalgae are not completely clear. Based on previous literature (Bashan et al., 2008;Chen et al., 2015;Giordano and Raven, 2014;Liu et al., 2015;Lu et al., 2018;Wu et al., 2016;Yamazaki et al., 2018;Yan et al., 2013;Zhang et al., 2017;Zuniga et al., 2016), the hypothetical pathway in mixotrophic C. pyrenoidosa was proposed as shown in Fig. 5. First of all, ammonium is primarily transported across the plasma membrane by a group of proteins belong to the ammonium transporter family (AMT) (Yan et al., 2013), and then assimilated via the direct amination pathway by glutamate dehydrogenase (GDH), or the GS-GOGAT pathway by the GS and GOGAT (Bashan et al., 2008;Wu et al., 2016). Autotrophic Chlorella cells can acclimate ammonium mainly through GDH and partially through GOGAT pathway, while the GS-GOGAT pathway has been demonstrated to be responsible for the ammonium uptake in Chlorella under absolute heterotrophic mode (Wu et al., 2016). ...
... Although the relevant metabolic pathway of ammonium, glucose and CO 2 in autotrophy or heterotrophy has been reported in some studies (Garcia et al., 2011;Liu et al., 2015;Wu et al., 2016), the underlying mechanisms related to high-efficient ammonium assimilation coupled with nutrient components biosynthesis in mixotrophic green microalgae are not completely clear. Based on previous literature (Bashan et al., 2008;Chen et al., 2015;Giordano and Raven, 2014;Liu et al., 2015;Lu et al., 2018;Wu et al., 2016;Yamazaki et al., 2018;Yan et al., 2013;Zhang et al., 2017;Zuniga et al., 2016), the hypothetical pathway in mixotrophic C. pyrenoidosa was proposed as shown in Fig. 5. First of all, ammonium is primarily transported across the plasma membrane by a group of proteins belong to the ammonium transporter family (AMT) (Yan et al., 2013), and then assimilated via the direct amination pathway by glutamate dehydrogenase (GDH), or the GS-GOGAT pathway by the GS and GOGAT (Bashan et al., 2008;Wu et al., 2016). Autotrophic Chlorella cells can acclimate ammonium mainly through GDH and partially through GOGAT pathway, while the GS-GOGAT pathway has been demonstrated to be responsible for the ammonium uptake in Chlorella under absolute heterotrophic mode (Wu et al., 2016). ...
To achieve ultrahigh-efficient ammonium removal and valuable biomass coproduction, Chlorella-mediated short-time acclimation was implemented in photo-fermentation. The results demonstrated short-time acclimation of mixotrophic Chlorella pyrenoidosa could significantly improve NH4⁺ removal and biomass production in shake flasks. After acclimation through two batch cultures in 5-L photo-fermenter, the maximum NH4⁺ removal rate (1,400 mg L⁻¹ d⁻¹) were achieved under high NH4⁺ level (4,750 mg L⁻¹) in batch 3. In 50-L photo-fermenter, through one batch acclimated culture, the maximum NH4⁺ removal rate (2,212 mg L⁻¹ d⁻¹) and biomass concentration (58.4 g L⁻¹) were achieved in batch 2, with the highest productivities of protein (5.56 g L⁻¹ d⁻¹) and total lipids (5.66 g L⁻¹ d⁻¹). The hypothetical pathway of nutrients assimilation in mixotrophic cells as cell factory was proposed with detailed discussion. This study provided a novel strategy for high-ammonium wastewater treatment without dilution, facilitating the algae-based “waste-to-treasure” bioconversion process for green manufacturing.
... Hydrophobicity analysis indicated that the CvNDI2 protein contains 11 transmembrane domains, which are generally conserved among the AMT proteins of a range of organisms ( Supplementary Fig. 3b). The AMT plays an important role in N metabolism by maintaining an optimal level of N in the cell 36,37 . Therefore, both CvNDI1 and CvNDI2 genes are expected to be involved in metabolism to support N sources for cell survival under N starvation conditions. ...
... Urea is converted into allophanate by CvNDI1 in the first step of urea metabolism 31 . AMT is known to play a vital role in ammonium uptake into a cell, and the transcription of AMT genes was found to be strongly induced under N starvation in Arabidopsis and Chlorella, suggesting that AMT might be an initial sensor of N deficiency 37,40,41 . CvNDI1 and CvNDI2 might be important for the survival of Chlorella under N starvation via the utilization of urea as N resource and the enhancing of ammonium transportation in the cell. ...
Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.
... In fact, this alga cannot grow on nitrate and uses ammonium as nitrogen source. Yan et al., identified and characterized an ammonium transporter gene in A. protothecoides (CpAMT1) and declared that nitrate could not be efficiently used by A. protothecoides as sole nitrogen source because of the lacking of NO 3 transporter genes [49]. ...
Low yield and inhibition of hydrogenase by oxygen are the main limitations for hydrogen production by microalgae. Considering the role of electron flow in the metabolism for hydrogen production, a genome-scale metabolic model (named iMM627) was reconstructed for Auxenochlorella protothecoides. iMM627 was evaluated using experimental data for growth and flux distribution. Then, considering the well-known degeneracy of FBA solutions, a new method of finding effective reactions based on multiple optimal solutions was developed. At a constant growth rate, flux distributions for maximal and minimal hydrogen production under anaerobiosis and for maximal oxygen production were compared to identify target reactions for improvement of hydrogen production and for providing anaerobiosis. Existing researches on some reactions truly confirm the predicted changes by iMM627. The main proposed strategies for improvement of hydrogen production include changes in metabolism to provide NADH. Consumption of oxygen by mitochondrial respiration and energy dissipation was proposed to provide anaerobiosis.
... However, light intensity had a smaller contribution to lutein accumulation than nutrients in the photoautotrophic stage of A. protothecoides (data not shown). Abundant nitrogen source is the key nutrient to photoautotrophic growth of A. protothecoides [30]. Three nitrogen sources-urea, NH 4 Cl, and glycine, were used for lutein accumulation after heterotrophic growth. ...
In order to enhance lutein accumulation and to explain the reasons for the difference in lutein accumulation under photoautotrophic and heterotrophic conditions, different culture modes and the associated transcriptome profiles were investigated in Auxenochlorella protothecoides. The heterotrophic-photoautotrophic transition culture mode was investigated for lutein accumulation, changing from organic carbon to increase biomass in dark fermentation to irradiation under nitrogen rich conditions. This strategy increased the lutein content 10 times along with chloroplast regeneration and little biomass loss in 48 h. The highest lutein productivity and production in the heterotrophic-photoautotrophic transition culture reached 12.36 mg/L/day and 34.13 mg/L respectively within seven days. Furthermore, compared to the photoautotrophic conditions, most genes involved in lutein biosynthesis and photosystem generation were down-regulated during heterotrophic growth. By contrast, two β-ring hydroxylases were transiently upregulated, while violaxanthin de-epoxidase and zeaxanthin epoxidase were mostly downregulated, which explained the extremely low lutein content of heterotrophic cells. Nevertheless, the lutein proportion in total carotenoids reached nearly 100%. This study is the first to our knowledge to report on a comparative transcriptome analysis of lutein biosynthesis, and it provides a promising strategy to boost lutein production in A. protothecoides.
... That is, BbAMT1;1 and 1;2 are most likely responsible for ammonium uptake when the alga is deprived of nitrogen, as being the case for Arabidopsis thaliana AMT1;1 [47,48]. Upregulation of AMT genes under nitrogen depletion have also been reported in several eukaryotes [26,32,49]. Recently it has also been shown through RNAseq analyses that expression of an ammonia permease gene was upregulated under nitrogen depletion in the A race of B. braunii, suggesting that adaptive mechanisms against this stress condition is conserved among different chemical races of B. braunii [50]. ...
As a prospective feedstock for sustainable biofuels, the green microalga Botryococcus braunii race B has attracted significant attention since this colony-forming alga has the ability to accumulate considerable amounts of triterpene hydrocarbons, botryococcenes, in its extracellular matrix. However, its primary metabolism, nutrient uptake and utilization relevant to growth, are still poorly understood in this alga. From this perspective, we investigated the utilization of ammonium by B. braunii as a more reduced form of nitrogen source compared to nitrate, which is generally used for cultures of the alga. Isotope-ratio mass spectrometry analyses indicated that the uptake rate of ammonium was higher than that of nitrate at a certain concentration. When ammonium was added to culture medium, however, it inhibited algal growth even in the presence of nitrate. Buffered culture media ameliorated the toxicity of ammonium, and the alga accumulated the same levels of hydrocarbons and secondary carotenoids, irrespective of nitrogen source. To characterize utilization of ammonium by B. braunii at the molecular level, putative ammonium transporter (AMT) genes were screened from a cDNA library and four isogenes (BbAMT1;1, 1;2, 1;3 and 1;4) were cloned. Two of them, BbAMT1;1 and 1;2, were upregulated under nitrogen deficient conditions. BbAMT1;1 complemented the growth of an ammonium uptake-defective yeast strain, showing it to be a functional gene coding for AMT. These results could pave the way for culturing B. braunii more efficiently using ammonium as a sole nitrogen source by improving ammonium uptake through our understanding of AMT.
... Wang et al. (2013) evaluated the removal of NH 4 + and TP in batch cultures with Oedogonium sp. to treat digested piggery wasterwater and found that high removal efficiency of nutrients was achieved (95.91 % NH 4 + and 92.92 % TP, respectively). Additionally, the removal efficiencies of NH 4 + and TP were closely related to the survival probability of algae in different nutrient conditions (Lee et al. 2012;Sanz-Luque et al. 2013;Yan et al. 2013). However, when the CTAB concentration was higher than 15 ng/L, the removal efficiency of both NH 4 + and TP significantly declined (p < 0.05), compared to that in the absence of CTAB. ...
Toxicants are generally harmful to biotechnology in wastewater treatment. However, trace toxicant can induce microbial hormesis, but to date, it is still unknown how this phenomenon affects nutrient removal during municipal wastewater treatment process. Therefore, this study focused on the effects of hormesis induced by cetyltrimethyl ammonium bromide (CTAB), a representative quaternary ammonium cationic surfactant, on nutrient removal by Chlorella vulgaris F1068. Results showed that when the concentration of CTAB was less than 10 ng/L, the cellular components chlorophyll a, proteins, polysaccharides, and total lipids increased by 10.11, 58.17, 38.78, and 11.87 %, respectively, and some enzymes in nutrient metabolism of algal cells, such as glutamine synthetase (GS), acid phosphatase (ACP), H+-ATPase, and esterase, were also enhanced. As a result, the removal efficiencies of ammonia nitrogen (NH4+) and total phosphorus (TP) increased by 14.66 and 8.51 %, respectively, compared to the control during a 7-day test period. The underlying mechanism was mainly due to an enhanced photosynthetic activity of C. vulgaris F1068 indicated by the increase in chlorophyll fluorescence parameters (the value of Fv/Fm, ΦII, Fv/Fo, and rETR increased by 12.99, 7.56, 25.59, and 8.11 %, respectively) and adenylate energy charge (AEC) (from 0.68 to 0.72). These results suggest that hormesis induced by trace toxicants could enhance the nutrient removal, which would be further considered in the design of municipal wastewater treatment processes.
Graphical abstract
The schematic mechanism of C. vulgaris F1068 under CTAB induced hormesis. Green arrows (
) represent the increase and the red arrow (
) represents the decrease.
... Therefore, despite little experimental evidence, it is plausible that similar transceptors (dual-function membrane proteins) also play a role in N signalling in microalgae. A study on Chlorella protothecoides, initially grown autotrophically and then transferred into a high C/N ratio medium as a way to trigger lipid production, revealed induction in the expression of the ammonium transporter CpAMT1 during lipid accumulation (Yan et al., 2013). While CpAMT1's expression was modulated by ammonium, glutamine and glutamic acid, it was not changed by nitrate availability. ...
... While CpAMT1's expression was modulated by ammonium, glutamine and glutamic acid, it was not changed by nitrate availability. The correlation in expression between CpAMT1 and glutamine synthetase/glutamate synthetase suggested that CpAMT1 might be one of the N sensors in C. protothecoides (Yan et al., 2013). This is consistent with the observation that glutamine, the primary metabolite derived from nitrogen assimilation in plants, along with ATP and 2-oxogluta- rate, controls the C/N sensing mechanism and homeostasis in cyanobacteria (Forchhammer, 2004 ). ...
The great need for more sustainable alternatives to fossil fuels has increased our research interests in algal biofuels. Microalgal cells, characterized by high photosynthetic efficiency and rapid cell division, are an excellent source of neutral lipids as potential fuel stocks. Various stress factors, especially nutrient-starvation conditions, induce an increased formation of lipid bodies filled with triacylglycerol in these cells. Here we review our knowledge base on glycerolipid synthesis in the green algae with an emphasis on recent studies on carbon flux, redistribution of lipids under nutrient-limiting conditions and its regulation. We discuss the contributions and limitations of classical and novel approaches used to elucidate the algal triacylglycerol biosynthetic pathway and its regulatory network in green algae. Also discussed are gaps in knowledge and suggestions for much needed research both on the biology of triacylglycerol accumulation and possible avenues to engineer improved algal strains.
... The ability of C. protothecoides to accumulate lipids to 60% DCW under heterotrophic conditions is remarkable, yet the mechanism underlying the production of such high oil content remains mostly unknown. A screen for genes selectively expressed when cells grown under autotrophic conditions are supplied with excess organic carbon identified an ammonium transport gene, CpAMT1 as a possible component of a C/N ratio sensory mechanism [22]. In support of their hypothesis that nitrogen availability regulates lipid accumulation, the authors of this study found that supplementation of various amino acids in the culture medium resulted in diminished oil content. ...
... For Cp0710, we showed in an earlier study that it prefers ammonium and amino acids as nitrogen sources, and it has lost the ability to grow on nitrate-or urea-containing medium. We also identified and characterized one ammonium transporter gene (CpAMT1) [22]. To further our understanding of the nitrogen preference of Cp0710, a survey of proteins involved in nitrogen transport and assimilation in the Cp0710 genome was performed, leading to the identification of 13 genes (Additional file 1 Table S7). ...
... Not surprisingly, both the nitrate and the urea transporter were missing from the Cp0710 genome but were present in other green algae. Meanwhile, three nitrogen-related transporters (the nitrite, ammonium, and amino acid transporters) were identified, consistent with our findings on the choice of nitrogen sources in Cp0710, as described previously [22]. The ornithine-urea cycle (OUC), a metabolic pathway that is important for detoxification of excessive ammonia in animals [23], is absent from green plants and algae, though it was recently identified in diatoms [24]. ...
... Another interesting finding after analyzing the Cp0710 genome was the lack of both nitrate and urea transporters, while other nitrogen-related transporters were present, including nitrite, ammonium, and amino acid transporters. This result was not a surprise to us since our previous study showed that this alga could not grow in medium using nitrate or urea as nitrogen sources [22]; however, no other green alga sequenced so far has lost both of these transporters. Since C. variabilis NC64A and C. subellipsoidea C-169 have both transporters, it is possible that the loss of these transporters from the Cp0710 genome might have happened recently, after the divergence of these species from their last common ancestor. ...
Background
Microalgae-derived biodiesel is a promising substitute for conventional fossil fuels. In particular, the green alga Chlorella protothecoides sp. 0710 is regarded as one of the best candidates for commercial manufacture of microalgae-derived biofuel. This is due not only to its ability to live autotrophically through photosynthesis, but also to its capacity to produce a large amount of biomass and lipid through fermentation of glucose. However, until the present study, neither its genome sequence nor the platform required for molecular manipulations were available.
Results
We generated a draft genome for C. protothecoides, and compared its genome size and gene content with that of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169. This comparison revealed that C. protothecoides has a reduced genome size of 22.9 Mbp, about half that of its close relatives. The C. protothecoides genome encodes a smaller number of genes, fewer multi-copy genes, fewer unique genes, and fewer genome rearrangements compared with its close relatives. In addition, three Chlorella-specific hexose-proton symporter (HUP)-like genes were identified that enable the consumption of glucose and, consequently, heterotrophic growth. Furthermore, through comparative transcriptomic and proteomic studies, we generated a global perspective regarding the changes in metabolic pathways under autotrophic and heterotrophic growth conditions. Under heterotrophic conditions, enzymes involved in photosynthesis and CO2 fixation were almost completely degraded, either as mRNAs or as proteins. Meanwhile, the cells were not only capable of quickly assimilating glucose but also showed accelerated glucose catabolism through the upregulation of glycolysis and the tricarboxylic acid (TCA) cycle. Moreover, the rapid synthesis of pyruvate, upregulation of most enzymes involved in fatty acid synthesis, and downregulation of enzymes involved in fatty acid degradation favor the synthesis of fatty acids within the cell.
Conclusions
Despite similarities to other Chlorella, C. protothecoides has a smaller genome than its close relatives. Genes involved in glucose utilization were identified, and these genes explained its ability to grow heterotrophically. Transcriptomic and proteomic results provided insight into its extraordinary ability to accumulate large amounts of lipid. The C. protothecoides draft genome will promote the use of this species as a research model.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-582) contains supplementary material, which is available to authorized users.
... The rapid exhaustion of ammonium in the medium during the heterotrophic cultivation could be due to high biomass demand as a result of the high cell density and rapid synthesis of new biomass when provided with organic carbon as well as by additional induction of the ammonium transport by glucose metabolic products (Schlee and Komor 1986). That uptake of ammonium nitrogen across algal plasmalemma is facilitated by the ammonium transporters (Fernandez and Galvan 2007) recently characterized in A. protothecoides (Yan et al. 2013). The biological uptake of the mineral supplements is more complex and may occur by several mechanisms. ...
A critical factor in implementing microalgal biofuels for mass production is the nutrient requirements. The current study investigated the fate of macro- and micronutrients and their availability in a sequential phototrophic-heterotrophic production process for the lipid rich microalga Auxenochlorella protothecoides. More than 99 % (by weight) of overall process nutrients were supplied during the initial photoautotrophic stage reflecting its significantly larger volume. Under photoautotrophic growth conditions only 9-35 % of supplied Mn, S, Fe, N, Mg, and Cu and less than 5 % of P, Mo, Co, B, Zn, and Ca were consumed by the algae. The rest of these nutrients remain in the spent growth media during the culture concentration-down from an 800 L phototrophic pond to a 5 L heterotrophic fermenter. In contrast, Zn, Mo, Mn, Mg, Ca, and N were exhausted (90-99 % removal) during the first 25 h of the heterotrophic growth stage. The depletion of these key nutrients may have ultimately limited the final biomass density and/or lipid productivity achieved. Approximately 10-20 % of the total supplied S, Mn, Fe, N, and Cu and 5 % of Ca and Zn were assimilated into algal biomass. Several elements including N, P, Mn, B, Cu, Ca, Mg, S, and Fe were released back into the liquid phase by anaerobic digestion (AD) of the residual biomass after lipid extraction. The nutrients recovered from the AD effluent and remaining in the spent medium should be recycled or their initial concentration to the phototrophic stage decreased to enhance process economics and sustainability for future commercialization of algal-derived biofuels.
