Development of a chloroplast expression system for Dunaliella salina.

Dunaliella salina is an innovative expression system due to its distinct advantages such as high salt tolerance, low susceptibility to contamination, and the absence of the cell wall. While nuclear transformation has been extensively studied, research on D. salina chloroplast transformation remains in the preliminary stages. In this study, we established an efficient chloroplast expression system for D. salina using Golden Gate assembly. We developed a D. salina toolkit comprising essential components such as chloroplast-specific promoters, terminators, homologous fragments, and various vectors. We confirmed its functionality by expressing the EGFP protein. Moreover, we detailed the methodology of the entire construction process. This expression system enables the specific targeting of foreign genes through simple homologous recombination, resulting in stable expression in chloroplasts. The toolkit achieved a relatively high transformation efficiency within a shorter experimental cycle. Consequently, the construction and utilization of this toolkit have the potential to enhance the efficiency of transgenic engineering in D. salina and advance the development of microalgal biofactories.

Enhancing astaxanthin accumulation through the expression of the plant-derived astaxanthin biosynthetic pathway in Dunaliella salina.

Dunaliella salina, a microalga that thrives under high-saline conditions, is notable for its high ß-carotene content and the absence of a polysaccharide cell wall. These unique characteristics render it a prime candidate as a cellular platform for astaxanthin production. In this study, our initial tests in an E. coli revealed that ß-ring-4-dehydrogenase (CBFD) and 4-hydroxy-ß-ring-4-dehydrogenase (HBFD) genes from Adonis aestivalis outperformed ß-carotene hydroxylase (BCH) and ß-carotene ketolase (BKT) from Haematococcus pluvialis counterparts by two-fold in terms of astaxanthin biosynthesis efficiency. Subsequently, we utilized electroporation to integrate either the BKT gene or the CBFD and HBFD genes into the genome of D. salina. In comparison to wild-type D. salina, strains transformed with BKT or CBFD and HBFD exhibited inhibited growth, underwent color changes to shades of red and yellow, and saw a nearly 50% decline in cell density. HPLC analysis confirmed astaxanthin synthesis in engineered D. salina strains, with CBFD + HBFD-D. salina yielding 134.88 ± 9.12 µg/g of dry cell weight (DCW), significantly higher than BKT-D. salina (83.58 ± 2.40 µg/g). This represents the largest amount of astaxanthin extracted from transgenic D. salina, as reported to date. These findings have significant implications, opening up new avenues for the development of specialized D. salina-based microcell factories for efficient astaxanthin production.

Functional Characterization of a CruP-Like Isomerase in Dunaliella.

Dunaliella bardawil is a marine unicellular green algal that produces large amounts of ß-carotene and is a model organism for studying the carotenoid synthesis pathway. However, there are still many mysteries about the enzymes of the D. bardawil lycopene synthesis pathway that have not been revealed. Here, we have identified a CruP-like lycopene isomerase, named DbLyISO, and successfully cloned its gene from D. bardawil. DbLyISO showed a high homology with CruPs. We constructed a 3D model of DbLyISO and performed molecular docking with lycopene, as well as molecular dynamics testing, to identify the functional characteristics of DbLyISO. Functional activity of DbLyISO was also performed by overexpressing gene in both E. coli and D. bardawil. Results revealed that DbLyISO acted at the C-5 and C-13 positions of lycopene, catalyzing its cis-trans isomerization to produce a more stable trans structure. These results provide new ideas for the development of a carotenoid series from engineered bacteria, algae, and plants.

Choline Dehydrogenase Contributes to Salt Tolerance in Dunaliella through Betaine Synthesis.

In Dunaliella tertiolecta, a microalga renowned for its extraordinary tolerance to high salinity levels up to 4.5 M NaCl, the mechanisms underlying its stress response have largely remained a mystery. In a groundbreaking discovery, this study identifies a choline dehydrogenase enzyme, termed DtCHDH, capable of converting choline to betaine aldehyde. Remarkably, this is the first identification of such an enzyme not just in D. tertiolecta but across the entire Chlorophyta. A 3D model of DtCHDH was constructed, and molecular docking with choline was performed, revealing a potential binding site for the substrate. The enzyme was heterologously expressed in E. coli Rosetta (DE3) and subsequently purified, achieving enzyme activity of 672.2 U/mg. To elucidate the role of DtCHDH in the salt tolerance of D. tertiolecta, RNAi was employed to knock down DtCHDH gene expression. The results indicated that the Ri-12 strain exhibited compromised growth under both high and low salt conditions, along with consistent levels of DtCHDH gene expression and betaine content. Additionally, fatty acid analysis indicated that DtCHDH might also be a FAPs enzyme, catalyzing reactions with decarboxylase activity. This study not only illuminates the role of choline metabolism in D. tertiolecta's adaptation to high salinity but also identifies a novel target for enhancing the NaCl tolerance of microalgae in biotechnological applications.

A molecular toolkit of cross-feeding strains for engineering synthetic yeast communities.

Engineered microbial consortia often have enhanced system performance and robustness compared with single-strain biomanufacturing production platforms. However, few tools are available for generating co-cultures of the model and key industrial host Saccharomyces cerevisiae. Here we engineer auxotrophic and overexpression yeast strains that can be used to create co-cultures through exchange of essential metabolites. Using these strains as modules, we engineered two- and three-member consortia using different cross-feeding architectures. Through a combination of ensemble modelling and experimentation, we explored how cellular (for example, metabolite production strength) and environmental (for example, initial population ratio, population density and extracellular supplementation) factors govern population dynamics in these systems. We tested the use of the toolkit in a division of labour biomanufacturing case study and show that it enables enhanced and tuneable antioxidant resveratrol production. We expect this toolkit to become a useful resource for a variety of applications in synthetic ecology and biomanufacturing.

Dual Roles of Two Malic Enzymes in Lipid Biosynthesis and Salt Stress Response in Dunaliella salina.

Triacylglycerols (TAG) from microalgae can be used as feedstocks for biofuel production to address fuel shortages. Most of the current research has focused on the enzymes involved in TAG biosynthesis. In this study, the effects of malic enzyme (ME), which provides precursor and reducing power for TAG biosynthesis, on biomass and lipid accumulation and its response to salt stress in Dunaliella salina were investigated. The overexpression of DsME1 and DsME2 improved the lipid production, which reached 0.243 and 0.253 g/L and were 30.5 and 36.3% higher than wild type, respectively. The transcript levels of DsME1 and DsME2 increased with increasing salt concentration (0, 1, 2, 3, and 4.5 mol/L NaCl), indicating that DsMEs participated in the salt stress response in D. salina. It was found that cis-acting elements associated with the salt stress response were present on the promoters of two DsMEs. The deletion of the MYB binding site (MBS) on the DsME2 promoter confirmed that MBS drives the expression of DsME2 to participate in osmotic regulation in D. salina. In conclusion, MEs are the critical enzymes that play pivotal roles in lipid accumulation and osmotic regulation.

Roles of Two Phytoene Synthases and Orange Protein in Carotenoid Metabolism of the ß-Carotene-Accumulating Dunaliella salina.

Phytoene synthase (PSY) is a key enzyme in carotenoid metabolism and often regulated by orange protein. However, few studies have focused on the functional differentiation of the two PSYs and their regulation by protein interaction in the ß-carotene-accumulating Dunaliella salina CCAP 19/18. In this study, we confirmed that DsPSY1 from D. salina possessed high PSY catalytic activity, whereas DsPSY2 almost had no activity. Two amino acid residues at positions 144 and 285 responsible for substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Moreover, orange protein from D. salina (DsOR) could interact with DsPSY1/2. DbPSY from Dunaliella sp. FACHB-847 also had high PSY activity, but DbOR could not interact with DbPSY, which might be one reason why it could not highly accumulate ß-carotene. Overexpression of DsOR, especially the mutant DsORHis, could significantly improve the single-cell carotenoid content and change cell morphology (with larger cell size, bigger plastoglobuli, and fragmented starch granules) of D. salina. Overall, DsPSY1 played a dominant role in carotenoid biosynthesis in D. salina, and DsOR promoted carotenoid accumulation, especially ß-carotene via interacting with DsPSY1/2 and regulating the plastid development. Our study provides a new clue for the regulatory mechanism of carotenoid metabolism in Dunaliella. IMPORTANCE Phytoene synthase (PSY) as the key rate-limiting enzyme in carotenoid metabolism can be regulated by various regulators and factors. We found that DsPSY1 played a dominant role in carotenogenesis in the ß-carotene-accumulating Dunaliella salina, and two amino acid residues critical in the substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Orange protein from D. salina (DsOR) can promote carotenoid accumulation via interacting with DsPSY1/2 and regulating the plastid development, which provides new insights into the molecular mechanism of massive accumulation of ß-carotene in D. salina.

DbMADS regulates carotenoid metabolism by repressing two carotenogenic genes in the green alga Dunaliella sp. FACHB-847.

MADS transcription factors are involved in the regulation of fruit development and carotenoid metabolism in plants. However, whether and how carotenoid accumulation is regulated by algal MADS are largely unknown. In this study, we first used functional complementation to confirm the functional activity of phytoene synthase from the lutein-rich Dunaliella sp. FACHB-847 (DbPSY), the key rate-limiting enzyme in the carotenoid biosynthesis. Promoters of DbPSY and DbLcyB (lycopene ß-cyclase) possessed multiple cis-acting elements such as light-, UV-B-, dehydration-, anaerobic-, and salt-responsive elements, W-box, and C-A-rich-G-box (MADS-box). Meanwhile, we isolated one nucleus-localized MADS transcription factor (DbMADS), belonging to type I MADS gene. Three carotenogenic genes, DbPSY, DbLcyB, and DbBCH (ß-carotene hydroxylase) genes were upregulated at later stages, which was well correlated with the carotenoid accumulation. In contrast, DbMADS gene was highly expressed at lag phase with low carotenoid accumulation. Yeast one-hybrid assay and dual-luciferase reporter assay demonstrated that DbMADS could directly bind to the promoters of two carotenogenic genes, DbPSY and DbLcyB, and repress their transcriptions. This study suggested that DbMADS may act as a negative regulator of carotenoid biosynthesis by repressing DbPSY and DbLcyB at the lag phase, which provide new insights into the regulatory mechanisms of carotenoid metabolism in Dunaliella.

Engineering the ß-Carotene Metabolic Pathway of Microalgae Dunaliella To Confirm Its Carotenoid Synthesis Pattern in Comparison To Bacteria and Plants.

Dunaliella salina is the most salt-tolerant eukaryote and has the highest ß-carotene content, but its carotenoid synthesis pathway is still unclear, especially the synthesis of lycopene, the upstream product of ß-carotene. In this study, DsGGPS, DsPSY, DsPDS, DsZISO, DsZDS, DsCRTISO, and DsLYCB genes were cloned from D. salina and expressed in Escherichia coli. A series of carotenoid engineering E. coli strains from phytoene to ß-carotene were obtained. ZISO was first identified from Chlorophyta, while CRTISO was first isolated from algae. It was found that DsZISO and DsCRTISO were essential for isomerization of carotenoids in photosynthetic organisms and could not be replaced by photoisomerization, unlike some plants. DsZDS was found to have weak beta cyclization abilities, and DsLYCB was able to catalyze 7,7',9,9'-tetra-cis-lycopene to generate 7,7',9,9'-tetra-cis-ß-carotene, which had not been reported before. A new carotenoid 7,7',9,9'-tetra-cis-ß-carotene, the beta cyclization product of prolycopene, was discovered. Compared with the bacterial-derived carotenoid synthesis pathway, there is higher specificity and greater efficiency of the carotenoid synthesis pathway in algae. This research experimentally confirmed that the conversion of phytoene to lycopene in D. salina was similar to that of plants and different from bacteria and provided a new possibility for the metabolic engineering of ß-carotene. IMPORTANCE The synthesis mode of all trans-lycopene in bacteria and plants is clear, but there are still doubts in microalgae. Dunaliella is the organism with the highest ß-carotene content, and plant-type and bacterial-type enzyme genes have been found in its carotenoid metabolism pathway. In this study, the entire plant-type enzyme gene was completely cloned into Escherichia coli, and high-efficiency expression was obtained, which proved that carotenoid synthesis of algae is similar to that of plants. In bacteria, CRT can directly catalyze 4-step continuous dehydrogenation to produce all trans-lycopene. In plants, four enzymes (PDS, ZISO, ZDS, and CRTISO) are involved in this process. Although a carotenoid synthetase similar to that of bacteria has been found in algae, it does not play a major role. This research reveals the evolutionary relationship of carotenoid metabolism in bacteria, algae, and plants and is of methodologically innovative significance for molecular evolution research.

Effects of Piperonyl Butoxide on the Accumulation of Lipid and the Transcript Levels of DtMFPα in Dunaliella tertiolecta.

As one of the sources of biodiesel, microalgae are expected to solve petroleum shortage. In this study, different concentrations of piperonyl butoxide were added to the culture medium to investigate their effects on the growth, pigment content, lipid accumulation, and content of carotenoids in Dunaliella tertiolecta. The results showed that piperonyl butoxide addition significantly decreased the biomass, chlorophyll content, and total carotenoid content but hugely increased the lipid accumulation. With the treatment of 150 ppm piperonyl butoxide combined with 8000 Lux light intensity, the final lipid accumulation and single-cell lipid content were further increased by 21.79 and 76.42% compared to those of the control, respectively. The lipid accumulation in D. tertiolecta is probably related to the increased expression of DtMFPα in D. tertiolecta under the action of piperonyl butoxide. The phylogenetic trees of D. tertiolecta and other oil-rich plants were constructed by multiple sequence alignment of DtMFPα, demonstrating their evolutionary relationship, and the tertiary structure of DtMFPα was predicted. In conclusion, piperonyl butoxide has a significant effect on lipid accumulation in D. tertiolecta, which provides valuable insights into chemical inducers to enhance biodiesel production in microalgae to solve the problem of diesel shortage.

A strategy to promote carotenoids production in Dunaliella bardawil by melatonin combined with photoinduction.

Microalgae are considered to be a very promising class of raw material for carotenoid production. In this study, melatonin (MLT), a widely used plant growth regulator, was added to the autotrophic medium of Dunaliella bardawil to explore its effects on the growth and pigment accumulation of Dunaliella bardawil. The results showed that the induction of exogenous MLT alone was not beneficial to the growth and pigment accumulation of Dunaliella bardawil, and the higher the concentration, the more obvious the inhibitory effect on the algal cells. Therefore, a strategy to promote carotenoid accumulation in Dunaliella bardawil by combining exogenous MLT and light induction was carried out. Under 4500 LUX light intensity, the content of zeaxanthin was significantly increased under exogenous MLT induction. In the 200 µg/mL, 300 µg/mL, and 400 µg/mL MLT-treated groups, the zeaxanthin single-cell content in the 300 µg/mL-treated group was as high as 0.38 ng/mL (0.17 ng/mL in the control group), which was 1.24-fold higher compared to the control. Under 9500 LUX light intensity, all carotenoids showed an increasing trend in all experimental groups, except for zeaxanthin, which showed a decreasing trend. The effect of 300 µg/mL showed the most obvious in the 200 µg/mL,300 µg/mL, and 400 µg/mL MLT treatment groups, where the lutein, α-carotene and ß-carotene contents were 1.24, 1.14 and 1.31 times higher than those of the control group, respectively. Overall, exogenous MLT at high light intensities had a significant effect on pigment accumulation in Dunaliella bardawil.

The expression pattern of ß-carotene ketolase gene restricts the accumulation of astaxanthin in Dunaliella under salt stress.

Dunaliella salina can accumulate a large amount of ß-carotene which is generally considered to be its terminal product of carotenoid metabolism. In this study, it was proved that D. salina has the ketolase (DsBKT) of catalyzing the synthesis of astaxanthin, the downstream products of ß-carotene. Therefore, the reason why D. salina does not synthesize astaxanthin is the purpose of this study. The enzymatic activity of DsBKT was detected by functional complementation assays in Escherichia coli, results showed that DsBKT had efficient ketolase activity toward ß-carotene and zeaxanthin to produce astaxanthin, indicating that there were complete astaxanthin-producing genes in Dunaliella. Unlike the induced expression of Lycopene cyclase (catalyzing ß-carotene synthesis) under salt stress, the expression of DsBKT was very low under both normal and stress conditions, which may be the main reason why D. salina cannot accumulate astaxanthin. On the contrary, with the astaxanthin-rich Haematococcus pluvialis as a control, its BKT gene was significantly upregulated under salt stress. Further study showed that DsBKT promoter had strong promoter ability and could stably drive the expression of ble-egfp in D. salina. Obviously, DsBKT promoter is not the reason of DsBKT not being expressed which may be caused by Noncoding RNA.

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We examined biomass characteristics and the potential driving factors of different forest types of Quercus spp. secondary forest in Hunan. A total of fifty plots were divided into five forest types: Castanopsis eyri - Rhododendron latoucheae mixed forest (CR), Fagus lucida - Fargesia spathacea mixed forest (FF), Lithocarpus glaber - Damnacanthus indicus + Camellia japonica mixed forest (LDC), C. eyri + Quercus serrata - R. latoucheae mixed forest (CQR), Cyclobalanopsis glauca - Camellia oleifera + R. latoucheae mixed forest (CCR). The biomass of understory vegetation was low in the five forest types, being smaller than 2.3 t·hm-2. There was no significant difference in the biomass of understory shrubs among the five forest types. The biomass of herbage layer in CR was significantly lower than that of the other four forest types. The factors affecting the biomass of understory vegetation varied in different forests types. In CR, biomass of herbaceous layer was negatively correlated with canopy and uniform angle index, whereas total understory biomass was positively correlated with opening degree index. In FF, biomass of shrub layer was negatively correlated with stand canopy density and uniform angle index, while herbaceous biomass and total understory biomass were positively correlated with stand closure. In LDC, herbaceous biomass was positively correlated with the stand aggregation index. In CQR, shrub biomass was negatively correlated with stand mingling index, while herbaceous biomass was positively related with stand density. In CCR, there was no significant correlation between stand structure and understory biomass. To adjust the understory biomass, we should first adjust the horizontal distribution pattern of stand and then adjust the degree of forest cover and tree species structure.

Functional Identification of Two Types of Carotene Hydroxylases from the Green Alga Dunaliella bardawil Rich in Lutein.

The salt-tolerant unicellular alga Dunaliella bardawil FACHB-847 can accumulate large amounts of lutein, but the underlying cause of massive accumulation of lutein is still unknown. In this study, genes encoding two types of carotene hydroxylases, i.e., ß-carotene hydroxylase (DbBCH) and cytochrome P450 carotenoid hydroxylase (DbCYP97s; DbCYP97A, DbCYP97B, and DbCYP97C), were cloned from D. bardawil. Their substrate specificities and enzyme activities were tested through functional complementation assays in Escherichia coli. It was showed that DbBCH could catalyze the hydroxylation of the ß-rings of both ß- and α-carotene, and displayed a low level of ε-hydroxylase. Unlike CYP97A from higher plants, DbCYP97A could not hydroxylate ß-carotene. DbCYP97A and DbCYP97C showed high hydroxylase activity toward the ß-ring and ε-ring of α-carotene, respectively. DbCYP97B displayed minor activity toward the ß-ring of α-carotene. The high accumulation of lutein in D. bardawil may be due to the multiple pathways for lutein biosynthesis generated from α-carotene with zeinoxanthin or α-cryptoxanthin as intermediates by DbBCH and DbCYP97s. Taken together, this study provides insights for understanding the underlying reason for high production of lutein in the halophilic green alga D. bardawil FACHB-847.

The bifunctional identification of both lycopene ß- and ε-cyclases from the lutein-rich Dunaliella bardawil.

The halophilic green alga Dunaliella bardawil FACHB-847 is rich in lutein and α-carotene, which has great potential for carotenoid production in open ponds. In this study, genes encoding lycopene ß- and ε-cyclases (DbLcyB and DbLcyE) from D. bardawil FACHB-847 were functionally identified by genetic complementation in E. coli. The bifunctional DbLcyB not only catalyzed the formation of both mono- and bi-cyclic ß-rings with a major ß-cyclase activity, but also possessed a weak ε-cyclase activity. In contrast, DbLcyE preferred to convert lycopene into monocyclic δ-carotene, and possessed a weak ß-monocyclase activity. Lutein and α-carotene were the prominent carotenoids in D. bardawil FACHB-847, which was in agreement with the result of genetic complementation of co-expression of DbLcyB and DbLcyE in E. coli with α-carotene as the prominent product. The bifunctional DbLcyB and DbLcyE may contribute to the high accumulation of α-carotene in D. bardawil FACHB-847. Interestingly, the accumulation of lutein in D. bardawil FACHB-847 was more sensitive to salt stress, while the accumulation of ß-carotene in D. salina CCAP 19/18 was induced by salt stress. In brief, the production of different carotenoid compositions from these two Dunaliella species can be induced by different growth conditions.

Sodium azide intervention, salinity stress and two-step cultivation of Dunaliella tertiolecta for lipid accumulation.

A two-step strategy was employed to culture Dunaliella tertiolecta, an oleaginous unicellular green alga, combined by the salt stress and sodium azide intervention, to observe their effects on its lipid accumulation. When the algae cultured at different salt concentrations reached the logarithmic growth phase, sodium azide was added. The results showed that the addition of sodium azide significantly increased the lipid content and had no significant effect on cell biomass. The lipid yield and single cell lipid content under 50 µM sodium azide increased by 10.4% and 21.7%. Under the two-step culture condition, combining of the treatment of 50 µM sodium azide and 2.5 M salt stress, the total lipid productivity and single-cell lipid content were 10% and 70.5% higher than that of the control. It seemed that sodium azide and salinity might have a synergistic effect on the lipid accumulation of D. tertiolecta. It can be concluded that sodium azide is an effective inducer of lipid accumulation in D. tertiolecta, and two-stage cultivation is a feasible way to improve lipid accumulation in microalgae.

Effects of triethylamine on the expression patterns of two G3PDHs and lipid accumulation in Dunaliella tertiolecta.

Glycerol-3-phosphate (G3P) is the important precursors for triacylglycerol synthesis, while glycerol-3-phosphate dehydrogenase (GPDH) determines the formation of G3P. In this study, two GDPH genes, Dtgdp1 and Dtgdp2 were isolated and identified from Dunaliella tertiolecta. The full-length Dtgdp1 and Dtgdp2 CDS were 2016 bp and 2094 bp, which encoded two putative protein sequences of 671 and 697 amino acids with predicted molecular weights of 73.64 kDa and 76.73 kDa, respectively. DtGDP1 and DtGDP2 both had a close relationship with those of algal and higher plants. DtGDP1 shared two conserved superfamily (A1 and A2) and four signature motifs (I-IV), and the DtGDP2 showed six signature domains (from motif I to VI) and DAO_C conserved family. Our previous work showed that the triethylamine intervention could greatly increase the triacylglycerol content (up to 80%) of D. tertiolecta. This study aims to investigate the effect of triethylamine on GPDH expression. Results showed that, when treated by triethylamine at 100 ppm and 150 ppm, the expression levels of Dtgdp1 and Dtgpd2 were increased to 5.121- and 56.964-fold compared with the control, respectively. Triethylamine seemed to enhance lipid metabolic flow by inducing the expressions of Dtgdp1 and Dtgdp2 to increase the lipid content, which provides a new insight into the desired pathway of lipid synthesis in algae through genetic engineering.

Comparative toxic effects of butylparaben sodium, sodium diacetate and potassium sorbate to Dunaliella tertiolecta and HL7702 cells.

Butylparaben sodium (BP), sodium diacetate (SDA) and potassium sorbate (PS) are safe and internationally recognized preservatives. The aim of this study is to further evaluate their toxicities using microalgae cells, and a comparison is made with their mammalian cell cytotoxicities. Unicellular Dunaliella tertiolecta, was employed to test the possible toxicities of BP, SDA and PS. The results show that the three preservatives have a negative effect on D. tertiolecta, as manifested by a strong decrease in chlorophyll and carotenoid content, viable algal cells, and superoxide dismutase (SOD) and catalase (CAT) activities. SDA and PS had small effects on the normal hepatocytes HL7702, but similar to that for MCF-10A cells, BP is toxic. The effective concentration (EC50) value for HL7702 is 215.97 mg L-1. It is concluded that BP, SDA and PS have low toxicities to D. tertiolecta under slightly alkaline conditions, while under acidic conditions, SDA has moderate toxicity and PS has high toxicity. The sensitivity of algal cells is higher than that of HL7702 cells under slightly alkaline conditions, and is even more sensitive under acidic conditions. D. tertiolecta can be used as a pre-screen for toxicity testing.

Lipid Accumulation Mechanisms in Auto- and Heterotrophic Microalgae.

Microalgae lipids have attracted great attention in the world as a result of their potential use for biodiesel productions. Microalgae are cultivated in photoautotrophic conditions in most cases, but several species are able to grow under heterotrophic conditions, in which microalgae are cultivated in the dark where the cell growth and reproduction are supported by organic carbons. This perspective is covering the related studies concerning the difference between hetero- and autotrophic cultivation of microalgae. The auto- and heterotrophic central carbon metabolic pathways in microalgae are described, and the catalyzing reactions of several key metabolic enzymes and their corresponding changes in the protein level are summarized. Under adverse environmental conditions, such as nutrient deprivation, microalgae have the ability to highly store energy by forming triacylglycerol (TAG), the reason for which is analyzed. In addition, the biosynthesis of fatty acids and TAGs and their difference between auto- and heterotrophic conditions are compared at the molecular level. The positive regulatory enzymes, such as glucose transporter protein, fructose-1,6-bisphosphate aldolase, and glycerol-3-phosphate dehydrogenase, and the negative regulation enzymes, such as triose phosphate isomerase, played a crucial role in the lipid accumulation auto- and heterotrophic conditions.

Implications of glycerol metabolism for lipid production.

Triacylglycerol (TAG) is an important product in oil-producing organisms. Biosynthesis of TAG can be completed through either esterification of fatty acids to glycerol backbone, or through esterification of 2-monoacylglycerol. This review will focus on the former pathway in which two precursors, fatty acid and glycerol-3-phosphate (G3P), are required for TAG formation. Tremendous progress has been made about the enzymes or genes that regulate the biosynthetic pathway of TAG. However, much attention has been paid to the fatty acid provision and the esterification process, while the possible role of G3P is largely neglected. Glycerol is extensively studied on its usage as carbon source for value-added products, but the modification of glycerol metabolism, which is directly associated with G3P synthesis, is seldom recognized in lipid investigations. The relevance among glycerol metabolism, G3P synthesis and lipid production is described, and the role of G3P in glycerol metabolism and lipid production are discussed in detail with an emphasis on how G3P affects lipid production through the modulation of glycerol metabolism. Observations of lipid metabolic changes due to glycerol related disruption in mammals, plants, and microorganisms are introduced. Altering glycerol metabolism results in the changes of final lipid content. Possible regulatory mechanisms concerning the relationship between glycerol metabolism and lipid production are summarized.