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.

Involvement of Transcription Factors and Regulatory Proteins in the Regulation of Carotenoid Accumulation in Plants and Algae.

Carotenoids are essential for photosynthesis and photoprotection in photosynthetic organisms, which are widely used in food coloring, feed additives, nutraceuticals, cosmetics, and pharmaceuticals. Carotenoid biofortification in crop plants or algae has been considered as a sustainable strategy to improve human nutrition and health. However, the regulatory mechanisms of carotenoid accumulation are still not systematic and particularly scarce in algae. This article focuses on the regulatory mechanisms of carotenoid accumulation in plants and algae through regulatory factors (transcription factors and regulatory proteins), demonstrating the complexity of homeostasis regulation of carotenoids, mainly including transcriptional regulation as the primary mechanism, subsequent post-translational regulation, and cross-linking with other metabolic processes. Different organs of plants and different plant/algal species usually have specific regulatory mechanisms for the biosynthesis, storage, and degradation of carotenoids in response to the environmental and developmental signals. In plants and algae, regulators such as MYB, bHLH, MADS, bZIP, AP2/ERF, WRKY, and orange proteins can be involved in the regulation of carotenoid metabolism. And many more regulators, regulatory networks, and mechanisms need to be explored. Our paper will provide a basis for multitarget or multipathway engineering for carotenoid biofortification in plants and algae.

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.

Genome-guided purification and characterization of polymyxin A1 from Paenibacillus thiaminolyticus SY20: A rarely explored member of polymyxins.

Polymyxin A1 was a rarely investigated member in the polymyxins family produced by Bacillus aerosporus. As a cyclic non-ribosomal lipopeptide, it was purified from Paenibacillus thiaminolyticus for the first time. The producing strain SY20 was screened from Chinese natural fermented bamboo shoots and identified as P. thiaminolyticus SY20 using 16S rRNA homology along with whole genome sequencing. The optimum incubation time was 32 h by the growth kinetics of antimicrobial agent production. The proteinaceous nature of antimicrobial agents was characterized according to the physicochemical properties of the cell-free supernatant. Subsequently, the active antimicrobial agent was purified from the supernatant using ammonium sulfate-graded precipitation, ion-exchange chromatography, and C18-H chromatography. The active agent was identified as polymyxin A1 with a molecular weight 1156.7 Da and antimicrobial activity mainly against Gram-negative bacteria. The molecular structure, a cyclic heptapeptide and a tripeptide side chain acylated by a fatty acid at the amino terminus, was elucidated using the combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), amino acid analysis, and whole genome mining tool. Meanwhile, the biosynthetic gene cluster of polymyxin A1 including five open reading frames (ORFs) was demonstrated in the genome. The compound should be further explored for its efficacy and toxicity in vivo to develop its application.

Protective Effect of Galangin Methylation Modification Based on Cell Imaging on Inflammatory Lung Injury and Its Molecular Mechanism.

Background: Recently, inflammation has become a major threat to human health. Studies have confirmed that some Chinese traditional medicine ingredients may effectively interfere with the expression of inflammatory mediators through epigenetic modification, showing a great potential of the application. Objective: To investigate the role of the PPAR/DNMT3A pathway in the reversal of galangin-mediated inflammatory lung injury, promote the development of new anti-inflammatory drugs, reduce the side effects of chemical synthetic drugs on the body, and prove the effectiveness and safety of galangin in inhibiting inflammatory response and injury. Methods: 120 rats were randomly divided into 6 groups: (Group 1) LPS group; (Group 2) LPS + galangin group; (Group 3) LPS + galangin + GW9662 group; (Group 4) LPS + galangin + DNMT3A siRNA group; (Group 5) LPS + galangin + siRNA negative group; (Group 6) control group. The model of inflammatory lung injury was established by intrathecal instillation of LPS in the first five groups and NS in the control group. SD survival rate was recorded every 24 hours after modeling, lasting for 168 hours. The lung tissues were taken 168 hours after the establishment of the model. The pathological morphology of lung tissue was observed after the staining under the light microscope, and the lung dry/wet weight ratio was calculated after drying. After NS was perfused into lung tissue, the lavage fluid was collected and the levels of IL-6 and TNF-a were measured by ELISA. The contents of PPAR, DNMT3A, phosphorylated p65, and ERK in monocytes were detected by the WB method, and the binding contents of p65 and AP-1 in the promoter regions of IL-6 and TNF-a genes were detected by the Chip-qPCR method. Results: Intraperitoneal injection of galangin could inhibit the synthesis of alveolar inflammatory factors (TFs) in the SD model of lung injury induced by LPS, reduce the degree of pathological injury of lung tissue, and improve the survival rate of the SD model. GW9662 can completely reverse the protective effect, while DNMT3A interference can only partially block its protective effect. In addition, galangin could significantly inhibit the LPS-induced expression of p65 and AP-1 in alveolar monocytes and their binding content in the promoter region of inflammatory genes by activating PPAR/DNMT3A pathway. GW9662 could completely reverse the inhibitory effect of galangin. DNMT3A interference could restore the binding content of transcription factors at the promoter of the inflammatory gene but had no significant effect on its synthesis. Conclusion: Galangin can interfere with the binding of transcription factors to inflammatory gene promoters through the methylation modification induced by PPAR/DNMT3A pathway, so as to inhibit the synthesis of inflammatory molecules and reverse inflammatory lung injury.

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.

Two Triacylglycerol Lipases Are Negative Regulators of Chilling Stress Tolerance in Arabidopsis.

Cold stress is one of the abiotic stress conditions that severely limit plant growth and development and productivity. Triacylglycerol lipases are important metabolic enzymes for the catabolism of triacylglycerols and, therefore, play important roles in cellular activities including seed germination and early seedling establishment. However, whether they play a role in cold stress responses remains unknown. In this study, we characterized two Arabidopsis triacylglycerol lipases, MPL1 and LIP1 and defined their role in cold stress. The expression of MPL1 and LIP1 is reduced by cold stress, suggesting that they may be negative factors related to cold stress. Indeed, we found that loss-of-function of MPL1 and LIP1 resulted in increased cold tolerance and that the mpl1lip1 double mutant displayed an additive effect on cold tolerance. We performed RNA-seq analysis to reveal the global effect of the mpl1 and lip1 mutations on gene expression under cold stress. The mpl1 mutation had a small effect on gene expression under both under control and cold stress conditions whereas the lip1 mutation caused a much stronger effect on gene expression under control and cold stress conditions. The mpl1lip1 double mutant had a moderate effect on gene expression under control and cold stress conditions. Together, our results indicate that MPL1 and LIP1 triacylglycerol lipases are negative regulators of cold tolerance without any side effects on growth in Arabidopsis and that they might be ideal candidates for breeding cold-tolerant crops through genome editing technology.

Quantitative proteomic analysis reveals the metabolic characteristics and adaptive mechanism of Cupriavidus oxalaticus T2 in the process of simultaneous nitrogen and phenol removal.

Phenol and ammonia in wastewater pose a serious threat to ecosystems and human health. However, the currently limited studies on single bacterium simultaneously removing phenol and nitrogen pollution have not fully elucidated the relevant metabolic mechanisms. The differences in proteomic profile after supplementing with phenol and ammonia for 6 and 24 h, respectively, were evaluated to explore the metabolic characteristics and adaptive mechanism of Cupriavidus oxalaticus T2 during the simultaneous removal process of phenol and nitrogen. Results revealed that a new potential phenol para-degradation pathway appeared in T2. Phenol induced changes in nitrogen metabolism, resulting in increased denitrification and decreased synthesis of glutamate from ammonia at 6 h. In addition, phenol exposure enhanced the expression of cytochrome oxidases with high oxygen affinity and increased ATP synthesis. The increase in chemotaxis and flagellar assembly was conducive to the uptake and utilization of phenol. The synthesis of lipoic acid and biotin was also promoted to resist phenol toxicity. Moreover, phenol triggered cellular stress response, thereby leading to the upregulation of anti-stress proteins, such as universal stress protein, iron­sulfur cluster protein, and chaperones. This study contributes to revealing the metabolic characteristics and adaptive mechanism of T2 during simultaneous nitrogen and phenol removal. SIGNIFICANCE: Phenol and ammonia often co-exist in wastewater, causing serious environmental problems. The information on the metabolic mechanism of simultaneously removing these two pollutants by bacteria is insufficient at present. Moreover, phenol is toxic to microbial and causes cells damage. Therefore, exploring the response mechanism of bacteria to phenol stress is conducive to understand their tolerance mechanism to aromatic compounds. In this study, the metabolic characteristics and adaptive mechanism of C. oxalaticus T2 during the simultaneous removal of phenol and nitrogen process were evaluated by comparing the proteome profiles at different stages. The results revealed the degradation pathways of phenol and nitrogen by strain T2. A variety of phenol response mechanisms were determined, including enhanced energy production, improved cell motility, increased the synthesis of lipoic acid and biotin, and combined action of multiple anti-stress proteins. This study is potentially useful to future phenol and nitrogen co-pollution bioremediation strategies and provides insight into the phenolic compound resistance mechanism in bacteria.

Lactiplantibacillus plantarum DMDL 9010 alleviates dextran sodium sulfate (DSS)-induced colitis and behavioral disorders by facilitating microbiota-gut-brain axis balance.

Previous studies have found that probiotic supplements can ameliorate mental behavioral disorders. This study investigated the effects of Lactiplantibacillus plantarum DMDL 9010 (LP9010) intake on the depression-like behavior induced by dextran sodium sulfate (DSS) and its possible mechanism. Male C57BL/6N mice were fed with DSS to establish the model of ulcerative colitis. LP9010 intake reduced the DSS-induced inflammatory response, and repaired intestinal barrier damage, as well as lightened depression-like behavior. LP9010 supplementation also inhibited neuroinflammation by up-regulating the levels of neurotransmitters, especially 5-HT, NE, DA, and 5-HIAA. Moreover, the intake of LP9010 reorganized the gut microbiome by increasing the relative abundance of Bacteroidetes and Firmicutes, and decreasing the relative abundance of Proteobacteria and Verrucomicrobia. Furthermore, treatment with LP9010 increased the levels of short-chain fatty acids, such as butyric acid and propionic acid. In conclusion, LP9010 intake was a promising probiotic intervention strategy for the prevention of colitis-induced behavioral disorders through the microbiota-gut-brain axis.

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.

Creatinine combined with light increases the contents of lutein and ß-carotene, the main carotenoids of Dunaliella bardawil.

Dunaliella bardawil, a unicellular green alga, can accumulate a large amount of lutein and ß-carotene under stresses. Using chemical inducers combined with abiotic stress to promote the accumulation of high value-added products such as lipids and carotenoids in microalgae has attracted more and more attention. In this study, creatinine was added into autotrophic medium to investigate its effects on the growth, chlorophyll content, and the ingredients and content of carotenoids in D. bardawil. The results showed that creatinine alone could significantly increase the biomass, chlorophyll and carotenoid contents of D. bardawil, among which the contents of lutein and ß-carotene were further increased, while the content of zeaxanthin was decreased. In order to further improve the content of the two carotenoids, different light intensities combined with creatinine have been adopted. Under 6.589 W/m2 light intensity, creatinine could effectively increase the production of lutein, zeaxanthin, α-carotene and ß-carotene. Compared with the control, the content of lutein increased by 46 % and the content of ß-carotene increased by 77 % when the concentration of creatinine was 500 µg/mL. In conclusion, creatinine can effectively improve the production lutein and ß-carotene in D. bardawil, which is more conducive under lower light intensity.

Stilbene glycoside upregulates SIRT3/AMPK to promotes neuronal mitochondrial autophagy and inhibit apoptosis in ischemic stroke.

BACKGROUND: Ischemic stroke, also known as cerebrovascular accident or cerebral stroke, occupies the first place in the world's top 10 causes of death, with high incidence, mortality and disability rates. OBJECTIVES: To investigate the effect of stilbene glycoside upregulated SIRT3/AMPK expression on neuronal mitochondrial autophagy and neuronal apoptosis in ischemic stroke. MATERIAL AND METHODS: The PC12 cells were cultured without serum to construct an ischemic neuron model. The cells were divided into 6 groups: normal group (untreated cells), model group (ischemic treated cells), TSG group (stilbene glycoside treatment), NC group (SIRT3 and AMPK negative control treatment), si-SIRT3 group (SIRT3 silencing treatment), TSG+si-SIRT3 group (joint treatment), and TSG+si-SIRT3+oe-AMPK group (joint treatment). Cell survival and the expression of related molecules were detected. RESULTS: Compared with normal group, the model group had significantly decreased cell survival rate, mitochondrial membrane potential, as well as the expression of Bcl-2, LC3II/I, P62, PINK1, Parkin, SIRT3, AMPK, and p-AMPK, while showing significantly increased proportion of apoptosis and the expression of caspase 3 and Bax. Compared with the model group, TSG treatment promoted cell survival rate and mitochondrial autophagy, and inhibited apoptosis, while SIRT3 silencing treatment reduced cell survival rate and mitochondrial autophagy, and increased apoptosis. The SIRT3 silencing could block the inhibitory effect of TSG on the apoptosis of ischemic PC12 cells and promote mitochondrial autophagy, and AMPK overexpression could save the apoptosis of ischemic PC12 cells caused by SIRT3 silencing, and promote mitochondrial autophagy. CONCLUSIONS: By promoting the expression of SIRT3/AMPK, TSG promotes mitochondrial autophagy in ischemic neurons and inhibits their apoptosis.

Regulation of carotenoid degradation and production of apocarotenoids in natural and engineered organisms.

Carotenoids are important precursors of a wide range of apocarotenoids with their functions including: hormones, pigments, retinoids, volatiles, and signals, which can be used in the food, flavors, fragrances, cosmetics, and pharmaceutical industries. This article focuses on the formation of these multifaceted apocarotenoids and their diverse biological roles in all living systems. Carotenoid degradation pathways include: enzymatic oxidation by specific carotenoid cleavage oxygenases (CCOs) or nonspecific enzymes such as lipoxygenases and peroxidases and non-enzymatic oxidation by reactive oxygen species. Recent advances in the regulation of carotenoid cleavage genes and the biotechnological production of multiple apocarotenoids are also covered. It is suggested that different developmental stages and environmental stresses can influence both the expression of carotenoid cleavage genes and the formation of apocarotenoids at multiple levels of regulation including: transcriptional, transcription factors, posttranscriptional, posttranslational, and epigenetic modification. Regarding the biotechnological production of apocarotenoids especially: crocins, retinoids, and ionones, enzymatic biocatalysis and metabolically engineered microorganisms have been a promising alternative route. New substrates, carotenoid cleavage enzymes, biosynthetic pathways for apocarotenoids, and new biological functions of apocarotenoids will be discussed with the improvement of our understanding of apocarotenoid biology, biochemistry, function, and formation from different organisms.

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.

Isolation, expression, and biochemical characterization: nitrite reductase from Bacillus cereus LJ01.

Biological remediation of toxic oxygen-containing anions such as nitrate that are common in the environment is of great significance. Therefore, it is necessary to understand the specific role of nitrate and nitrite reductase in the bioremediation process. Bacillus cereus LJ01, which was isolated from traditional Chinese soybean paste, effectively degraded nitrite (such as NaNO2) at 0-15 mmol L-1 in LB medium. Moreover, the nitrite-degrading active substance (ASDN) was isolated and purified from B. cereus LJ01. The nitrite-degrading activity of nitrite reductase (named LJ01-NiR) was 4004.89 U mg-1. The gene encoding the assimilation of nitrite reductase in B. cereus LJ01 was cloned and overexpressed in E. coli. The purified recombinant LJ01-NiR has a wide range of activities under temperature (20-60 °C), pH (6.5-8.0) and metal ions (Fe3+, Fe2+, Cu2+, Mn2+, and Al3+). Kinetic parameters of LJ01-NiR, including the values of K m and V max were 1.38 mM and 2.00 µmol g-1 min-1, respectively. The results showed that LJ01-NiR could degrade nitrite with or without an electron donor. In addition, sequence analysis revealed that LJ01-NiR was a ferredoxin-dependent nitrite reductase given the presence of conserved [Fe4-S4] cluster and heme-binding domain. The nitrite ion binds to the LJ01-NiR active site by forming three hydrogen bonds with the residues ASN72, ALA133 and ASN140. Due to its high nitrite-degrading activity, LJ01-NiR could potentially be used for environmental pollution treatment.

Structural characterization of a novel Lactarius volemus Fr. polysaccharide and its immunity activity in BALB/c mice.

Lactarius volemus Fr. has been regarded as a great edible medicinal fungal resource in China. In this study, L. volemus Fr. polysaccharide (LVP) with an average molecular weight of 16.842 kDa was obtained by water extraction. The structure of LVP was characterized to be mannan, and the linkages in the mannan were found to comprise the Manp, (1→4)-α-Man and (1→4,6)-α-Man. Furthermore, intraperitoneal administration of LVP increased the thymus, spleen and liver indices, dose-dependently. Additionally, LVP enhanced the immune response and the phagocytic activities. Pathological evaluations showed that LVP in mice increased the proliferation of red medullary lymphocytes (60-70%). Collectively, these results indicated that LVP might be a potential resource of raw material for further investigations of functional foods.

Transgenic microalgae as bioreactors.

Microalgae are unicellular organisms that act as the crucial primary producers all over the world, typically found in marine and freshwater environments. Most of them can live photo-autotrophically, reproduce rapidly, and accumulate biomass in a short period efficiently. To adapt to the uninterrupted change of the environment, they evolve and differentiate continuously. As a result, some of them evolve special abilities such as toleration of extreme environment, generation of sophisticated structure to adapt to the environment, and avoid predators. Microalgae are believed to be promising bioreactors because of their high lipid and pigment contents. Genetic engineering technologies have given revolutions in the microalgal industry, which decoded the secrets of microalgal genes, express recombinant genes in microalgal genomes, and largely soar the accumulation of interested components in transgenic microalgae. However, owing to several obstructions, the industry of transgenic microalgae is still immature. Here, we provide an overview to emphasize the advantage and imperfection of the existing transgenic microalgal bioreactors.