Quantitative analysis on photon numbers received per cell for triggering β-carotene accumulation in Dunaliella salina
Ontology highlight
ABSTRACT:
SUBMITTER: Xi Y
PROVIDER: S-EPMC10992135 | biostudies-literature | 2021 Oct
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.
| S-EPMC8185118 | biostudies-literature
Project description:Strains of Dunaliella salina microalgae are of considerable research and industrial interest because they hyper-accumulate β-carotene as well as produce high-quality protein. To explore the co-production of valuable compounds in D. salina, this study compared the production of β-carotene, phytoene and amino acids in two strains cultivated under white, red or blue light until no further nitrogen was available. D. salina DF15 (CCAP 19/41 (PLY DF15)) produced more than 12% β-carotene (ash-free dry weight (AFDW) basis), and red light triggered the production of 9-cis β-carotene at a 9-cis/all-trans β-carotene ratio of 1.5. Phytoene production was also evident in D. salina DF15 under all conditions, particularly under blue light. However, the profile of essential amino acids (EAAs) and calculation of the essential amino acid index (EAAI) was less than ideal in terms of protein quality, for both strains. Umami compounds, quantified as monosodium glutamate (MSG) equivalents, indicated a higher equivalent umami concentration (EUC) in D. salina DF15 under red light (3.2 g MSG/100 g AFDW) than in D. salina CCAP19/30. Overall, D. salina DF15 demonstrates valuable traits for further exploration and product optimisation.
| S-EPMC8617983 | biostudies-literature
Project description:Batch growth and β-carotene production of Dunaliella salina CCAP19/18 was investigated in flat-plate gas-lift photobioreactors with a light path of 2 cm, operated in physically simulated outdoor conditions. Dunaliella salina CCAP19/18 showed robust growth with respect to pH 8.0-9.0 and 15-35°C at increasing salinity, simulating the evaporation of open photobioreactors. The highest β-carotene concentration of 25 mg L-1 (3 mg gCDW -1) was observed in batch processes at pH 8.5, 15-30°C and increasing salinity up to 110 g L-1, simulating a typical Mediterranean summer climate. Intracellular β-carotene accumulation of D. salina CCAP19/18 was shown to be independent of light availability, although nutrient limitation (K2HPO4, MgSO4, and/or ammonium ferric citrate) seems to enable stable β-carotene content in the algal cells despite increasing cell densities in the photobioreactor. Fully controlled, lab-scale photobioreactors simulating typical climate conditions of any region of interest are valuable tools for enabling a realistic characterization of microalgae on a laboratory scale, for production processes projected in open photobioreactor systems (e.g. thin-layer cascade photobioreactors).
| S-EPMC7923581 | biostudies-literature
Project description:The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially for production of beta-carotene (=pro-vitamin A). The presented draft genome of the genuine strain CCAP19/18 will allow investigations into metabolic processes involved in regulation of stress responses, including carotenogenesis and adaptations to life in high-salinity environments.
| S-EPMC5658486 | biostudies-literature
Project description:Carotenoids, which are natural pigments found abundantly in wide-ranging species, have diverse functions and high industrial potential. The carotenoid biosynthesis pathway is very complex and has multiple branches, while the accumulation of certain metabolites often affects other metabolites in this pathway. The DsLCYB gene that encodes lycopene cyclase was selected in this study to evaluate β-carotene production and the accumulation of β-carotene in the alga Dunaliella salina. Compared with the wild type, the transgenic algal species overexpressed the DsLCYB gene, resulting in a significant enhancement of the total carotenoid content, with the total amount reaching 8.46 mg/g for an increase of up to 1.26-fold. Interestingly, the production of α-carotene in the transformant was not significantly reduced. This result indicated that the regulation of DsLCYB on the metabolic flux distribution of carotenoid biosynthesis is directional. Moreover, the effects of different light-quality conditions on β-carotene production in D. salina strains were investigated. The results showed that the carotenoid components of β-carotene and β-cryptoxanthin were 1.8-fold and 1.23-fold higher than that in the wild type under red light stress, respectively. This suggests that the accumulation of β-carotene under red light conditions is potentially more profitable.
| S-EPMC9843872 | biostudies-literature
Project description: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 β-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 β-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 β-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 β-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 β-carotene in D. salina.
| S-EPMC10269666 | biostudies-literature
Project description:To study the relationship between β-carotene synthesis and geranylgeranyl pyrophosphate synthase (GGPS) activity, 15 species of Dunaliella were used to determine the changes in photosynthetic pigment contents, chlorophyll fluorescence parameters, β-carotene content, and GGPS activity. By observing the morphology and size of 15 species of Dunaliella, D8 has the largest individual algal cell and D9 has the smallest individual. Growth was relatively slow during days one through seven. After about eight days, the cells entered the logarithmic growth period and grew rapidly to a high density. After about 45 days, they entered a mature period, and growth slowed down. The contents of chlorophyll, carotenoids, and β-carotene increased during growth. D1 has the highest accumulation of β-carotene, and GGPS enzyme activity has a positive linear relationship with the β-carotene synthesis content. Phylogenetic analysis showed that the GGPS proteins of the 15 species were highly homologous, and the GGPS protein was not part of the membrane.
| S-EPMC8747272 | biostudies-literature
Project description:Vitamin B12 is one of nature's complex metabolite which is industrially produced using certain bacteria. Algae could be an alternative source of vitamin B12 and in this study, vitamin B12 from a halotolerant green alga, Dunaliella salina V-101 was purified and characterized. The extract of Dunaliella was purified by passing through Amberlite XAD-2 and EASI-extract vitamin B12 immunoaffinity column. The total vitamin B12 content in purified sample fractions was 42 ± 2 μg/100 g dry weight as determined by the chemiluminescence method which was almost close to 49 ± 2 μg/100 g dry weight as estimated by microbiological method. Further quantification of total vitamin B12 using gold nanoparticle (AUNPs) based aptamer showed 40 ± 0.8/100 g dry weight. There was a good correlation among all the methods of quantification. Adenosylcobalamin, a form of vitamin B12 which is a cofactor for methylmalonyl CoA mutase was identified by HPLC. Upon quantification, Dunaliella was found to contain 34 ± 4 μg of adenosylcobalamin for 100 g dry biomass. Authenticity of adenosylcobalmin was confirmed by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) studies.
| S-EPMC4711437 | biostudies-literature
Project description:Green algae offer sustainable, clean and eco-friendly energy resource. However, production efficiency needs to be improved. Increasing cellular lipid levels by nitrogen depletion is one of the most studied strategies. Despite this, the underlying physiological and biochemical mechanisms of this response have not been well defined. Algae species adapted to hypersaline conditions can be cultivated in salty waters which are not useful for agriculture or consumption. Due to their inherent extreme cultivation conditions, use of hypersaline algae species is better suited for avoiding culture contamination issues. In this study, we identified a new halophilic Dunaliella salina strain by using 18S ribosomal RNA gene sequencing. We found that growth and biomass productivities of this strain were directly related to nitrogen levels, as the highest biomass concentration under 0.05 mM or 5 mM nitrogen regimes were 495 mg/l and 1409 mg/l, respectively. We also confirmed that nitrogen limitation increased cellular lipid content up to 35% under 0.05 mM nitrogen concentration. In order to gain insight into the mechanisms of this phenomenon, we applied fluorometric, flow cytometric and spectrophotometric methods to measure oxidative stress and enzymatic defence mechanisms. Under nitrogen depleted cultivation conditions, we observed increased lipid peroxidation by measuring an important oxidative stress marker, malondialdehyde and enhanced activation of catalase, ascorbate peroxidase and superoxide dismutase antioxidant enzymes. These observations indicated that oxidative stress is accompanied by increased lipid content in the green alga. In addition, we also showed that at optimum cultivation conditions, inducing oxidative stress by application of exogenous H2O2 leads to increased cellular lipid content up to 44% when compared with non-treated control groups. Our results support that oxidative stress and lipid overproduction are linked. Importantly, these results also suggest that oxidative stress mediates lipid accumulation. Understanding such relationships may provide guidance for efficient production of algal biodiesels.
| S-EPMC3961284 | biostudies-literature
Project description:Illumina HiseqTM2000 RNA-sequencing of Dunaliella salina under nitrogen deprivation condition was performed.
2019-02-23 | GSE97378 | GEO