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Proteomic Analysis of the Chlorophyta Dunaliella New Strain AL-1 Revealed Global Changes of Metabolism during High Carotenoid Production.

ABSTRACT: The green microalgae Dunaliella genus is known for the production of high added value molecules. In this study, strain AL-1 was isolated from the Sebkha of Sidi El Hani (Sousse, Tunisia). This isolate was identified both morphologically and genetically via 18S rRNA gene sequence as a member of the genus Dunaliella. Strain AL-1 was found to be closely related to Dunaliella salina, Dunaliella quartolecta and Dunaliella polymorpha with more than 97% similarity. Response surface methodology was used to maximize carotenoid production by strain AL-1 by optimizing its growth conditions. The highest carotenoid content was obtained at salinity: 51, light intensity: 189.89 ?mol photons·m-2·s-1, and nitrogen: 60 mg·L-1. Proteomic profiling, using two-dimensional gel electrophoresis, was performed from standard and optimized cultures. We detected 127 protein spots which were significantly differentially expressed between standard and optimized cultures. Among them 16 protein spots were identified with mass spectrometry and grouped into different functional categories using KEGG (Kyoto Encyclopedia of Genes and Genomes) such as photosynthetic Calvin cycle, regulation/defense, energy metabolism, glycolysis, and cellular processes. The current study could be of great interest in providing information on the effect of stressful conditions in microalgae carotenoid production.

SUBMITTER: Ben Amor F

PROVIDER: S-EPMC5618432 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Proteomic Analysis of the Chlorophyta Dunaliella New Strain AL-1 Revealed Global Changes of Metabolism during High Carotenoid Production.

Ben Amor Faten F Elleuch Fatma F Ben Hlima Hajer H Garnier Matthieu M Saint-Jean Bruno B Barkallah Mohamed M Pichon Chantal C Abdelkafi Slim S Fendri Imen I

Marine drugs 20170920 9

The green microalgae Dunaliella genus is known for the production of high added value molecules. In this study, strain AL-1 was isolated from the Sebkha of Sidi El Hani (Sousse, Tunisia). This isolate was identified both morphologically and genetically via 18S rRNA gene sequence as a member of the genus Dunaliella. Strain AL-1 was found to be closely related to Dunaliella salina, Dunaliella quartolecta and Dunaliella polymorpha with more than 97% similarity. Re ...[more]

PMID: 28930152

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Project description:BackgroundThe genus Dunaliella (Class - Chlorophyceae) is widely studied for its tolerance to extreme habitat conditions, physiological aspects and many biotechnological applications, such as a source of carotenoids and many other bioactive compounds. Biochemical and molecular characterization is very much essential to fully explore the properties and possibilities of the new isolates of Dunaliella. In India, hyper saline lakes and salt pans were reported to bloom with Dunaliella spp. However, except for the economically important D. salina, other species are rarely characterized taxonomically from India. Present study was conducted to describe Dunaliella strains from Indian salinas using a combined morphological, physiological and molecular approach with an aim to have a better understanding on the taxonomy and diversity of this genus from India.ResultsComparative phenotypic and genetic studies revealed high level of diversity within the Indian Dunaliella isolates. Species level identification using morphological characteristics clearly delineated two strains of D. salina with considerable β-carotene content (>20 pg/cell). The variation in 18S rRNA gene size, amplified with MA1-MA2 primers, ranged between ~1800 and ~2650 base pairs, and together with the phylogeny based on ITS gene sequence provided a pattern, forming five different groups within Indian Dunaliella isolates. Superficial congruency was observed between ITS and rbcL gene phylogenetic trees with consistent formation of major clades separating Indian isolates into two distinct clusters, one with D. salina and allied strains, and another one with D. viridis and allied strains. Further in both the trees, few isolates showed high level of genetic divergence than reported previously for Dunaliella spp. This indicates the scope of more numbers of clearly defined/unidentified species/sub-species within Indian Dunaliella isolates.ConclusionPresent work illustrates Indian Dunaliella strains phenotypically and genetically, and confirms the presence of not less than five different species (or sub-species) in Indian saline waters, including D. salina and D. viridis. The study emphasizes the need for a combined morphological, physiological and molecular approach in the taxonomic studies of Dunaliella.

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.

Project description:Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

Dataset Information

Proteomic Analysis of the Chlorophyta Dunaliella New Strain AL-1 Revealed Global Changes of Metabolism during High Carotenoid Production.

Publications

Proteomic Analysis of the Chlorophyta Dunaliella New Strain AL-1 Revealed Global Changes of Metabolism during High Carotenoid Production.

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