Dataset Information

Comparative Analysis of ?-Carotene Production by Mucor circinelloides Strains CBS 277.49 and WJ11 under Light and Dark Conditions.

ABSTRACT: Carotenoids are natural potent antioxidants and free radical scavengers which are able to modulate the pathogenesis of some cancers and heart diseases in human, indicating their importance in being provided through the diet. Mucor circinelloides accumulates ?-carotene as the main carotenoid compound and has been used as a model organism in carotenogenic studies. In the present study, the potential of two M. circinelloides strains to accumulate ?-carotene was investigated under light and dark conditions. The results, which were quantitated by HPLC, showed that CBS 277.49 accumulated higher pigment in comparison to WJ11 under both conditions. Continuous illumination triggered the pigment accumulation up to 2.7-fold in strain CBS 277.49 and 2.2-fold in strain WJ11 in comparison to dark. The mRNA analysis of the four key genes involved in isoprenoid pathway by RT-qPCR showed higher transcriptional levels in CBS 277.49 as compared to WJ11, indicating that the pigment production metabolic machinery is more active in CBS 277.49 strain. A new scope for further research was established by this work for improved ?-carotene production in the high producing strain CBS 277.49.

SUBMITTER: Naz T

PROVIDER: S-EPMC7022308 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Comparative Analysis of β-Carotene Production by Mucor circinelloides Strains CBS 277.49 and WJ11 under Light and Dark Conditions.

Naz Tahira T Nosheen Shaista S Li Shaoqi S Nazir Yusuf Y Mustafa Kiren K Liu Qing Q Garre Victoriano V Song Yuanda Y

Metabolites 20200116 1

Carotenoids are natural potent antioxidants and free radical scavengers which are able to modulate the pathogenesis of some cancers and heart diseases in human, indicating their importance in being provided through the diet. Mucor circinelloides accumulates β-carotene as the main carotenoid compound and has been used as a model organism in carotenogenic studies. In the present study, the potential of two M. circinelloides strains to accumulate β-carotene was investigated under ligh ...[more]

PMID: 31963282

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Project description:BackgroundMucor circinelloides WJ11 is a high-lipid producing strain and an excellent producer of γ-linolenic acid (GLA) which is crucial for human health. We have previously identified genes that encode for AMP-activated protein kinase (AMPK) complex in M. circinelloides which is an important regulator for lipid accumulation. Comparative transcriptional analysis between the high and low lipid-producing strains of M. circinelloides showed a direct correlation in the transcriptional level of AMPK genes with lipid metabolism. Thus, the role of Snf-β, which encodes for β subunit of AMPK complex, in lipid accumulation of the WJ11 strain was evaluated in the present study.ResultsThe results showed that lipid content of cell dry weight in Snf-β knockout strain was increased by 32 % (from 19 to 25 %). However, in Snf-β overexpressing strain, lipid content of cell dry weight was decreased about 25 % (from 19 to 14.2 %) compared to the control strain. Total fatty acid analysis revealed that the expression of the Snf-β gene did not significantly affect the fatty acid composition of the strains. However, GLA content in biomass was increased from 2.5 % in control strain to 3.3 % in Snf-β knockout strain due to increased lipid accumulation and decreased to 1.83 % in Snf-β overexpressing strain. AMPK is known to inactivate acetyl-CoA carboxylase (ACC) which catalyzes the rate-limiting step in lipid synthesis. Snf-β manipulation also altered the expression level of the ACC1 gene which may indicate that Snf-β control lipid metabolism by regulating ACC1 gene.ConclusionsOur results suggested that Snf-β gene plays an important role in regulating lipid accumulation in M. circinelloides WJ11. Moreover, it will be interesting to evaluate the potential of other key subunits of AMPK related to lipid metabolism. Better insight can show us the way to manipulate these subunits effectively for upscaling the lipid production. Up to our knowledge, it is the first study to investigate the role of Snf-β in lipid accumulation in M. circinelloides.

Project description:The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of -4.87 kcal/mol to citric acid, while -3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.

Project description:BackgroundCarotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as β-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce β-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate β-carotene, possesses a vast collection of genetic tools with which to manipulate its genome.ResultsThis work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of β-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of β-carotene resulted in a further increase in β-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of β-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA.ConclusionsThe use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of β-carotene and are candidates for further improvements in biotechnological β-carotene production.

Dataset Information

Comparative Analysis of ?-Carotene Production by Mucor circinelloides Strains CBS 277.49 and WJ11 under Light and Dark Conditions.

Publications

Comparative Analysis of β-Carotene Production by <i>Mucor circinelloides</i> Strains CBS 277.49 and WJ11 under Light and Dark Conditions.

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