Metabolic mechanism of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to sodium citrate treatment
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SUBMITTER: Pan X
PROVIDER: S-EPMC10992204 | biostudies-literature | 2023 Apr
REPOSITORIES: biostudies-literature
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Project description:Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one-hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two-hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild-type strain did not largely modify the carotenoid profile, indicating translational and/or post-translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild-type strain and the XdWC2-disrupted strain apparently improved the production of monocyclic carotenoid 3-hydroxy-3', 4'-didehydro-β, ψ-carotene-4-one (HDCO) rather than β-carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over-producing strains.
| S-EPMC9518987 | biostudies-literature
Project description:BackgroundThe yeast Xanthophyllomyces dendrorhous is one of the most promising and economically attractive natural sources of astaxanthin. The biosynthesis of this valuable carotenoid is a complex process for which the regulatory mechanisms remain mostly unknown. Several studies have shown a strong correlation between the carbon source present in the medium and the amount of pigments synthesized. Carotenoid production is especially low when high glucose concentrations are used in the medium, while a significant increase is observed with non-fermentable carbon sources. However, the molecular basis of this phenomenon has not been established.ResultsIn this work, we showed that glucose caused transcriptional repression of the three genes involved in the synthesis of astaxanthin from geranylgeranyl pyrophosphate in X. dendrorhous, which correlates with a complete inhibition of pigment synthesis. Strikingly, this regulatory response was completely altered in mutant strains that are incapable of synthesizing astaxanthin. However, we found that addition of ethanol caused the induction of crtYB and crtS gene expression and promoted de novo synthesis of carotenoids. The induction of carotenogenesis was noticeable as early as 24 h after ethanol addition.ConclusionFor the first time, we demonstrated that carbon source-dependent regulation of astaxanthin biosynthesis in X. dendrorhous involves changes at the transcriptional level. Such regulatory mechanism provides an explanation for the strong and early inhibitory effect of glucose on the biosynthesis of this carotenoid.
| S-EPMC3184065 | biostudies-literature
Project description:BackgroundThe yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid with high commercial interest. The proposed biosynthetic route in this organism is isopentenyl-pyrophosphate (IPP) --> geranyleranyl pyrophosphate (GGPP) --> phytoene --> lycopene --> beta-carotene --> astaxanthin. Recently, it has been published that the conversion of beta-carotene into astaxanthin requires only one enzyme, astaxanthin synthase or CrtS, encoded by crtS gene. This enzyme belongs to the cytochrome P450 protein family.ResultsIn this work, a crtR gene was isolated from X. dendrorhous yeast, which encodes a cytochrome P450 reductase (CPR) that provides CrtS with the necessary electrons for substrate oxygenation. We determined the structural organization of the crtR gene and its location in the yeast electrophoretic karyotype. Two transformants, CBSTr and T13, were obtained by deleting the crtR gene and inserting a hygromycin B resistance cassette. The carotenoid composition of the transformants was altered in relation to the wild type strain. CBSTr forms yellow colonies because it is unable to produce astaxanthin, hence accumulating beta-carotene. T13 forms pale colonies because its astaxanthin content is reduced and its beta-carotene content is increased.ConclusionIn addition to the crtS gene, X. dendrorhous requires a novel gene, crtR, for the conversion of beta-carotene to astaxanthin.
| S-EPMC2575211 | biostudies-literature
Project description:Astaxanthin is one of the most attractive carotenoids due to its high antioxidant activity and beneficial biological properties, while Xanthophyllomyces dendrorhous is one of its main microbial sources. Since astaxanthin is synthesized as a response to oxidative stress, several oxidative agents have been evaluated to increase X. dendrorhous astaxanthin yields. However, the extent of the stimulation is determined by the cellular damage caused by the applied oxidative agent. Phytohormones have also been reported as stimulants of astaxanthin biosynthesis acting directly on its metabolic pathway and indirectly promoting cellular resistance to reactive oxygen species. We reasoned that both oxidative agents and phytohormones lead to increased astaxanthin synthesis, but the latter could mitigate the drawbacks of the former. Thus, here, the stimulation on astaxanthin biosynthesis, as well as the cellular and transcriptional responses of wild type X. dendrorhous to phytohormones (6-benzylaminopurine, 6-BAP; abscisic acid, ABA; and indole-3-acetic acid, IAA), and oxidative agents (glutamate, menadione, H2O2, and/or Fe2+) were evaluated as a single or combined treatments. ABA and 6-BAP were the best individual stimulants leading to 2.24- and 2.60-fold astaxanthin biosynthesis increase, respectively. Nevertheless, the effect of combined 6-BAP and H2O2 led to a 3.69-fold astaxanthin synthesis increase (0.127 ± 0.018 mg astaxanthin/g biomass). Moreover, cell viability (> 82.75%) and mitochondrial activity (> 82.2%) remained almost intact in the combined treatment (6-BAP + H2O2) compared to control (< 52.17% cell viability; < 85.3% mitochondrial activity). On the other hand, mRNA levels of hmgR, idi, crtYB, crtR, and crtS, genes of the astaxanthin biosynthetic pathway, increased transiently along X. dendrorhous fermentation due to stimulations assayed in this study. KEY POINTS: • Combined 6-BAP and H2O2 is the best treatment to increase astaxanthin yields in X. dendrorhous. • 6-BAP preserves cell integrity under oxidative H2O2 stress conditions. • 6-BAP and H2O2 increase transcriptional responses of hmgR, idi, and crt family genes transiently.
| S-EPMC10803577 | biostudies-literature
Project description:Xanthophyllomyces dendrorhous is a promising source of natural astaxanthin due to its ability to accumulate high amounts of astaxanthin. This study showed that 6-benzylaminopurine (6-BAP) is an effective substrate that enhances cell biomass and astaxanthin accumulation in X. dendrorhous. In the current study, the biomass and astaxanthin content in X. dendrorhous were determined to be improved by 21.98% and 24.20%, respectively, induced by 6-BAP treatments. To further understand the metabolic responses of X. dendrorhous to 6-BAP, time-course metabolomics and gene expression levels of X. dendrorhous cultures with and without 6-BAP feeding were investigated. Metabolome analysis revealed that 6-BAP facilitated glucose consumption, promoted the glycolysis, suppressed the TCA cycle, drove carbon flux of acetyl-CoA into fatty acid and mevalonate biosynthesis, and finally facilitated the formation of astaxanthin. ROS analysis suggested that the antioxidant mechanism in X. dendrorhous can be induced by 6-BAP. Additionally, the process of 6-BAP significantly upregulated the expression of six key genes involved in pathways related to astaxanthin biosynthesis. This research demonstrates the metabolomic mechanism of phytohormone stimulation of astaxanthin production iNn X. dendrorhous and presents a new strategy to improve astaxanthin production to prevent the dilemma of choosing between accumulation of astaxanthin and cell biomass.
| S-EPMC7415379 | biostudies-literature
Project description:BackgroundXanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous.ResultsIn this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced.ConclusionsThis methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods.
| S-EPMC3852557 | biostudies-literature
Project description:The red yeast Xanthophyllomyces dendrorhous is an established platform for the synthesis of carotenoids. It was used for the generation of novel multi oxygenated carotenoid structures. This was achieved by a combinatorial approach starting with the selection of a β-carotene accumulating mutant, stepwise pathway engineering by integration of three microbial genes into the genome and finally the chemical reduction of the resulting 4,4'-diketo-nostoxanthin (2,3,2',3'-tetrahydroxy-4,4'-diketo-β-carotene) and 4-keto-nostoxanthin (2,3,2',3'-tetrahydroxy-4-monoketo-β-carotene). Both keto carotenoids and the resulting 4,4'-dihydroxy-nostoxanthin (2,3,4,2',3',4'-hexahydroxy-β-carotene) and 4-hydroxy-nostoxanthin (2,3,4,2'3'-pentahydroxy-β-carotene) were separated by high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry. Their molecular masses and fragmentation patterns allowed the unequivocal identification of all four carotenoids.
| S-EPMC5715969 | biostudies-literature
Project description:Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma, and at a concentration of 1000 mg/L of TiO2 for 105 days, the optimal strain JMU-ALE105 for astaxanthin production was obtained. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that the astaxanthin synthesis pathway, and fatty acid, pyruvate, and nitrogen metabolism pathway of the ALE105 strain were significantly upregulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content, reaching 8.36 mg/g. This study provides a technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma.One-sentence summaryA high-yield astaxanthin strain (ALE105) was obtained through TiO2 domestication, and its metabolic mechanism was analysed by transcriptomics, which combined with nitrogen source regulation to further improve astaxanthin yield.
| S-EPMC10448994 | biostudies-literature
Project description:BackgroundOccurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation in viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNAs are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed.ResultsThe complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totiviridae family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain.ConclusionsThree totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.
| S-EPMC3561658 | biostudies-literature
Project description:BackgroundThe yeast Xanthophyllomyces dendrorhous is used for the microbiological production of the antioxidant carotenoid astaxanthin. In this study, we established an optimal protocol for protein extraction and performed the first proteomic analysis of the strain ATCC 24230. Protein profiles before and during the induction of carotenogenesis were determined by two-dimensional polyacrylamide gel electrophoresis and proteins were identified by mass spectrometry.ResultsAmong the approximately 600 observed protein spots, 131 non-redundant proteins were identified. Proteomic analyses allowed us to identify 50 differentially expressed proteins that fall into several classes with distinct expression patterns. These analyses demonstrated that enzymes related to acetyl-CoA synthesis were more abundant prior to carotenogenesis. Later, redox- and stress-related proteins were up-regulated during the induction of carotenogenesis. For the carotenoid biosynthetic enzymes mevalonate kinase and phytoene/squalene synthase, we observed higher abundance during induction and/or accumulation of carotenoids. In addition, classical antioxidant enzymes, such as catalase, glutathione peroxidase and the cytosolic superoxide dismutases, were not identified.ConclusionsOur results provide an overview of potentially important carotenogenesis-related proteins, among which are proteins involved in carbohydrate and lipid biosynthetic pathways as well as several redox- and stress-related proteins. In addition, these results might indicate that X. dendrorhous accumulates astaxanthin under aerobic conditions to scavenge the reactive oxygen species (ROS) generated during metabolism.
| S-EPMC3224108 | biostudies-literature