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  • CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA .

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  • The substitution of synthetic food dyes with natural colorants continues to be assiduously pursued. The current list of natural carotenoid colorants consists of plant-derived annatto (bixin and norbixin), paprika (capsanthin and capsorubin), saffron (crocin), tomato and gac fruit lycopene, marigold lutein, and red palm oil (α- and β-carotene), along with microalgal β-carotene and astaxanthin and fungal β-carotene and lycopene. Potential microalgal sources are being sought, especially in relation to lutein, for which commercial plant sources are lacking. Research efforts, manifested in numerous reviews and research papers published in the last decade, have been directed to green extraction, microencapsulation/nanoencapsulation, and valorization of processing by-products. Extraction is shifting from conventional extraction with organic solvents to supercritical CO extraction and different types of assisted extraction. Initially intended for the stabilization of the highly degradable carotenoids, additional benefits of encapsulation have been demonstrated, especially the improvement of carotenoid solubility and bioavailability. Instead of searching for new higher plant sources, enormous effort has been directed to the utilization of by-products of the fruit and vegetable processing industry, with the application of biorefinery and circular economy concepts. Amidst enormous research activities, however, the gap between research and industrial implementation remains wide.

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  • Concomitant Production of Erythritol and β-Carotene by Engineered .

    While the expansion of the erythritol production industry has resulted in unprecedented production of yeast cells, it also suffers from a lack of effective utilization. β-Carotene is a value-added compound that can be synthesized by engineered . Here, we first evaluated the production performance of erythritol-producing yeast strains under two different morphologies and then successfully constructed a chassis with yeast-like morphology by deleting and genes. Subsequently, β-carotene synthesis pathway genes, and from were introduced to construct the β-carotene and erythritol coproducing strain ylmcc. The rate-limiting genes and were overexpressed to increase the β-carotene yield by 45.32-fold compared with the strain ylmcc. However, increased β-carotene accumulation led to prolonged fermentation time; therefore, transporter engineering through overexpression of and genes was used to alleviate fermentation delays. Using batch fermentation in a 3 L bioreactor, this engineered strain produced erythritol with production, yield, and productivity values of 171 g/L, 0.56 g/g glucose, and 2.38 g/(L·h), respectively, with a concomitant β-carotene yield of 47.36 ± 0.06 mg/g DCW. The approach presented here improves the value of erythritol-producing cells and offers a low-cost technique to obtain hydrophobic terpenoids.

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  • The intranasal route has been suggested as a promising alternative to improve the direct transport of molecules to the brain, avoiding the need to cross the blood-brain barrier (BBB). In this area, the use of lipid nanoparticles, namely solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), has been highlighted as a promising strategy to improve the treatment of neurodegenerative diseases. In this work, formulations containing SLN and NLC that were loaded with astaxanthin that was obtained from different sources (astaxanthin extract (AE) from the algae and pure astaxanthin (PA) from the fungi ) were prepared for nose-to-brain administration, and comparative in vitro experiments were performed to evaluate the biocompatibility of the formulations with nasal (RPMI 2650) and neuronal (SH-SY5Y) cells. Afterwards, the antioxidant activity of the formulations was evaluated for its potential neuroprotective effects, using different chemical aggressors. Finally, the cellular uptake of the astaxanthin was evaluated for the formulations that showed the greatest neuroprotection of the neuronal cells against chemical-induced damage. On the production day, all the formulations showed a particle size, a high encapsulation efficiency (EE), the presence of nanoparticles with a typical spherical shape, and a polydispersity index (PDI) and zeta potential (ZP) that are suitable for nose-to-brain administration. After three months of storage at room temperature, no significant changes were observed in the characterization parameters, predicting a good long-term stability. Furthermore, these formulations were shown to be safe with concentrations of up to 100 µg/mL in differentiated SH-SY5Y and RPMI 2650 cells. Regarding neuroprotection studies, the PA-loaded SLN and NLC formulations showed an ability to counteract some mechanisms of neurodegeneration, including oxidative stress. Moreover, when compared with the PA-loaded SLN, the PA-loaded NLC showed greater neuroprotective effects against the cytotoxicity induced by aggressors. In contrast, the AE-loaded SLN and NLC formulations showed no significant neuroprotective effects. Although further studies are needed to confirm these neuroprotective effects, the results of this study suggest that the intranasal administration of PA-loaded NLC may be a promising alternative to improve the treatment of neurodegenerative diseases.

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  • Trisporic acids are considered to be key regulators of carotenoid biosynthesis and sexual reproduction in zygomycetes, but the mechanisms underlying this regulation have not been fully elucidated.

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  • β-Carotene, as a kind of potent antioxidant compounds, has gained extensive attention. Blakeslea trispora, a filiform aerobic fungus, has been proposed as a natural source of β-carotene for commercial exploitation. However, it has not yet been investigated whether β-carotene extracted from Blakeslea trispora can attenuate oxidative stress, inflammatory, liver injury and immune damage of zebrafish (Danio rerio) exposed to copper sulfate (CuSO). In this study, we evaluated the effects of β-carotene on migration of GFP-labeled neutrophils, histological changes of liver, markers of oxidative, inflammatory cytokines and transaminase analysis, as well as the expression and activities of apoptosis, immune-related certain genes in zebrafish treated with different concentrations of β-carotene (0, 10, 20, 40 μg/mL) after exposure to CuSO. The results indicated that β-carotene reduced migration of neutrophils and released liver damage. What's more, β-carotene was found to reduce the index levels of oxidative stress response (HMOX-1, reactive oxygen species (ROS), NADPH, MDA), inflammatory factors (interleukine-1β (IL-1β), interleukine-6 (IL-6), interleukine-8 (IL-8), tumor necrosis factor-α (TNF-α)), liver function protein (AST, ALT) which increased by CuSO. β-Carotene also promoted the activities of SOD, GSH-Px, ACP, AKP and LZM and increased the protein of immune-related factors, IgM and IFN-γ after exposure to CuSO. Thus, our results demonstrate that β-carotene has an antioxidant, anti-inflammatory and hepatoprotective activity and participation in immunoregulation.

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  • Carotenoids (C40H56) including lycopene and beta-carotene are relatively strong antioxidants that provide benefits to human health. Here, we screened highly efficient crt variants from red yeasts to improve lycopene and beta-carotene production in Saccharomyces cerevisiae. We identified that crt variants from Sporidiobolus pararoseus TBRC-BCC 63403 isolated from rice leaf in Thailand exhibited the highest activity in term of lycopene and beta-carotene production in the context of yeast. Specifically, the phytoene desaturase SpCrtI possessed up to 4-fold higher in vivo activity based on lycopene content than the benchmark enzyme BtCrtI from Blakeslea trispora in our engineered WWY005 strain. Also, the geranylgeranyl pyrophosphate (GGPP) synthase SpCrtE, the bifunctional phytoene synthase-lycopene cyclase SpCrtYB, and SpCrtI when combined led to 7-fold improvement in beta-carotene content over the benchmark enzymes from Xanthophyllomyces dendrorhous in the laboratory strain CEN.PK2-1C. Sucrose as an alternative to glucose was found to enhance lycopene production in cells lacking GAL80. Lastly, we demonstrated a step-wise improvement in lycopene production from shake-flasks to a 5-L fermenter using the strain with GAL80 intact. Altogether, our study represents novel findings on more effective crt genes from Sp. pararoseus over the previously reported benchmark genes and their potential applications in scale-up lycopene production.

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  • Blakeslea trispora has great potential uses in industrial production because of the excellent capability of producing a large quantity of carotenoids. However, the mechanisms of light-induced carotenoid biosynthesis even the structural and regulatory genes in pathways remain unclear. In this paper, we reported the first transcriptome study in B. trispora in which we have carried out global survey of expression changes of genes participated in blue light response. We verified that the yield of β-carotene increased 3-fold when transferred from darkness to blue light for 24 h and the enhancement of transcription levels of carRA and carB presented a positive correlation with the increase in carotenoid production. RNA-seq analysis revealed that 1124 genes were upregulated and 740 genes were downregulated respectively after blue light exposure. Annotation through GO, KEGG, Swissprot, and COG databases showed 11119 unigenes compared well with known gene sequences, 5514 unigenes were classified into Gene Ontology, and 4675 unigenes were involved in distinct pathways. Among the blue light-responsive genes, 4 genes (carG1, carG3, carRA and carB) identified to function in carotenoid metabolic pathways were dominantly upregulated. We also discovered that 142 TF genes belonging to 45 different superfamilies showed significant differential expression (p≤ 0.05), 62 of which were obviously repressed by blue light. The detailed profile of transcription data will not only allow us to conduct further functional genomics study in B. trispora, but also enhance our understanding of potential metabolic pathway and regulatory network involved in light-regulated carotenoid synthesis.

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  • Although has been used for industrial production of β-carotene, the effects of light and oxidative stress on its synthesis have not been fully clarified. The present study focuses on the effects of light and reactive oxygen species (ROS) on carotenoid synthesis and their multilevel regulation in . Blue light significantly influenced the intracellular ROS levels, carotenoid contents, and transcription of carotenoid structural genes, while ROS levels were positively correlated with intracellular carotenoid contents and transcriptional levels of carotenoid structural genes. Blue light and ROS were both significant factors affecting carotenoid synthesis with a significant interaction between them. Irradiation by pulsed blue light and (or) addition of generating agents for active oxygen could partially compensate for the inhibition derived from the transcription inhibitor (dactinomycin) and translation inhibitor (cycloheximide) on the multilevel phenotype. Therefore, blue light and ROS act on the transcription and translation of carotenoid structural genes to promote the accumulation of carotenoid in .

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  • There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi.

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