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Liver cancer is one of the most frequent causes of cancer-related deaths worldwide. The current study sought to screen, for the first time, a Ficus carica variety from the Shandong province of China against hepatocellular (HepG2) cells. Crude extracts of F. carica with various polarities were prepared and screened against HepG2 cells. The acetone extract of the leaf (FLA) exhibited significant antiproliferative effects against HepG2 cells (IC50 = 0.179 mg/mL) while producing the least toxicity on normal human umbilical vein endothelial cells (HUVECs). Biochemical evidence of apoptosis was confirmed by staining with DAPI and AO/EB. FLA also exhibited moderate anti-colonization potential against the population of HepG2 cells. It selectively arrested the cell cycle of HepG2 cells at the S phase and exerted anticancer activity by significantly increasing the reactive oxygen species (1.59 fold) inside the cells, which subsequently resulted in loss of the mitochondrial membrane potential as revealed by JC-1 probe assay. FLA downregulated the expression of tumor-promoter Transcription Factor (TP53), anti-apoptotic proteins (Bcl-2), and cell cycle regulatory kinases (CDK1, 5, 9) with respect to untreated cells for 24 h, 48 h, and 72 h. High-performance liquid chromatography (HPLC) of FLA revealed the presence of gallic acid and quercetin as major antiproliferative compounds along with trace amounts of rutin, luteolin, apigenin, benzoic acid, and β-sitosterol. Notably, our study showed FLA operating via novel mechanisms involving reactive oxygen species (ROS) generation and CDKs involvement in HepG2 cells. Thus, it suggests that FLA may be a potential source of new drugs in the treatment of liver cancer.

Background Mucor 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. Results The 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. Conclusions Our 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.

AMP-activated protein kinase (AMPK) is an important regulator for lipid accumulation, potentially known to have an inhibitory role in lipid synthesis. It inactivates acetyl-CoA carboxylase (ACC), an important regulatory enzyme required for lipid synthesis. However, in Mucor circinelloides, AMPK and its association with lipid accumulation has not been studied yet. To identify AMPK genes in M. circinelloides and to compare their expression levels in high and low lipid-producing strains of M. circinelloides to predict the possible roles of AMPK in lipid metabolism and to select candidate genes for further studies to enhance lipid accumulation. Two genes for α-subunit, one for β-subunit and six for γ-subunit were identified and annotated. Bioinformatic analysis confirmed the presence of typical conserved domains in these genes. Furthermore, transcriptional profiling displayed marked differences in expression kinetics of subunits among the selected strains. The expression of AMPK genes decreased rapidly in WJ11, high lipid producer strain during the lipid accumulation phase while contrasting profile of expression was observed in CBS 277.49, low lipid producer strain. The present study has shown the association of AMPK genes with lipid metabolism at the transcriptional level. The involvement of Snf-α1, Snf-α2, Snf-β, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

10 pags., 5 figs., 2 tabs., Stearidonic Acid (SDA; 18:4, n-3) is a ω-3 polyunsaturated fatty acid which is nutritionally important and has pharmaceutical applications. Hence, scientists are trying to construct SDA producing oleaginous microorganisms by genetic modification. Two enzymes, Delta-6 Desaturase (D6D) and ω-3 desaturase catalyze the reactions to produce SDA from Linoleic Acid (LA; 18:2, n-6). But the key enzyme, ω-3 desaturase converts LA to ALA and GLA to SDA is absent in high lipid accumulating Mucor circinelloides WJ11, retarding its ability to produce SDA. Therefore, in this study, we overexpressed fad3 gene from Arabidopsis thaliana in M. circinelloides WJ11 to produce SDA. Overexpression of fad3 gene in M. circinelloides WJ11 resulted in the production of 340 mg/l SDA. This research opened a new opportunity to make use of this fungus for industrial production of SDA., This work was supported by the National Natural Science Foundation of China (Grant Nos. 31670064 and 31972851), Natural Science Foundation of Shandong province (ZR2018BC058) Science and Technology Project of Shandong College (J16LE20 and J17KA133), Taishan Industry Leading Talent Project and starting grant from Shandong University of Technology.

11 pags, 4 figs, 1 tab. -- he Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnut.2021.756218/full#supplementary-materia, Canthaxanthin is a reddish-orange xanthophyll with strong antioxidant activity and higher bioavailability than carotenes, primarily used in food, cosmetics, aquaculture, and pharmaceutical industries. The spiking market for natural canthaxanthin promoted researchers toward genetic engineering of heterologous hosts for canthaxanthin production. Mucor circinelloides is a dimorphic fungus that produces β-carotene as the major carotenoid and is considered as a model organism for carotenogenic studies. In this study, canthaxanthin-producing M. circinelloides strain was developed by integrating the codon-optimized β-carotene ketolase gene (bkt) of the Haematococcus pluvialis into the genome of the fungus under the control of strong promoter zrt1. First, a basic plasmid was constructed to disrupt crgA gene, a negative regulator of carotene biosynthesis resulted in substantial β-carotene production, which served as the building block for canthaxanthin by further enzymatic reaction of the ketolase enzyme. The genetically engineered strain produced a significant amount (576 ± 28 μg/g) of canthaxanthin, which is the highest amount reported in Mucor to date. Moreover, the cell dry weight of the recombinant strain was also determined, producing up to more than 9.0 g/L, after 96 h. The mRNA expression level of bkt in the overexpressing strain was analyzed by RT-qPCR, which increased by 5.3-, 4.1-, and 3-folds at 24, 48, and 72 h, respectively, compared with the control strain. The canthaxanthin-producing M. circinelloides strain obtained in this study provided a basis for further improving the biotechnological production of canthaxanthin and suggested a useful approach for the construction of more valuable carotenoids, such as astaxanthin., This work was supported by the National Natural Science Foundation of China (Grant Nos. 31670064 and 31972851), the Tai Shan Industrial Experts Program tscy 20160101, and the Shandong provincial key technology R&D plan (2018GNC110039, 2018GSF121013).

Continuous decline of earth’s natural resources and increased use of hazardous chemical fertilizers pose a great concern for the future of agriculture. Biofertilizers are a promising alternative to hazardous chemical fertilizers and are gaining importance for attaining sustainable agriculture. Biofertilizers play a key role in increasing crop yield and maintaining long-term soil fertility, which is essential for meeting global food demand. Microbes can interact with the crop plants and enhance their immunity, growth, and development. Nitrogen, phosphorous, potassium, zinc, and silica are the essential nutrients required for the proper growth of crops, but these nutrients are naturally present in insolubilized or complex forms. Certain microorganisms render them soluble and make them available to the plants. The potential microbes, their mode of action, along with their effect on crops, are discussed in this review. Biofertilizers, being cost effective, non-toxic, and eco-friendly, serve as a good substitute for expensive and harmful chemical fertilizers. The knowledge gained from this review can help us to understand the importance of microbes in agriculture and the ways to formulate these microbes as biofertilizers for sustainable crop production.

Concerns about global warming, fossil-fuel depletion, food security, and human health have promoted metabolic engineers to develop tools/strategies to overproduce microbial functional oils directly from renewable resources. Medium-chain fatty acids (MCFAs, C8&ndash, C12) have been shown to be important sources due to their diverse biotechnological importance, providing benefits ranging from functional lipids to uses in bio-fuel production. However, oleaginous microbes do not carry native pathways for the production of MCFAs, and therefore, diverse approaches have been adapted to compensate for the requirements of industrial demand. Mucor circinelloides is a promising organism for lipid production (15&ndash, 36% cell dry weight, CDW) and the investigation of mechanisms of lipid accumulation, however, it mostly produces long-chain fatty acids (LCFAs). To address this challenge, we genetically modified strain M. circinelloides MU758, first by integrating heterologous acyl-ACP thioesterase (TE) into fatty acid synthase (FAS) complex and subsequently by modifying the &beta, oxidation pathway by disrupting the acyl-CoA oxidase (ACOX) and/or acyl-CoA thioesterase (ACOT) genes with a preference for medium-chain acyl-CoAs, to elevate the yield of MCFAs. The resultant mutant strains (M-1, M-2, and M-3, respectively) showed a significant increase in lipid production in comparison to the wild-type strain (WT). MCFAs in M-1 (47.45%) was sharply increased compared to the wild type strain (2.25%), and it was further increased in M-2 (60.09%) suggesting a negative role of ACOX in MCFAs production. However, MCFAs in M-3 were much decreased compared to M-1,suggesting a positive role of ACOT in MCFAs production. The M-2 strain showed maximum lipid productivity (~1800 milligram per liter per day or mg/L.d) and MCFAs productivity (~1100 mg/L.d). Taken together, this study elaborates on how the combination of two multidimensional approaches, TE gene over-expression and modification of the &beta, oxidation pathway via substantial knockout of specific ACOX gene, significantly increased the production of MCFAs. This synergistic approach ultimately offers a novel opportunity for synthetic/industrial biologists to increase the content of MCFAs.

In the present study, the impact of eight phytohormones from six different classes on the growth, lipid and docosahexaenoic acid (DHA) biosynthetic capacity of Aurantiochytrium sp. SW1 (SW1) was evaluated. Kinetin (KIN), jasmonic acid (JA) and gibberellic acid (GA) significantly enhanced the growth and DHA production of SW1 by 16%&ndash, 28% and 66%&ndash, 84% in comparison to the control, respectively. The synergistic effect of these three phytohormones, evaluated by the response surface methodology (RSM), showed that a combination of 3.6 mg/L GA, 2.0 mg/L KIN and 20.0 mg/L JA further increased the growth and DHA production of SW1 by 16% to 28% and 22% to 36%, respectively, in comparison to the individual supplementation. The synergistic effect of these phytohormones was also shown to be time-dependent, where feeding at 24 h of cultivation led to 15%, 26% and 35% further increments in the biomass, lipid and DHA production in comparison to that of 0 h, respectively. The determination of stress markers, antioxidant enzymes and key enzymes involved in fatty acid biosynthesis aided to elucidate the potential mechanism underlying the improvement of growth and DHA production by SW1 at various times of feeding. Supplementation with the phytohormones at 24 h exhibited the maximum impact on reducing the level of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as augmented the antioxidants (superoxide dismutase and catalase) and key metabolic enzymes involved in lipogenesis (malic, glucose-6-phosphate dehydrogenase and ATP-citrate lyase) in comparison to the control and other time points. This study signifies the potential application of phytohormones for improving the growth, lipid and DHA production in Aurantiochytrium spp.

Human liver cancer has emerged as a serious health concern in the world, associated with poorly available therapies. The Berberis genus contains vital medicinal plants with miraculous healing properties and a wide range of bioactivities. In this study, different crude extracts of B. lycium Royle were prepared and screened against Human Hepatocarcinoma (HepG2) cell lines. The water/ethanolic extract of B. lycium Royle (BLE) exhibited significant antiproliferative activity against the HepG2 cancer cell line with an IC50 value of 47 μg/mL. The extract decreased the clonogenic potential of HepG2 cells in a dose-dependent manner. It induced apoptotic cell death in HepG2 cells that were confirmed by cytometric analysis and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by the loss of mitochondrial membrane potential. The mechanism of apoptosis was further confirmed by gene expression analysis using RT-qPCR that revealed the decline in Bcl-2 independent of p53 mRNA and a rise in CDK1 while downregulating CDK5, CDK9, and CDK10 mRNA levels at 48 h of BLE treatment. The most active fraction was subjected to HPLC which indicated the presence of berberine (48 μg/mL) and benzoic acid (15.8 μg/mL) as major compounds in BLE and a trace amount of luteolin, rutin, and gallic acid. Our study highlighted the importance of the most active BLE extract as an excellent source of nutraceuticals against Human Hepatocarcinoma that can serve as an herbal natural cure against liver cancer.

Carotenoids produced by microbial sources are of industrial and medicinal importance due to their antioxidant and anticancer properties. In the current study, optimization of β-carotene production in M. circinelloides strain 277.49 was achieved using response surface methodology (RSM). Cerulenin and ketoconazole were used to inhibit fatty acids and the sterol biosynthesis pathway, respectively, in order to enhance β-carotene production by diverting metabolic pool towards the mevalonate pathway. All three variables used in screening experiments were found to be significant for the production of β-carotene. The synergistic effect of the C/N ratio, cerulenin, and ketoconazole was further evaluated and optimized for superior β-carotene production using central composite design of RSM. Our results found that the synergistic combination of C/N ratios, cerulenin, and ketoconazole at different concentrations affected the β-carotene productions significantly. The optimal production medium (std. order 11) composed of C/N 25, 10 μg/mL cerulenin, and 150 mg/L ketoconazole, producing maximum β-carotene of 4.26 mg/L (0.43 mg/g) which was 157% greater in comparison to unoptimized medium (1.68 mg/L, 0.17 mg/g). So, it was concluded that metabolic flux had been successfully redirected towards the mevalonate pathway for enhanced β-carotene production in CBS 277.49.

This study aimed at increasing the stability of heat-labile and pH-sensitive microbial antioxidants by the microencapsulation. Microbial antioxidants from Mucor circinelloides were microencapsulated. The physico-chemical and powder flowing properties of resulting microcapsules were evaluated. The initial safety studies were evaluated by in vivo acute oral toxicity tests. The bio-accessibility of powders vs. extracts was analyzed in in vitro digestion models with further application of microcapsules to model food system. Physico-chemical properties were significantly different (p 0.05) in powder flowing properties. The microencapsulation of extract with 5% whey protein hydrogels (WPHG) + 5% pectin (TA) showed higher retain-ability of polyphenols accompanying low degradation in gastric and intestinal digestion and with no major toxicity signs. The addition of TA microcapsule did not produce any nutritional, physico-chemical, compositional, and nutritional distinctions in cheese. Microencapsulation proved to be appropriate approach for not only protecting the thermo-labile and pH-sensitive microbial antioxidants but also for enhanced bioavailability, and targeted release of bioactive extracts.

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 &beta, 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 &beta, 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 &beta, carotene production in the high producing strain CBS 277.49.

Natural and de-novo biosynthesized phyto-compounds have gained much significance because of their non-controversial nutritional, health and safety benefits as compared with chemically synthesized commercially rivalry antioxidants. However, none of natural de-novo biosynthesized phyto-compounds has been commercially available and used in customary food business and processing. In this study, efficacy of sesame seed extracts (SSEs) in stabilizing sunflower oil during storage has been studied. Fine powder of sesame seed was extracted in different solvents. The results showed that significant differences in extractability of different solvents and maximum extraction yield (29.48%) were achieved with methanol. The antioxidant components and capability of different extracts were further investigated and evaluated via total phenolic contents, DPPH radical scavenging activity and β-carotene/linoleic acid calorimetric assays respectively. Being highest in yield and antioxidant potential, methanolic extract was used; three different concentrations of SSE (500, 750, and 1000 μL) were added in 100 mL of sunflower oil to further evaluate its oxidative stability. Sensory and oxidative analysis of baked product from these groups was also evaluated. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-018-3336-2) contains supplementary material, which is available to authorized users.