Role of Snf-ß in lipid accumulation in the high lipid-producing fungus Mucor circinelloides WJ11.

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-ß, which encodes for ß 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-ß knockout strain was increased by 32 % (from 19 to 25 %). However, in Snf-ß 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-ß 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-ß knockout strain due to increased lipid accumulation and decreased to 1.83 % in Snf-ß overexpressing strain. AMPK is known to inactivate acetyl-CoA carboxylase (ACC) which catalyzes the rate-limiting step in lipid synthesis. Snf-ß manipulation also altered the expression level of the ACC1 gene which may indicate that Snf-ß control lipid metabolism by regulating ACC1 gene. CONCLUSIONS: Our results suggested that Snf-ß 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-ß in lipid accumulation in M. circinelloides.

Synergistic efficacy of alkyltrimethylammonium bromide, chlorhexidine digluconate on diverse bacterial strains causing red-heat and purple-stain deteriorations of leather.

This investigation assessed the synergistic effectiveness of alkyltrimethylammonium bromide (ATMB) and chlorhexidine digluconate (CDG) on selected microbes causing red-heat and purple stain degradations on salt-cured hides and leather products. This biological phenomenon ultimately deteriorates the finished leather quality and grounds considerable economic losses for leather industry. In tryptic soy broth, combination of ATMB and/CDG at the concentration of 900/90 ppm for predefined time interval of 1, 3, and 6 min caused in 0.73, 2.45, and 2.95 log CFU/mL reductions of bacterial cocktail population cell survival, respectively. Whereas in saturated brine solution (SBS), the bacterial cocktail treated with 600/60 and 900/90 ppm of ATMB/CDG for 18 h resulted ~ 74% and 98% reduction, respectively, in comparison to their respective controls. Furthermore, lessening of individual bacterial cultivations by combined ATMB and CDG in SBS was also elucidated.

Increased Accumulation of Medium-Chain Fatty Acids by Dynamic Degradation of Long-Chain Fatty Acids in Mucor circinelloides.

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-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 (15-36% 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 ß-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 ß-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.

Draft Genome Sequence of Red-Heat-Causing Halomonas eurihalina MS1, a Moderately Halophilic Bacterium Isolated from Saline Soil in Alicante, Spain.

Here, we report the draft genome sequence of Halomonas eurihalina MS1, which was isolated from saline soil in Alicante, Spain, and causes the condition known as "red heat" in salt-packed cured hides, decreasing their commercial value for leather production.

Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides.

BACKGROUND: Dihomo-gamma linolenic acid (DGLA, 20:3, n-6) is the elongated product of Gamma linolenic acid (GLA, 18:3, n-6) catalyzed by the enzyme delta-6 elongase (D6E) or gamma linolenic acid elongase (GLELO). Construction of engineered oleaginous microbes have been attracting significant interest to produce DGLA because of its nutritional value and medicinal applications. Mucor circinelloides is a GLA producing filamentous fungus which can be a useful tool to produce DGLA. We have, therefore, overexpressed the D6E (GLELO) gene in this fungus to construct DGLA producing cell factory. RESULT: To produce DGLA in M. circinelloides, homologous overexpression of D6E (GLELO) gene was analyzed. When the gene was overexpressed in M. circinelloides CBS277.49, up to 5.72% DGLA was produced in this strain. CONCLUSION: To our knowledge, this is the first report describing the overexpression of D6E (GLELO) gene in M. circinelloides to construct DGLA producing cell factory. A new scope for further research has been established by this work for improved production of DGLA in this fungus, specifically in its high lipid-producing strain, WJ11.

Genetic Modification of Mucor circinelloides to Construct Stearidonic Acid Producing Cell Factory.

Stearidonic acid (SDA; 18:4, n-3) is the delta 15-desaturase product of gamma linolenic acid (GLA; 18:3, n-6) and delta 6-desaturase product of alpha linolenic acid (ALA; 18:3, n-3). Construction of engineered oleaginous microbes have been attracting significant interest in producing SDA because of its nutritional value and pharmaceutical applications. Mucor circinelloides is a GLA producing filamentous fungus, which can be a useful tool to produce SDA. This study has, therefore, overexpressed the delta-15 desaturase (D15D) gene from Mortierella alpina in this fungus to construct a SDA-producing cell factory. To produce SDA in M. circinelloides, the homologous overexpression of D15D gene was analyzed. When the gene was overexpressed in M. circinelloides CBS 277.49, up to 5.0% SDA was accumulated in this strain. According to current knowledge, this is the first study describing the construction of a SDA-producing cell factory by overexpression of D15D gene in oleaginous fungus M. circinelloides. A new scope for further research has been established by this work to improve SDA production in this fungus, specifically in its high lipid-producing strain, WJ11.

Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides.

Increasing energy demands and health-related concerns worldwide have motivated researchers to adopt diverse strategies to improve medium-chain fatty acid (MCFA) biosynthesis for use in the functional food and aviation industries. The abundance of naturally produced MCFAs from botanical sources (i.e., coconut fruit/seeds and palm tree) has been observed to be insufficient compared with the various microorganisms used to cope with industrial demands. Mucor circinelloides is one of many promising microorganisms; it exhibits diverse biotechnological importance ranging from the production of functional lipids to applications in the manufacture of bio-fuel. Thus, research was conducted to acquire the desired elevated amounts of MCFAs (i.e., C8⁻C12) from metabolically engineered strains of M. circinelloides M65. To achieve this goal, four different acyl-acyl carrier protein (ACP) thioesterase (TE)-encoding genes exhibiting a substrate preference for medium-chain acyl-ACP molecules were expressed in M. circinelloides M65, resulting in the generation of C8⁻C12 fatty acids. Among all the engineered strains, M65-TE-03 and M65-TE-04 demonstrated the highest production of non-native C8⁻C10 and C12 fatty acids, respectively, in comparison to the control. These recombinant strains biosynthesized MCFAs de novo within the range from 28 to 46% (i.e., 1.14 to 2.77 g/L) of total cell lipids. Moreover, the reduction in chain length eventually resulted in a 1.5⁻1.75-fold increase in total lipid productivity in the engineered strains. The MCFAs were also found to be integrated into all lipid classes. This work illustrates how the integration of heterologous enzymes in M. circinelloides can offer a novel opportunity to edit the fatty acid synthases (FAS) complex, resulting in increased production of microbial MFCAs.

Antioxidant Potential and the Characterization of Arachis hypogaea Roots.

Arachis hypogaea roots are used as traditional Chinese medicine to treat different ailments, and the present study involves the exploration and comparison of phenolic profile and antioxidant activities (ABTS+ and DPPH assay) of A. hypogaea root extract in different solvents. 70% aqueous acetone and 70% aqueous ethanol were proved to be the best solvents to recover total phenolic compounds, with a yield of 42.59 ± 1.96 and 41.34 ± 0.92 mg/g dry weight of extract, respectively. ABTS+ radical scavenging activity was the highest in 70% aqueous ethanol, while the absolute methanol extract showed the highest DPPH radical scavenging activity (29.50 ± 2.19 µg/mL). Furthermore, phytochemical profiling of 70% acetone extract of A. hypogaea roots was performed by LC-ESI-TOF-MS analysis which in turn indicated the presence of diverse compounds in the A. hypogaea root extract, namely, quinones, stilbenoids, and flavones and flavonoid glucosides.

Probiotics and Their Potential Preventive and Therapeutic Role for Cancer, High Serum Cholesterol, and Allergic and HIV Diseases.

The potential health benefits of probiotics have long been elucidated since Metchnikoff and his coworkers postulated the association of probiotic consumption on human's health and longevity. Since then, many scientific findings and research have further established the correlation of probiotic and gut-associated diseases such as irritable bowel disease and chronic and antibiotic-associated diarrhea. However, the beneficial impact of probiotic is not limited to the gut-associated diseases alone, but also in different acute and chronic infectious diseases. This is due to the fact that probiotics are able to modify the intestinal microbial ecosystem, enhance the gut barrier function, provide competitive adherence to the mucosa and epithelium, produce antimicrobial substances, and modulate the immune activity by enhancing the innate and adaptive immune response. Nevertheless, the current literature with respect to the association of probiotic and cancer, high serum cholesterol, and allergic and HIV diseases are still scarce and controversial. Therefore, in the present work, we reviewed the potential preventive and therapeutic role of probiotics for cancer, high serum cholesterol, and allergic and HIV diseases as well as providing its possible mechanism of actions.

Effects of sesame seed extract as a natural antioxidant on the oxidative stability of sunflower oil.

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 ß-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.

Evaluation of the Spermatogenic Activity of Polyherbal Formulation in Oligospermic Males.

The therapeutic use of natural herbs is an ancient human civilization act and the numbers of people have reliance on their pharmacological properties and preferred to use the natural herbs. People also use to consume these herbs as supplements to energize, bolster, and eventually enhance sexual ability. Polyherbal formulation (PHF) is one of these herbal amalgams that can be used to treat sexual dysfunction including erectile dysfunction, impotence, ejaculation dysfunction, and hypogonadism. The pilot study was aimed at evaluating the capacity of PHF in enhancing the spermatogenic potential of oligospermic patients. Thirty-six male patients with oligospermia were enrolled and randomized either to treatment (n = 23) with PHF (750 mg/d in three doses for 90 days) or to placebo (n = 13) in the same protocol. The preintervention semen analysis was compared with posttreatment semen analysis. Based on the postintervention semen analysis, patients were advised to undergo either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) to assess their fertility status. After polyherbal treatment, there was a 256% increase in sperm concentration (9.59 ± 4.37 × 106/mL to 25.61 ± 8.6 × 106/mL; P ≤0.001), 154% increase in semen volume (1.7 ± 0.14 mL to 4.32 ± 0.38 mL; P ≤0.001), and 215% increase in sperm motility (15.43 ± 2.40% to 48.65 ± 5.10%; P ≤ 0.001) on day 90 from baseline. Furthermore, a significant improvement and regulation were also observed in serum hormone levels with PHF treatment as compared to the placebo group. The present study demonstrated the evidence on synergistic spermatogenic effect of PHF as attributed in ayurveda for the treatment of oligospermia leading to infertility.

Microencapsulation and the Characterization of Polyherbal Formulation (PHF) Rich in Natural Polyphenolic Compounds.

Microencapsulation of polyherbal formulation (PHF) extract was carried out by freeze drying method, by employing gum arabic (GA), gelatin (GE), and maltodextrin (MD) with their designated different combinations as encapsulating wall materials. Antioxidant components (i.e., total phenolic contents (TPC), total flavonoids contents (TFC), and total condensed tannins (TCT)), antioxidant activity (i.e., DPPH, β-carotene & ABTS⁺ assays), moisture contents, water activity (aw), solubility, hygroscopicity, glass transition temperature (Tg), particle size, morphology, in vitroα-amylase and α-glucosidase inhibition and bioavailability ratios of the powders were investigated. Amongst all encapsulated products, TB (5% GA & 5% MD) and TC (10% GA) have proven to be the best treatments with respect to the highest preservation of antioxidant components. These treatments also exhibited higher antioxidant potential by DPPH and β-carotene assays and noteworthy for an ABTS⁺ assays. Moreover, the aforesaid treatments also demonstrated lower moisture content, aw, particle size and higher solubility, hygroscopicity and glass transition temperature (Tg). All freeze dried samples showed irregular (asymmetrical) microcrystalline structures. Furthermore, TB and TC also illustrated the highest in vitro anti-diabetic potential due to great potency for inhibiting α-amylase and α-glucosidase activities. In the perspective of bioavailability, TA, TB and TC demonstrated the excellent bioavailability ratios (%). Furthermore, the photochemical profiling of ethanolic extract of PHF was also revealed to find out the bioactive compounds.

Farm to Consumer: Factors Affecting the Organoleptic Characteristics of Coffee. II: Postharvest Processing Factors.

The production and consumption of coffee are increasing despite the roadblocks to its agriculture and global trade. The unique, refreshing, and stimulating final cupping quality of coffee is the only reason for this rising production and consumption. Coffee quality is a multifaceted trait and is inevitably influenced by the way it is successively processed after harvesting. Reportedly, 60% of the quality attributes of coffee are governed by postharvest processing. The current review elaborates and establishes for the first time the relationship between different methods of postharvest processing of coffee and its varying organoleptic and sensory quality attributes. In view of the proven significance of each processing step, this review has been subdivided into three sections, secondary processing, primary processing, and postprocessing variables. Secondary processing addresses the immediate processing steps on the farm after harvest and storage before roasting. The primary processing section adheres specifically to roasting, grinding and brewing/extraction, topics which have been technically addressed more than any others in the literature and by industry. The postprocessing attribute section deals generally with interaction of the consumer with products of different visual appearance. Finally, there are still some bottlenecks which need to be addressed, not only to completely understand the relationship of varying postharvest processing methods with varying in-cup quality attributes, but also to devise the next generation of coffee processing technologies.

Antioxidants Potential of the Filamentous Fungi (Mucor circinelloides).

Three important strains of Mucorcircinelloides grown in complete and minimal media for specified period (72 h, 120 h and 168 h) under submerged fermentation conditions were investigated for their potential antioxidants/secondary metabolite production. All mycelial extracts demonstrated effective antioxidant activities in terms of ß-carotene/linoleic acid bleaching, radical scavenging, reduction of metal ions and chelating abilities against ferrous ions. Different extraction methods and solvent systems affected the recovery yield and antioxidant activities of the extracts significantly (p ≤ 0.05). Ethanolic extracts were found to be rich source of antioxidant components and subsequently more effective in antioxidant properties. Fermentation period and media used also significantly affected (p ≤ 0.05) the antioxidant production and the resulting antioxidant properties. The (ethanolic) extracts of all the strains from late exponential growth phase (120 h) showed highest antioxidant production with topmost reducing, chelating and radical scavenging capabilities. Strain MC277.49 was found to be the highest producer of antioxidants followed by MC108.16 and WJ11. Phenolic compounds were detected significantly in higher (p ≤ 0.05) amount succeeded by the condensed tannins and flavonoids. Total phenol content of each extract was attributed to overall antioxidant capacity. Submerged fermentation with nutritional stress conditions were found to be excellent way of producing surplus amount of natural antioxidants/secondary metabolites with their vast potential commercial application in food and pharmaceutical industries.