Conservation of Endangered Cordyceps sinensis Through Artificial Cultivation Strategies of C. militaris, an Alternate.

Cordyceps, an entomopathogenic fungus belonging to the Ascomycota phylum, is a familiar remedial mushroom that is extensively used in the traditional medicinal system, especially in South Asian nations. The significance of this genus' members in a range of therapeutic and biotechnological applications has long been acknowledged. The exceedingly valuable fungus Ophiocordyceps sinensis (Cordyceps sinensis) is found in the alpine meadows of Bhutan, Nepal, Tibet, and India, where it is severely harvested. Driven by market demand and ecological concerns, the study highlights challenges in natural C. sinensis collection and emphasizes the shift towards sustainable artificial cultivation methods. This in-depth review navigates Cordyceps cultivation strategies, focusing on C. sinensis and the viable alternative, C. militaris. The escalating demand for Cordyceps fruiting bodies and bioactive compounds prompts a shift toward sustainable artificial cultivation. While solid-state fermentation on brown rice remains a traditional method, liquid culture, especially submerged and surface/static techniques, emerges as a key industrial approach, offering shorter cultivation periods and enhanced cordycepin production. The review accentuates the adaptability and scalability of liquid culture, providing valuable insights for large-scale Cordyceps production. The future prospects of Cordyceps cultivation require a holistic approach, combining scientific understanding, technological innovation, and sustainable practices to meet the demand for bioactive metabolites while ensuring the conservation of natural Cordyceps populations.

A review of alternative proteins for vegan diets: Sources, physico-chemical properties, nutritional equivalency, and consumer acceptance.

Alternate proteins are gaining popularity as a more sustainable and environmentally friendly alternative to animal-based proteins. These proteins are often considered healthier and are suitable for people following a vegetarian or vegan diet. Alternative proteins can be recovered from natural sources like legumes, grains, nuts, and seeds, while single cell proteins (mycoproteins), and algal proteins are being developed using cutting-edge technology to grow fungus, yeast and algal cells in a controlled environment, creating a more sustainable source of protein. Although, the demand for alternative protein products is increasing, there still happens to be a large gap in use among the general consumers mainly stemming from its lower bioavailability, lack of nutritional equivalency and reduced digestibility compared to animal proteins. The focus of the review is to emphasize on various sources and technologies for recovering alternative proteins for vegan diets. The review discusses physicochemical properties of alternative proteins and emphasise on the role of various processing technologies that can change the digestibility and bioavailability of these proteins. It further accentuates the nutritional equivalency and environmental sustainability of alternative protein against the conventional proteins from animals. The food laws surrounding alternative proteins as well as the commercial potential and consumer acceptance of alternative protein products are also highlighted. Finally, key challenges to improve the consumer acceptability and market value of plant-based proteins would be in achieving nutrient equivalency and enhance bioavailability and digestibility while maintaining the same physicochemical properties, taste, texture, as animal proteins, has also been highlighted.

Bioactive compounds from Cordyceps and their therapeutic potential.

The Clavicipitaceae family's largest and most diverse genus is Cordyceps. They are most abundant and diverse in humid temperate and tropical forests and have a wide distribution in: Europe, North America, and East and Southeast Asian countries, particularly: Bhutan, China, Japan, Nepal, Korea, Thailand, Vietnam, Tibet, and the Himalayan region of India, and Sikkim. It is a well-known parasitic fungus that feeds on insects and other arthropods belonging to 10 different orders. Over 200 bioactive metabolites, that include: nucleotides and nucleosides, polysaccharides, proteins, polypeptides, amino acids, sterols, and fatty acids, among others have been extracted from Cordyceps spp. demonstrating the phytochemical richness of this genus. These components have been associated with a variety of pharmacological effects, including: anti-microbial, anti-apoptotic, anti-cancer, anti-inflammatory, antioxidant, and immunomodulatory activities. In this paper, the bioactivity of various classes of metabolites produced by Cordyceps spp., and their therapeutic properties have been reviewed in an attempt to update the existing literature. Furthermore, one of its nucleoside and a key bioactive compound, cordycepin has been critically elaborated with regard to its biosynthesis pathway and the recently proposed protector-protégé mechanism as well as various biological and pharmacological effects, such as: suppression of purine and nucleic acid biosynthesis, induction of apoptosis, and cell cycle regulation with their mechanism of action. This review provides current knowledge on the bioactive potential of Cordyceps spp.

In-silico validation of novel therapeutic activities of withaferin a using molecular docking and dynamics studies.

Withaferin A is a bioactive molecule of W. somnifera. We access its efficacy against various target proteins associated with Cancer, Type-II Diabetes and hypercholesterolemia using molecular docking. Although it's efficacy against some of these targets have been reported earlier, we validate each mechanism in order to report the most appropriate mechanism of action. We explain the anti-cancer activity of Withaferin A by inhibition of Mortalin (mtHsp70) and Nrf2 protein with binding energies -8.85 kcal/mol and -12.59 kcal/mol respectively. Similarly, the anti-diabetic activity could be explained by inhibition of alpha and betα-glucosidase with binding energies -6.44 and -4.43 kcal/mol respectively and the cholesterol reduction could be explained by its ability to inhibition of NPC1 and SRB1 with binding energies -5.73 and -7.16 kcal/mol respectively. The molecular dynamics of the apoprotein and the protein-ligand complex simulated for the best targets of each activity namely Nrf2 protein for anti-cancer, α-glucosidase for anti-diabetic and SR-B1 for anti-hypercholesterolemia activity indicated the formation of stable complexes due to low RMSD deviations, low RMSF fluctuations and low RG values after the docking simulation. Finally, an ADME + T (Adsorption, distribution, metabolism, excretion and toxicity) prediction on Withaferin A showed that it obeyed all the Lipinsky's rules and qualified the drug-like criteria. All these results validate that Withaferin A possess potential anti-cancer, anti-diabetic and cholesterol reducing properties. This is the first report that indicates the possibility of Withaferin A binding and inhibiting SR-B1 as a mechanism of its anti-hypercholesterolemia activity.

An updated review on advancement in fermentative production strategies for biobutanol using Clostridium spp.

A significant concern of our fuel-dependent era is the unceasing exhaustion of petroleum fuel supplies. In parallel to this, environmental issues such as the greenhouse effect, change in global climate, and increasing global temperature must be addressed on a priority basis. Biobutanol, which has fuel characteristics comparable to gasoline, has attracted global attention as a viable green fuel alternative among the many biofuel alternatives. Renewable biomass could be used for the sustainable production of biobutanol by the acetone-butanol-ethanol (ABE) pathway. Non-extinguishable resources, such as algal and lignocellulosic biomass, and starch are some of the most commonly used feedstock for fermentative production of biobutanol, and each has its particular set of advantages. Clostridium, a gram-positive endospore-forming bacterium that can produce a range of compounds, along with n-butanol is traditionally known for its biobutanol production capabilities. Clostridium fermentation produces biobased n-butanol through ABE fermentation. However, low butanol titer, a lack of suitable feedstock, and product inhibition are the primary difficulties in biobutanol synthesis. Critical issues that are essential for sustainable production of biobutanol include (i) developing high butanol titer producing strains utilizing genetic and metabolic engineering approaches, (ii) renewable biomass that could be used for biobutanol production at a larger scale, and (iii) addressing the limits of traditional batch fermentation by integrated bioprocessing technologies with effective product recovery procedures that have increased the efficiency of biobutanol synthesis. Our paper reviews the current progress in all three aspects of butanol production and presents recent data on current practices in fermentative biobutanol production technology.

Synthetic Biology Tools for Genome and Transcriptome Engineering of Solventogenic Clostridium.

Strains of Clostridium genus are used for production of various value-added products including fuels and chemicals. Development of any commercially viable production process requires a combination of both strain and fermentation process development strategies. The strain development in Clostridium sp. could be achieved by random mutagenesis, and targeted gene alteration methods. However, strain improvement in Clostridium sp. by targeted gene alteration method was challenging due to the lack of efficient tools for genome and transcriptome engineering in this organism. Recently, various synthetic biology tools have been developed to facilitate the strain engineering of solventogenic Clostridium. In this review, we consolidated the recent advancements in toolbox development for genome and transcriptome engineering in solventogenic Clostridium. Here we reviewed the genome-engineering tools employing mobile group II intron, pyrE alleles exchange, and CRISPR/Cas9 with their application for strain development of Clostridium sp. Next, transcriptome engineering tools such as untranslated region (UTR) engineering and synthetic sRNA techniques were also discussed in context of Clostridium strain engineering. Application of any of these discussed techniques will facilitate the metabolic engineering of clostridia for development of improved strains with respect to requisite functional attributes. This might lead to the development of an economically viable butanol production process with improved titer, yield and productivity.

Economic and sustainable management of wastes from rice industry: combating the potential threats.

Rice is one of the imperative staple foods, particularly in the developing countries. The exponential boom in human population has resulted in the continuous expansion in the rice industry in order to meet the food demands. The various stages of paddy processing release huge quantity of solid wastes, mainly rice husk, rice husk ash and liquid wastes in the form of rice industry wastewater. The discharge of the rice industry wastewater imparts a substantial threat to the aquatic bodies and the nearby surrounding and, thus, consequently demands eco-benign treatment plan. As a result, different strategies are needed to enhance the effluent quality and minimize the operational cost of the treatment process. Therefore, efficient technological approach targeting the minimization of pollution as well as assuring the economic prosperity should be implemented. In this review article, several aspects related to the rice industry discussing the significant challenges involved in the generation of both solid and liquid wastes, mitigation experiments and future prospects have been meticulously elaborated. Furthermore, the article also focuses on the various processes utilized for reducing the pollution load and promoting the practice of reuse and recycle of waste rather than the discharge action for the sake of sustainability and the emergence of novel techniques for the generation of energy and value-added products.

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol-butanol-ethanol fuel mixture.

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone-butanol-ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol-butanol-ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab-scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot-scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab-scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production.

Acetone-butanol-ethanol production with high productivity using Clostridium acetobutylicum BKM19.

Conventional acetone-butanol-ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L(-1) of ABE (17.6 g L(-1) butanol, 10.5 g L(-1) ethanol, and 4.4 g L(-1) acetone) from 85.2 g L(-1) glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell-recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L(-1) h(-1) , respectively, could be achieved at the dilution rate of 0.85 h(-1) . Further cell recycling experiments were carried out with controlled cell-bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h(-1) with the bleeding rate of 0.04 h(-1) . Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L(-1) h(-1) , and the yields of 0.17 and 0.34 g g(-1) , respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known-processes.

Butanol production from renewable biomass by clostridia.

Global energy crisis and limited supply of petroleum fuels have rekindled the worldwide focus towards development of a sustainable technology for alternative fuel production. Utilization of abundant renewable biomass offers an excellent opportunity for the development of an economical biofuel production process at a scale sufficiently large to have an impact on sustainability and security objectives. Additionally, several environmental benefits have also been linked with the utilization of renewable biomass. Butanol is considered to be superior to ethanol due to its higher energy content and less hygroscopy. This has led to an increased research interest in butanol production from renewable biomass in recent years. In this paper, we review the various aspects of utilizing renewable biomass for clostridial butanol production. Focus is given on various alternative substrates that have been used for butanol production and on fermentation strategies recently reported to improve butanol production.

Butanol production from renewable biomass: rediscovery of metabolic pathways and metabolic engineering.

Biofuel from renewable biomass is one of the answers to help solve the problems associated with limited fossil resources and climate change. Butanol has superior liquid-fuel characteristics, with similar properties to gasoline, and thus, has the potential to be used as a substitute for gasoline. Clostridia are recognized as a good butanol producers and are employed in the industrial-scale production of solvents. Due to the difficulty of performing genetic manipulations on clostridia, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered the development of engineered clostridia strains with highly efficient and selective butanol-production capabilities. In recent years, the butanol-producing characteristics in clostridia have been further characterized and alternative pathways discovered. More recently, systems-level metabolic engineering approaches were taken to develop superior strains. Herein, we review recent discoveries of metabolic pathways for butanol production and the metabolic engineering strategies being developed.

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of Clostridium pasteurianum.

Butanol, a four-carbon primary alcohol (C(4)H(10)O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l(-1) butanol from 80 g l(-1) glycerol as compared to 7.6 g l(-1) butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l(-1), 0.30 g g(-1), and 0.43 g l(-1) h(-1) could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone-butanol-ethanol productivity and the butanol productivity of 8.3 and 7.8 g l(-1) h(-1), respectively, could be achieved at the dilution rate of 0.9 h(-1). This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.

Rapid screening and isolation of a fungus for sitosterol to androstenedione biotransformation.

Androstenedione (AD) is a steroid intermediate used in the pharmaceutical industry for the production of several important anabolic drugs. An important route for producing AD is by the microbial transformation of sterols. Microbes capable of efficiently transforming sterols to AD are few and newer strains need to be isolated. Conventional procedures for screening and isolation are time consuming. A new procedure was used for screening and isolation of fungal microorganisms capable of biotransformation of sitosterol to androstenedione. In this procedure, Basic Alignment Search Tool (BLAST) and Position-Specific Iterative BLAST were employed to obtain a parent set of candidate microorganisms. The parent set was reduced using heuristics and constraints to obtain a manageable number of microorganisms that may be tested experimentally. For this work, screening of the entire NCBI database yielded a parent set containing 64 microorganisms. Among these, only two microorganisms, Aspergillus oryzae and Aspergillus nidulans FGSC A4, qualified to the experimental stage. Sitosterol biotransformation experiments were carried out using A. oryzae and the production of AD in culture medium was confirmed.

Nutrient broth/PEG200/TritonX114/Tween80/Chloroform microemulsion as a reservoir of solubilized sitosterol for biotransformation to androstenedione.

Microemulsions (ME) can act as a reservoir of solubilized hydrophobic substrates. The biotransformation of hydrophobic sitosterol to androstenedione (AD) with MEs prepared from nutrient broth and PEG 200 (1:1) as aqueous phase, 40 g/l sitosterol dissolved in chloroform as organic phase, Triton X114 and Tween 80 (1:1) as surfactant phase, was investigated. The phase behavior of this system was studied for ten different ratios(w/w), 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10 of the organic phase and surfactant at 30 degrees C. A pseudoternary phase diagram was constructed to demarcate the region giving stable MEs. The maximum solubility of sitosterol in ME medium was observed to be 8 g/l, which is 3 orders of magnitude higher than the reported sitosterol solubility of 2-4 mg/l in aqueous medium. The ME medium was used for biotransformation studies and a comparative result has been reported. Transmission electron microscopy of cells grown in ME having oil, surfactant and aqueous phase in the ratio of 6:14:80 showed a weakened cell wall structure that permitted production of 465.86 mg/l AD.

Enhanced biotransformation of sitosterol to androstenedione by Mycobacterium sp. using cell wall permeabilizing antibiotics.

Mycobacterial cell wall is rigid and offers a high resistance to the transport of sitosterol into cytosol. The effect of ethambutol, penicillin, polymixin and bacitracin on biotransformation of sitosterol to androstenedione by modification of cell wall permeability was examined. Drug sensitivity assay results established that bacitracin increased the permeability of the cell wall to hydrophobic compounds. Growth inhibitory study of bacitracin and rifamycin, individually as well as in combination showed that these two antibiotics act synergistically to reduce cell growth. A comparison of transmission electron micrograph results of the bacitracin-treated cells with untreated cells, revealed deformities caused in the cell wall structure by bacitracin treatment. These deformities increased the cell wall permeability and transport of sitosterol inside the cell, and thus enhanced androstenedione (AD) production. A maximum of 1.37, 1.44, 1.65 and 1.76 g AD per gram dry cell weight of mycobacterial cells was produced in the presence of ethambutol, penicillin, polymixin and bacitracin, respectively. Below the minimum inhibitory concentration, bacitracin can be used as potent enhancer of permeability of hydrophobic substances across the mycobacterial cell wall.

Androstenedione production by biotransformation of phytosterols.

Androstenedione is a key intermediate of microbial steroid metabolism. It belongs to the 17-keto steroid family and is used as starting material for the preparation of different steroids. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis. In this review, various methods of biotransformation of sterols to androstenedione are surveyed. It begins with the history and current research status in this field. The existing methods of chemical and biochemical synthesis are examined. Various issues related to these methods and how researchers have addressed them is presented. Among these, the low solubility of sterols in aqueous systems is a critical problem since it limits the product yield. The main content of this review focuses on new methods of biotransformation that are being investigated. Recent biotechnological advances in this field are presented. The review ends with a note on future perspectives.