Microbial production of nutraceuticals: Metabolic engineering interventions in phenolic compounds, poly unsaturated fatty acids and carotenoids synthesis.

Nutraceuticals have attained substantial attention due to their health-boosting or disease-prevention characteristics. Growing awareness about the potential of nutraceuticals for the prevention and management of diseases affecting human has led to an increase in the market value of nutraceuticals in several billion dollars. Nevertheless, limitations in supply and isolation complications from plants, animals or fungi, limit the large-scale production of nutraceuticals. Microbial engineering at metabolic level has been proved as an environment friendly substitute for the chemical synthesis of nutraceuticals. Extensively used microbial systems such as E. coli and S. cerevisiae have been modified as versatile cell factories for the synthesis of diverse nutraceuticals. This review describes current interventions in metabolic engineering for synthesising some of the therapeutically important nutraceuticals (phenolic compounds, polyunsaturated fatty acids and carotenoids). We focus on the interventions in enhancing product yield through engineering at gene level or pathway level.

Customized yeast cell factories for biopharmaceuticals: from cell engineering to process scale up.

The manufacture of recombinant therapeutics is a fastest-developing section of therapeutic pharmaceuticals and presently plays a significant role in disease management. Yeasts are established eukaryotic host for heterologous protein production and offer distinctive benefits in synthesising pharmaceutical recombinants. Yeasts are proficient of vigorous growth on inexpensive media, easy for gene manipulations, and are capable of adding post translational changes of eukaryotes. Saccharomyces cerevisiae is model yeast that has been applied as a main host for the manufacture of pharmaceuticals and is the major tool box for genetic studies; nevertheless, numerous other yeasts comprising Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Yarrowia lipolytica have attained huge attention as non-conventional partners intended for the industrial manufacture of heterologous proteins. Here we review the advances in yeast gene manipulation tools and techniques for heterologous pharmaceutical protein synthesis. Application of secretory pathway engineering, glycosylation engineering strategies and fermentation scale-up strategies in customizing yeast cells for the synthesis of therapeutic proteins has been meticulously described.

Acetylation of Isoniazid Is a Novel Mechanism of Isoniazid Resistance in Mycobacterium tuberculosis.

Isoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a prodrug which is activated by the intracellular KatG enzyme of Mycobacterium tuberculosis The activated drug hinders cell wall biosynthesis by inhibiting the InhA protein. INH-resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH-resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study, we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze the transfer of the acetyl group to INH from acetyl coenzyme A (acetyl-CoA). High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. A drug susceptibility assay and confocal analysis showed that Mycobacterium smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Mutant proteins of Rv2170 failed to acetylate INH. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at MICs that inhibited wild-type strains. Besides, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity, although this needs to be tested in INH-resistant clinical strains.

A comparative study to elucidate the biological activities of crude extracts from rice bran and wheat bran in cell line models.

The present study investigated the nutritional composition of bran from rice (RB) and wheat (WB) and compared the natural virtues of crude extracts based on phenolic composition, antidiabetic and anticancer activities. The profiling of phenolic-rich ethyl acetate extracts (RBE and WBE) confirms that RBE is rich in catechol (0.122 mg/g dw), p-coumaric acid (0.159 mg/g dw), kaempferol (0.374 mg/g dw) and apigenin (0.399 mg/g dw); and WBE is affluent with catechol (0.144 mg/g dw), ferulic acid (0.160 mg/g dw), caffeic acid (0.083 mg/g dw) and ellagic acid (0.074 mg/g dw). RBE exhibited better antioxidant activity, inhibited the activity of α-amylase (IC50-353.41 µg/mL) and α-glucosidase (IC50-314.22 µg/mL), hindered glycation process (IC50-451.11 µg/mL), and enhanced glucose uptake in L6 muscle cells (20.4%) indicating its potential in diabetic management. RBE was toxic to HT29 colon cancer cells and decreased cell membrane integrity. RBE and WBE arrested cell-cycle transition in HT29 cells from G0 to G1 and G2 to M phase respectively and induced apoptosis (27.15% and 5.9%, respectively for RBE and WBE) suggesting anticancer activities of the extract. The study indicates that bran from rice and wheat are a potential source of dietary fibre and phytochemicals with antidiabetic and anticancer properties for developing value-added products with nutraceutical benefits.

Nutraceutical properties of cumin residue generated from Ayurvedic industries using cell line models.

Spent cumin (SC), generated from Ayurvedic industry, was evaluated for its nutraceutical potential in terms of antioxidant, antidiabetic and anticancer properties, and compared with that of the raw cumin (RC). SC and RC seeds were extracted with ethyl acetate (E) and methanol (M). SCM (methanol extract) were rich in p-coumaric acid, ferulic acid, ellagic acid and cinnamic acid (6.4445, 5.8286, 2.1519, 4.3085 mg/g dry extract). SCM reduced Fe2+ ion (89.68 µM AA/g dry weight), scavenged DPPH radical (IC50-238.6 µg/mL), better α-amylase inhibition (IC50-337.22 µg/mL) and glucose uptake activity in 30.7% of L6 cells. SCM inhibited viability, retarded migration area up to 41.02%, arrested cell cycle at S phase and induced apoptosis in 2.45% of HT29 colon cancer cells. The results indicated that dietary interventions using nutraceutical food formulation made out of SC can play a significant role in the prevention and management of degenerative diseases.

Plantain peel - a potential source of antioxidant dietary fibre for developing functional cookies.

Plantain cultivar Nendran is popular as a staple food in many parts of India and deep fried chips made from raw matured Nendran are one of the popular snack items in India. This study aims to utilize peel from Nendran variety- the main byproduct of banana chips industry- to develop high fibre cookies with enhanced bioactive content. Proximate analysis indicated that peels are rich in total dietary fibre (64.33 g/100 g), vitamins (Folic acid- 33.12 mg/100 g) and minerals (Potassium- 35.61 mg/100 g). Nendran Peel Flour (NPF) was extracted with hexane, ethyl acetate and methanol. Phenolic and flavonoid content was high for ethyl acetate extract (15.21 and 9.39 mg QE/g dry weight). Methanol extract was more potent in reducing Copper ion (2.36 µM TR/g dry weight) and scavenging NO (IC50-381.71 µg/mL). Ethyl acetate extract was capable of scavenging DPPH and hydroxyl radical. HPLC profiling showed presence of gallic acid, protocatechuic acid, rutin hydrate and quercetin in ethyl acetate extract and gallic acid, chlorogenic acid and vanillic acid in methanol extract. Cookies prepared with NPF possess higher total dietary fibre content. There was a decrease in spread ratio, breaking strength and browning index of cookies as the percentage of NPF increased. NPF incorporation gradually increased the phenolic content from 4.36 to 5.28 mg GAE, compared to control cookie (3.21 mg GAE). DPPH scavenging activity also increased with increase in NPF. Hence NPF is a very good source of antioxidant dietary fibre and acceptable cookies can be produced by replacing wheat flour with 10 % NPF.

Lignin nanoparticles from Ayurvedic industry spent materials: Applications in Pickering emulsions for curcumin and vitamin D3 encapsulation.

Lignin nanoparticles (LNP), extracted from spent materials of Dashamoola Arishta (Ayurvedic formulation), shared a molecular weight of 14.42 kDa with commercial lignin. Processed into LNPs (496.43 ± 0.54 nm) via planetary ball milling, they demonstrated stability at pH 8.0 with a zeta potential of -32 ± 0.27 mV. Operating as Pickering particles, LNP encapsulated curcumin and vitamin D3 in sunflower oil, forming LnE + Cu + vD3 nanoemulsions (particle size: 347.40 ± 0.71 nm, zeta potential: -42.27 ± 0.72 mV) with high encapsulation efficiencies (curcumin: 87.95 ± 0.21%, vitamin D3: 72.66 ± 0.11%). The LnE + Cu + vD3 emulsion exhibited stability without phase separation over 90 days at room (27 ± 2 °C) and refrigeration (4 ± 1 °C) temperatures. Remarkably, LnE + Cu + vD3 exhibited reduced toxicity, causing 29.32% and 34.99% cell death in L6 and RAW264.7 cells respectively, at the highest concentration (50 µg/mL). This underscores the potential valorization of Ayurvedic industry spent materials for diverse industrial applications.

Design and genome engineering of microbial cell factories for efficient conversion of lignocellulose to fuel.

The gradually increasing need for fossil fuels demands renewable biofuel substitutes. This has fascinated an increasing investigation to design innovative energy fuels that have comparable Physico-chemical and combustion characteristics with fossil-derived fuels. The efficient microbes for bioenergy synthesis desire the proficiency to consume a large quantity of carbon substrate, transfer various carbohydrates through efficient metabolic pathways, capability to withstand inhibitory components and other degradation compounds, and improve metabolic fluxes to synthesize target compounds. Metabolically engineered microbes could be an efficient methodology for synthesizing biofuel from cellulosic biomass by cautiously manipulating enzymes and metabolic pathways. This review offers a comprehensive perspective on the trends and advances in metabolic and genetic engineering technologies for advanced biofuel synthesis by applying various heterologous hosts. Probable technologies include enzyme engineering, heterologous expression of multiple genes, CRISPR-Cas technologies for genome editing, and cell surface display.

Lignin-based nanomaterials for food and pharmaceutical applications: Recent trends and future outlook.

Small particles of size ranging from 1 to 100 nm are referred to as nanoparticles. Nanoparticles have tremendous applications in various sectors, including the areas of food and pharmaceutics. They are being prepared from multiple natural sources widely. Lignin is one such source that deserves special mention due to its ecological compatibility, accessibility, abundance, and low cost. This amorphous heterogeneous phenolic polymer is the second most abundant molecule in nature after cellulose. Apart from being used as a biofuel source, lignin is less explored for its potential at a nano-level. In plants, lignin exhibits cross-linking structures with cellulose and hemicellulose. Numerous advancements have taken place in synthesizing nanolignins for manufacturing lignin-based materials to benefit from the untapped potential of lignin in high-value-added applications. Lignin and lignin-based nanoparticles have numerous applications, but in this review, we are mainly focusing on the applications in the food and pharmaceutical sectors. The exercise we undertake has great relevance as it helps scientists and industries gain valuable insights into lignin's capabilities and exploit its physical and chemical properties to facilitate the development of future lignin-based materials. We have summarized the available lignin resources and their potential in the food and pharmaceutical industries at various levels. This review attempts to understand various methods adopted for the preparation of nanolignin. Furthermore, the unique properties of nano-lignin-based materials and their applications in fields including the packaging industry, emulsions, nutrient delivery, drug delivery hydrogels, tissue engineering, and biomedical applications were well-discussed.

Synthesis of C2-C4 diols from bioresources: Pathways and metabolic intervention strategies.

Diols are important platform chemicals with extensive industrial applications in biopolymer synthesis, cosmetics, and fuels. The increased dependence on non-renewable sources to meet the energy requirement of the population raised issues regarding fossil fuel depletion and environmental impacts. The utilization of biological methods for the synthesis of diols by utilizing renewable resources such as glycerol and agro-residual wastes gained attention worldwide because of its advantages. Among these, biotransformation of 1,3-propanediol (1,3-PDO) and 2,3-butanediol (2,3-BDO) were extensively studied and at present, these diols are produced commercially in large scale with high yield. Many important isomers of C2-C4 diols lack natural synthetic pathways and development of chassis strains for the synthesis can be accomplished by adopting synthetic biology approaches. This current review depicts an overall idea about the pathways involved in C2-C4 diol production, metabolic intervention strategies and technologies in recent years.

Engineering interventions in industrial filamentous fungal cell factories for biomass valorization.

Filamentous fungi possess versatile capabilities for synthesizing a variety of valuable bio compounds, including enzymes, organic acids and small molecule secondary metabolites. The advancements of genetic and metabolic engineering techniques and the availability of sequenced genomes discovered their potential as expression hosts for recombinant protein production. Remarkably, plant-biomass degrading filamentous fungi show the unique capability to decompose lignocellulose, an extremely recalcitrant biopolymer. The basic biochemical approaches have motivated several industrial processes for lignocellulose biomass valorisation into fermentable sugars and other biochemical for biofuels, biomolecules, and biomaterials. The review gives insight into current trends in engineering filamentous fungi for enzymes, fuels, and chemicals from lignocellulose biomass. This review describes the variety of enzymes and compounds that filamentous fungi produce, engineering of filamentous fungi for biomass valorisation with a special focus on lignocellulolytic enzymes and other bulk chemicals.

Integrated biorefinery development for pomegranate peel: Prospects for the production of fuel, chemicals and bioactive molecules.

Current experimental evidence has revealed that pomegranate peel is a significant source of essential bio compounds, and many of them can be transformed into valorized products. Pomegranate peel can also be used as feedstock to produce fuels and biochemicals. We herein review this pomegranate peel conversion technology and the prospective valorized product that can be synthesized from this frequently disposed fruit waste. The review also discusses its usage as a carbon substrate to synthesize bioactive compounds like phenolics, flavonoids and its use in enzyme biosynthesis. Based on reported experimental evidence, it is apparent that pomegranate peel has a large number of applications, and therefore, the development of an integrated biorefinery concept to use pomegranate peel will aid in effectively utilizing its significant advantages. The biorefinery method displays a promising approach for efficiently using pomegranate peel; nevertheless, further studies should be needed in this area.

Transcription Repressor Protein ZBTB25 Associates with HDAC1-Sin3a Complex in Mycobacterium tuberculosis-Infected Macrophages, and Its Inhibition Clears Pathogen by Autophagy.

Downregulation of host gene expression is a key strategy employed by intracellular pathogens for their survival in macrophages and subsequent pathogenesis. In a previous study, we have shown that histone deacetylase 1 (HDAC1) levels go up in macrophages infected with Mycobacterium tuberculosis, and it hypoacetylates histone H3 at the promoter of IL-12B gene, leading to its downregulation. We now show that after infection with M. tuberculosis, HDAC1 is phosphorylated, and the levels of phosphorylated HDAC1 (pHDAC1) increase significantly in macrophages. We found that transcriptional repressor protein zinc finger and BTB domain 25 (ZBTB25) and transcriptional corepressor Sin3a associate with the HDAC1 silencing complex, which is recruited to the promoter of IL-12B to downregulate its expression in infected macrophages. Knocking down of ZBTB25 enhanced release of IL-12p40 from infected macrophages. Inhibition of HDAC1 and ZBTB25 promoted colocalization of M. tuberculosis and LC3 (microtubule-associated protein 1A/1B-light chain 3) in autophagosomes. Induction of autophagy resulted in the killing of intracellular M. tuberculosis Enhanced phosphorylation of JAK2 and STAT4 was observed in macrophages upon treatment with HDAC1 and ZBTB inhibitors, and inhibition of JAK2/STAT4 negated the killing of the intracellular pathogen, suggesting their role in the autophagy-mediated killing of intracellular M. tuberculosis In view of the emergence of drug resistance in M. tuberculosis, host-directed therapy is an attractive alternative strategy to combat tuberculosis (TB). HDACs have been proposed to be host targets for TB treatment. Our study indicates that ZBTB25, a functional subunit of the HDAC1/Sin3a repressor complex involved in IL-12B suppression, could be an alternative target for host-directed anti-TB therapy.IMPORTANCE Following infection with M. tuberculosis, levels of HDAC1 go up in macrophages, and it is recruited to the promoter of IL-12B where it hypoacetylates histone H3, leading to the downregulation of the gene. Here, we show that host transcriptional repressor protein ZBTB25 and transcriptional corepressor Sin3a associate with HDAC1 in the silencing complex. Knocking down of ZBTB25 prevented the recruitment of the complex to the promoter and consequently enhanced the gene expression and the release of IL-12p40 from infected macrophages. Pharmacological inhibition of ZBTB25 in infected macrophages resulted in the induction of autophagy and killing of intracellular M. tuberculosis Drug-resistant TB is a serious challenge to TB control programs all over the world which calls for finding alternative therapeutic methods. Host-directed therapy is gaining significant momentum in treating infectious diseases. We propose that ZBTB25 is a potential target for host-directed treatment of TB.

Design of novel enzyme biocatalysts for industrial bioprocess: Harnessing the power of protein engineering, high throughput screening and synthetic biology.

Biocatalysts have wider applications in various industries. Biocatalysts are generating bigger attention among researchers due to their unique catalytic properties like activity, specificity and stability. However the industrial use of many enzymes is hindered by low catalytic efficiency and stability during industrial processes. Properties of enzymes can be altered by protein engineering. Protein engineers are increasingly study the structure-function characteristics, engineering attributes, design of computational tools for enzyme engineering, and functional screening processes to improve the design and applications of enzymes. The potent and innovative techniques of enzyme engineering deliver outstanding opportunities for tailoring industrially important enzymes for the versatile production of biochemicals. An overview of the current trends in enzyme engineering is explored with important representative examples.

Metabolic circuits and gene regulators in polyhydroxyalkanoate producing organisms: Intervention strategies for enhanced production.

Worldwide worries upsurge concerning environmental pollutions triggered by the accumulation of plastic wastes. Biopolymers are promising candidates for resolving these difficulties by replacing non-biodegradable plastics. Among biopolymers, polyhydroxyalkanoates (PHAs), are natural polymers that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and other physico-chemical properties comparable to those of synthetic plastics. Consequently, considerable research have been attempted to advance a better understanding of mechanisms related to the metabolic synthesis and characteristics of PHAs and to develop native and recombinant microorganisms that can proficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent developments in metabolic engineering and synthetic biology applied to enhance PHA synthesis include, promoter engineering, ribosome-binding site (RBS) engineering, development of synthetic constructs etc. This review gives a brief overview of metabolic routes and regulators of PHA production and its intervention strategies.

Thermophilic Chitinases: Structural, Functional and Engineering Attributes for Industrial Applications.

Chitin is the second most widely found natural polymer next to cellulose. Chitinases degrade the insoluble chitin to bioactive chitooligomers and monomers for various industrial applications. Based on their function, these enzymes act as biocontrol agents against pathogenic fungi and invasive pests compared with conventional chemical fungicides and insecticides. They have other functional roles in shellfish waste management, fungal protoplast generation, and Single-Cell Protein production. Among the chitinases, thermophilic and thermostable chitinases are gaining popularity in recent years, as they can withstand high temperatures and maintain the enzyme stability for longer periods. Not all chitinases are thermostable; hence, tailor-made thermophilic chitinases are designed to enhance their thermostability by direct evolution, genetic engineering involving mutagenesis, and proteomics approach. Although research has been done extensively on cloning and expression of thermophilic chitinase genes, there are only few papers discussing on the mechanism of chitin degradation using thermophiles. The current review discusses the sources of thermophilic chitinases, improvement of protein stability by gene manipulation, metagenomics approaches, chitin degradation mechanism in thermophiles, and their prospective applications for industrial, agricultural, and pharmaceutical purposes.

Probiotics and gut microbiome - Prospects and challenges in remediating heavy metal toxicity.

The gut microbiome, often referred to as "super organ", comprises up to a hundred trillion microorganisms, and the species diversity may vary from person to person. They perform a decisive role in diverse biological functions related to metabolism, immunity and neurological responses. However, the microbiome is sensitive to environmental pollutants, especially heavy metals. There is continuous interaction between heavy metals and the microbiome. Heavy metal exposure retards the growth and changes the structure of the phyla involved in the gut microbiome. Meanwhile, the gut microbiome tries to detoxify the heavy metals by altering the physiological conditions, intestinal permeability, enhancing enzymes for metabolizing heavy metals. This review summarizes the effect of heavy metals in altering the gut microbiome, the mechanism by which gut microbiota detoxifies heavy metals, diseases developed due to heavy metal-induced dysbiosis of the gut microbiome, and the usage of probiotics along with advancements in developing improved recombinant probiotic strains for the remediation of heavy metal toxicity.

Nanobiocatalysts: Advancements and applications in enzyme technology.

Nanobiocatalysts are one of the most promising biomaterials produced by synergistically integrating advanced biotechnology and nanotechnology. These have a lot of potential to improve enzyme stability, function, efficiencyand engineering performance in bioprocessing. Functional nanostructures have been used to create nanobiocatalystsbecause of their specific physicochemical characteristics and supramolecular nature. This review covers a wide range of nanobiocatalysts including polymeric, metallic, silica and carbon nanocarriers as well as their recent developments in controlling enzyme activity. The enormous potential of nanobiocatalysts in bioprocessing in designing effective laboratory trials forapplications in various fields such as food, pharmaceuticals, biofuel, and bioremediation is also discussed extensively.

Valorization of food and kitchen waste: An integrated strategy adopted for the production of poly-3-hydroxybutyrate, bioethanol, pectinase and 2, 3-butanediol.

The present investigation gives an insight on the potential of food and kitchen waste as a suitable feed stock for the production of biopolymer, biofuels, enzymes and chemicals. Media engineering improved poly-3-hydroxybutyrate (PHB) production from 0.91 g/L to 5.132 g/L. There is a five-fold increase in PHB production. The food and kitchen waste was also evaluated for the production of bioethanol, 2, 3 - butanediol, and pectinase. Saccharomyces cerevisiae produced 0.316 g of bioethanol, Bacillus sonorensis MPTD1 produced 2.47 (µM/mL)/min of pectinase and Enterobacter cloacae SG1 produced 3 g/L of 2, 3-butanediol with a productivity of 0.03 g/L/h using food and kitchen waste as carbon source. Targeting on multiple value added products will improve the overall process economics.

Zerumbone, a Cyclic Sesquiterpene from Zingiber zerumbet Induces Apoptosis, Cell Cycle Arrest, and Antimigratory Effects in SW480 Colorectal Cancer Cells.

Zerumbone isolated from the rhizomes of Zingiber zerumbet was investigated for the mechanisms by which it exhibits antiproliferative activity in colorectal cancer cells (SW480). The results indicated that the zerumbone suppressed cell growth and enhanced cell apoptosis. Exposure to zerumbone induced generation of reactive oxygen species, reduced the cellular antioxidant status, decreased mitochondrial membrane potential, and activated caspase 3, caspase 8, and caspase 9 (p < 0.001). It was also found that there was a decrease in the expression of Bcl 2 and elevation of Bax (p < 0.001) on exposure to zerumbone. Furthermore, treatment with 50, 75, and 100 µM zerumbone resulted in cell cycle arrest at the G2/M phase with a value of 17.2 ± 0.1, 19.63 ± 0.25, and 26.66 ± 0.25, respectively, and also distorted the microfilament network and effectively inhibited cellular migration.