Rebooting life: engineering non-natural nucleic acids, proteins and metabolites in microorganisms.

The surging demand of value-added products has steered the transition of laboratory microbes to microbial cell factories (MCFs) for facilitating production of large quantities of important native and non-native biomolecules. This shift has been possible through rewiring and optimizing different biosynthetic pathways in microbes by exercising frameworks of metabolic engineering and synthetic biology principles. Advances in genome and metabolic engineering have provided a fillip to create novel biomolecules and produce non-natural molecules with multitude of applications. To this end, numerous MCFs have been developed and employed for production of non-natural nucleic acids, proteins and different metabolites to meet various therapeutic, biotechnological and industrial applications. The present review describes recent advances in production of non-natural amino acids, nucleic acids, biofuel candidates and platform chemicals.

Enhanced production of violacein by Chromobacterium violaceum using agro-industrial waste soybean meal.

AIMS: The research is aimed at developing an economic and sustainable growth medium using abundantly available and highly nutritive agro-industrial waste soybean meal as the substrate for the production of violacein by Chromobacterium violaceum. METHODS AND RESULTS: Violacein produced using soybean meal medium was compared with the commercial complex growth media. Upon utilization of 2% w/v soybean meal (SM2 ) medium, 496 mg/L crude violacein was achieved after 48-hr incubation time, which was 1.62-fold higher than the crude violacein produced in Luria-Bertani (LB) broth. Additionally, supplementation of 100 mg/L L-tryptophan to 1% and 2% w/v soybean meal (SMT1 and SMT2 ) medium yielded 1217 mg/L (3.96-fold higher as compared to LB) and 1198 mg/L (3.90-fold higher as compared to LB) crude violacein respectively. Optimization of culture conditions and concentration of L-tryptophan using Box-Behnken design (BBD) model produced as high as 1504.5 mg/L crude violacein. To the best of our knowledge, this is the highest crude violacein produced to date using agro-industrial-based waste as a substrate with minimal supplementation in a shake flask. CONCLUSIONS: The study signifies the potentiality of soybean meal as a cost-effective growth medium for the production of violacein. Optimization of the fermentation parameters clearly demonstrated a surge in violacein production. SIGNIFICANCE AND IMPACT OF THE STUDY: Utilization of soybean meal as an alternative to the expensive commercial media would surely promote the large-scale synthesis of this multifaceted compound.

Synergistic bactericidal profiling of prodigiosin extracted from Serratia marcescens in combination with antibiotics against pathogenic bacteria.

The emergence of multidrug-resistant (MDR) bacteria is on the rise and the situation has been worsening with each passing day, which is evident from the outpouring number of reports about how more and more pathogens are becoming resistant to even the third and fourth generations of antibiotics. Lately, combination therapies or drug synergy have been giving promising results in curbing infections since it delineates its action on multiple aspects as compared to monotherapies. In this study, we used prodigiosin, a bacterial pigment endowed with magnificent biological properties, in combination with six antibiotics to study its effect on Pseudomonas aeruginosa, Staphylococcus aureus and Chromobacterium violaceum. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of prodigiosin against the test organisms was determined and a checkerboard assay of prodigiosin with various antibiotic combinations was performed with an aim to abate antimicrobial resistance. MIC and MBC of prodigiosin was obtained in the range of 4-16 µg/mL, which was lower than that of most test antibiotics. Coupling prodigiosin with other test antibiotics exhibited an excellent synergy profile against all test organisms and the effects were reported to be either synergistic or additive. In the case of S. aureus and C. violaceum, all combinations were found to be synergic, and remarkably for S. aureus, FBC index was reported to be as low as ≤0.25 with all of the test antibiotics. Therefore, it is deduced that prodigiosin augments and intensifies the action of antibiotics, and results in a double-whammy against the MDR strains.

New insight into the control of peptic ulcer by targeting the histamine H2 receptor.

Peptic ulcer disease is one of the major challenges in public health globally and new evidence shows that it can be controlled by targeting the histamine H2 receptor (H2 R). Recently, a number of H2 R antagonists have been synthesized and used to block the action of histamine on the parietal cells in the stomach and decrease the acid production. In this study, we modeled the H2 R by homology modeling using the 3-D crystal structure and this model was validated based on free energy and amino acid residues present in the allowed regions of a Ramachandran plot. We used this 3-D model for screening of highly potent drugs using molecular docking. We found cimetidine, cimetex, and famotidine as the most potent drugs based on the binding affinity of drug-protein interactions. We also generated a cellular network for H2 R that could be useful for better understanding of cellular mechanism and drug targets. These findings provide a new insight into the development of suitable, specific, and effective anti-ulcer drugs for a most effective treatment of ulcerous diseases.

Synthetic small RNAs: Current status, challenges, and opportunities.

Small regulatory RNAs act at the levels of transcription, posttranscription, and translation, with numerous roles that include binding to protein targets, protein modification, binding to messenger RNA targets, and regulation of gene expression. We discuss the development of a number of riboregulators and riboswitches, highlighting their use in metabolic engineering and genetic control. Riboregulators and riboswitches are self-assembled RNA molecules that can dynamically change their conformation, acting as regulatory switches that affect biological changes. They have currently been designed, characterized, and implemented in a wide range of organisms and cell types, including bacteria, yeast, and mammalian cells. We have identified and examined the recent advances in RNA synthetic biology, underlining the potential future development of their use and capabilities, noting how these can be ultimately expanded and improved into novel biotechnological, biomedical, and industrial applications.

Recent Advances in CRISPR-Cas9 Genome Editing Technology for Biological and Biomedical Investigations.

The Type II CRISPR-Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single-mutation genetic diseases, and multiple-site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR-Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81-94, 2018. © 2017 Wiley Periodicals, Inc.

Exploring the potential of phage and their applications.

Antibiotic resistant microorganisms are significantly increasing due to horizontal gene transfer, mutation and overdose of antibiotics leading to serious health conditions globally. Several multidrug resistant microorganisms have shown resistance to even the last line of antibiotics making it very difficult to treat them. Besides using antibiotics, an alternative approach to treat such resistant bacterial pathogens through the use of bacteriophage (phage) was used in the early 1900s which however declined and vanished after the discovery of antibiotics. In recent times, phage has emerged and gained interest as an alternative approach to antibiotics to treat MDR pathogens. Phage can self-replicate by utilizing cellular machinery of bacterial host by following lytic and lysogenic life cycles and therefore suitable for rapid regeneration. Application of phage for detection of bacterial pathogens, elimination of bacteria, agents for controlling food spoilage, treating human disease and several others entitles phage as a futuristic antibacterial armamentarium.

Microalgal lutein: Advancements in production, extraction, market potential, and applications.

Lutein, a bioactive xanthophyll, has recently attracted significant attention for numerous health benefits, e.g., protection of eye health, macular degeneration, and acute and chronic syndromes etc. Microalgae have emerged as the best platform for high-value lutein production with high productivity, lutein content, and scale-up potential. Algal lutein possesses numerous bioactivities, hence widely used in pharmaceuticals, nutraceuticals, aquaculture, cosmetics, etc. This review highlights advances in upstream lutein production enhancement and feasible downstream extraction and cell disruption techniques for a large-scale lutein biorefinery. Besides bioprocess-related advances, possible solutions for existing production challenges in microalgae-based lutein biorefinery, market potential, and emerging commercial scopes of lutein and its potential health applications are also discussed. The key enzymes involved in the lutein biosynthesizing Methyl-Erythritol-phosphate (MEP) pathway have been briefly described. This review provides a comprehensive updates on lutein research advancements covering scalable upstream and downstream production strategies and potential applications for researchers and industrialists.

Low density lipoprotein receptor endocytosis in cardiovascular disease and the factors affecting LDL levels.

Cardiovascular disease (CVD) is the one of major global health issues with approximately 30% of the mortality reported in the mid-income population. Low-density lipoprotein (LDL) plays a crucial role in development of CVD. High LDL along with others forms a plaque and blocks arteries, resulting in CVD. The present chapter deals with the mechanism of receptor-mediated endocytosis of LDL and its management by drugs such as statins and PCSK9 inhibitors along with dietary supplementation for health improvements.

Amyloid precursor protein in Alzheimer's disease.

Amyloid precursor protein (APP) is a membrane protein expressed in several tissues. The occurrence of APP is predominant in synapses of nerve cells. It acts as a cell surface receptor and plays a vital role as a regulator of synapse formation, iron export and neural plasticity. It is encoded by the APP gene that is regulated by substrate presentation. APP is a precursor protein activated by proteolytic cleavage and thereby generating amyloid beta (Aß) peptides which eventually form amyloid plaques that accumulate in Alzheimer's disease patients' brains. In this chapter, we highlight basic mechanism, structure, expression patterns and cleavage of amyloid plaques, and its diagnosis and potential treatment for Alzheimer's disease.

CRISPR-dCas9 system for epigenetic editing towards therapeutic applications.

In the past few decades, epigenetics has emerged as an important area of study to enable a better understanding of gene expression and its regulation. Due to epigenetics, stable phenotypic changes have been possible without alterations in DNA sequences. Epigenetic changes may occur due to DNA methylation, acetylation, phosphorylation and other such mechanisms which alter the level of gene expression without making any difference to DNA sequences. In this chapter, CRISPR-dCas9 used to bring about epigenome modifications for regulating gene expression towards a therapeutic approaches for treating human diseases have been discussed.

Rewiring of metabolic pathways in yeasts for sustainable production of biofuels.

The ever-increasing global energy demand has led world towards negative repercussions such as depletion of fossil fuels, pollution, global warming and climate change. Designing microbial cell factories for the sustainable production of biofuels is therefore an active area of research. Different yeast cells have been successfully engineered using synthetic biology and metabolic engineering approaches for the production of various biofuels. In the present article, recent advancements in genetic engineering strategies for production of bioalcohols, isoprenoid-based biofuels and biodiesels in different yeast chassis designs are reviewed, along with challenges that must be overcome for efficient and high titre production of biofuels.

Advanced approaches for resource recovery from wastewater and activated sludge: A review.

Due to resource scarcity, current industrial systems are switching from waste treatment, such as wastewater treatment and biomass, to resource recovery (RR). Biofuels, manure, pesticides, organic acids, and other bioproducts with a great market value can be produced from wastewater and activated sludge (AS). This will not only help in the transition from a linear economy to a circular economy, but also contribute to sustainable development. However, the cost of recovering resources from wastewater and AS to produce value-added products is quite high as compared to conventional treatment methods. In addition, most antioxidant technologies remain at the laboratory scale that have not yet reached the level at industrial scale. In order to promote the innovation of resource recovery technology, the various methods of treating wastewater and AS to produce biofuels, nutrients and energy are reviewed, including biochemistry, thermochemistry and chemical stabilization. The limitations of wastewater and AS treatment methods are prospected from biochemical characteristics, economic and environmental factors. The biofuels derived from third generation feedstocks, such as wastewater are more sustainable. Microalgal biomass are being used to produce biodiesel, bioethanol, biohydrogen, biogas, biooils, bioplastics, biofertilizers, biochar and biopesticides. New technologies and policies can promote a circular economy based on biological materials.

Phage engineering and phage-assisted CRISPR-Cas delivery to combat multidrug-resistant pathogens.

Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.

Design and fabrication of microfluidics devices for molecular biology applications.

In the past decade, microfluidics has emerged as a rapidly growing area with potential to reduce cost and reagent consumption. It has been used for detection of nucleic acids and high-throughput screening of cells and metabolites. It is extensively used for extraction of DNA, RNA, proteins, biomolecules, as well as for cloning and transformation of plasmid into cells. Microfluidics is made up of polydimethylsiloxane (PDMS) polymer which is transparent and is used for preparation of a wide range of devices and systems. In this chapter, we discuss advances and challenges of using microfluidics in molecular biology and its biomedical applications.

Microfluidics based point-of-care for disease diagnostics.

Microfluidics platform is widely used for several basic biological to advanced biotechnological applications. It reduces the expenditure of reagent consumption by readily reducing the volume of the reaction system. It is being used for early diagnosis of diseases, detection of pathogens, cancer markers, high-throughput screening and many such applications. Currently, microfluidics and lab-on-chip is integrated together with sample preparation, extraction, analysis and detection of biomarkers for disease diagnosis. This technology offers low-cost, rapid, sensitive and paper-based lateral flow mode of detection which is user-friendly and scalable. In this chapter, we highlight recent developments in microfluidics platform for disease diagnosis.

Microfluidics device for drug discovery, screening and delivery.

Microfluidics and lab-on-chip are two progressive technologies widely used for drug discovery, screening and delivery. It has been designed in a way to act as a platform for sample preparations, culturing, incubation and screening through multi-channels. These devices require a small amount of reagent in about micro- to nanolitre volume. Microfluidics has the capacity to perform operations in a programmable manner and is easy to fine tune the size, shape and composition of drugs by changing flow rate and precise manipulations. Microfluidics platform comes with the advantage of mixing fluid in droplet reactors. Microfluidics is used in the field of chemistry, biomedical, biology and nanotechnology due to its high-throughput performance in various assays. It is potent enough to be used in microreactors for synthesis of particles and encapsulation of many biological entities for biological and drug delivery applications. Microfluidics therefore has the scope to be uplifted from basic to advanced diagnostic and therapeutic applications.

Microfluidics for single cell analysis.

Cells have several internal molecules that are present in low amounts and any fluctuation in its number drives a change in cell behavior. These molecules present inside the cells are continuously fluctuating, thus producing noises in the intrinsic environment and thereby directly affecting the cellular behavior. Single-cell analysis using microfluidics is an important tool for monitoring cell behavior by analyzing internal molecules. Several gene circuits have been designed for this purpose that are labeled with fluorescence encoding genes for monitoring cell dynamics and behavior. We discuss herewith designed and fabricated microfluidics devices that are used for trapping and tracking cells under controlled environmental conditions. This chapter highlights microfluidics chip for monitoring cells to promote their basic understanding.

Gut microbiota in gastrointestinal diseases.

Gut microbiota is a highly dense population of different kinds of bacteria residing in the gut which co-evolves with the host. It engages in a number of metabolic and immunological activities. Gut microbiota is associated with maintenance of health, and unbalanced microbiota contributes in the development of several diseases. Alteration of beneficial gut microbiota population triggers gastrointestinal diseases including irritable bowel syndrome, inflammatory bowel disease, celiac disease, colorectal cancer, and many others. Gut microbiota can be affected by multiple factors such as diet, stress, genetic variations. In this chapter, we highlight how gut microbiota plays a key role in pathogenesis of gastrointestinal disease.

Advances in microfluidics devices and its applications in personalized medicines.

Microfluidics is an exponentially growing area and is being used for numerous applications from basic science to advanced biotechnology and medicines. Microfluidics provides a platform to the research community for studying and building new strategies for the diagnosis and therapeutics applications. In the last decade, microfluidic have enriched the field of diagnostics by providing new solutions which was not possible with conventional detection and treatment methods. Microfluidics has the ability to precisely control and perform high-throughput functions. It has been proven as an efficient and rapid method for biological sample preparation, analysis and controlled drug delivery system. Microfluidics plays significant role in personalized medicine. These personalized medicines are used for medical decisions, practices and other interventions as well as for individual patients based on their predicted response or risk of disease. This chapter highlights microfluidics in developing personalized medical applications for its applications in diseases such as cancer, cardiovascular disease, diabetes, pulmonary disease and several others.