Insulin evolution: A holistic view of recombinant production advancements.

The increased prevalence of diabetes and the growing popularity of non-invasive methods of recombinant human insulin uptake, such as oral insulin, have increased insulin demand, further limiting the affordability of insulin. Over 40 years have passed since the development of engineered microorganisms that replaced the animal pancreas as the primary source of insulin. To stay ahead of the need for insulin in the present and the future, a few drawbacks with the existing expression systems need to be alleviated, including the inclusion body formation, the use of toxic inducers, and high process costs. To address these bottlenecks and improve insulin production, a variety of techniques are being used in bacteria, yeasts, transgenic plants and animals, mammalian cell lines, and cell-free expression systems. Different approaches for the production of insulin, including two-chain, proinsulin or mini-proinsulin, preproinsulin coupled with fusion protein, chaperone, signal peptide, and purification tags, are explored in upstream, whereas downstream processing takes into account the recovery of intact protein in its bioactive form and purity. This article focuses on the strategies used in the upstream and downstream phases of the bioprocess to produce recombinant human insulin. This review also covers a range of analytical methods and tools employed in investigating the genuity of recombinant human insulin.

diaPASEF Enables High-Throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development.

Monitoring and quantifying host cell proteins (HCPs) in biotherapeutic production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs, such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.

Sustainable biorefinery approach by utilizing xylose fraction of lignocellulosic biomass.

Lignocellulosic biomass (LCB) has been a lucrative feedstock for developing biochemical products due to its rich organic content, low carbon footprint and abundant accessibility. The recalcitrant nature of this feedstock is a foremost bottleneck. It needs suitable pretreatment techniques to achieve a high yield of sugar fractions such as glucose and xylose with low inhibitory components. Cellulosic sugars are commonly used for the bio-manufacturing process, and the xylose sugar, which is predominant in the hemicellulosic fraction, is rejected as most cell factories lack the five­carbon metabolic pathways. In the present review, more emphasis was placed on the efficient pretreatment techniques developed for disintegrating LCB and enhancing xylose sugars. Further, the transformation of the xylose to value-added products through chemo-catalytic routes was highlighted. In addition, the review also recapitulates the sustainable production of biochemicals by native xylose assimilating microbes and engineering the metabolic pathway to ameliorate biomanufacturing using xylose as the sole carbon source. Overall, this review will give an edge on the bioprocessing of microbial metabolism for the efficient utilization of xylose in the LCB.

Recombinant monoclonal antibody production in yeasts: Challenges and considerations.

Monoclonal antibodies (mAbs) are laboratory-based engineered protein molecules with a monovalent affinity or multivalent avidity towards a specific target or antigen, which can mimic natural antibodies that are produced in the human immune systems to fight against detrimental pathogens. The recombinant mAb is one of the most effective classes of biopharmaceuticals produced in vitro by cloning and expressing synthetic antibody genes in a suitable host. Yeast is one of the potential hosts among others for the successful production of recombinant mAbs. However, there are very few yeast-derived mAbs that got the approval of the regulatory agencies for direct use for treatment purposes. Certain challenges encountered by yeasts for recombinant antibody productions need to be overcome and a few considerations related to antibody structure, host engineering, and culturing strategies should be followed for the improved production of mAbs in yeasts. In this review, the drawbacks related to the metabolic burden of the host, culturing conditions including induction mechanism and secretion efficiency, solubility and stability, downstream processing, and the pharmacokinetic behavior of the antibody are discussed, which will help in developing the yeast hosts for the efficient production of recombinant mAbs.

Insights into the management of food waste in developing countries: with special reference to India.

Up to one third of the food that is purposely grown for human sustenance is wasted and never consumed, with adverse consequences for the environment and socio-economic aspects. In India, managing food waste is a significant environmental concern. Food waste output is increasing in Indian cities and towns as a result of the country's urban expansion, modernization, and population growth. Poor management of food waste can have negative consequences for the environment and pose a risk to the public's health issues. This review focuses on the current challenges, management strategies, and future perspectives of food waste management in India. The efficient management of food waste involves a comprehensive study regarding the characterization of food waste and improved waste management methods. In addition, the government policies and rules for managing food waste that is in effect in India are covered in this review.

Role of Elevated Ozone on Development and Metabolite Contents of Lemongrass [Cymbopogon flexuosus (Steud.) (Wats.)].

The present study was conducted to assess the effect of elevated ozone stress on the development and metabolite contents of lemongrass, a medicinal plant. The experimental plant was exposed to two elevated ozone concentrations (ambient + 15 ppb, and ambient + 30 ppb) using open-top chambers. Samplings were carried out at 45 and 90 days after transplantation (DAT), for the analysis of different characteristics, while the metabolite contents of leaves and essential oils were analyzed at 110 DAT. Both the doses of elevated ozone had notable negative effects on the carbon fixation efficiency of plants, resulting in a significant reduction in plant biomass. Enzymatic antioxidant activity increased during the second sampling, which suggests that the scavenging of reactive oxygen species was more prominent in lemongrass during the later developmental stage. The results of the present study showed a stimulated diversion of resources towards the phenylpropanoid pathway, which is made evident by the increase in the number and contents of metabolites in foliar extract and essential oils of plants grown at elevated ozone doses, as compared to ambient ozone. Elevated ozone not only upregulated the contents of medicinally important components of lemongrass, it also induced the formation of some pharmaceutically active bio compounds. On the basis of this study, it is expected that increasing ozone concentrations in near future will enhance the medicinal value of lemongrass. However, more experiments are required to validate these findings.

Combating ozone stress through N fertilization: A case study of Indian bean (Dolichos lablab L.).

The present study investigates the efficiency of nitrogen (N) amendments in the management of ozone (O3) stress in two varieties (Kashi Sheetal and Kashi Harittima) of Indian bean (Dolichos lablab L.). Two O3 concentrations, ambient (44.9 ppb) and elevated (74.64 ppb) were used, and each O3 concentration has 3 nitrogen (N) dose treatments viz recommended (N1), 1.5 times recommended (N2), 2 times recommended (N3) and no nitrogen, which served as control (C). The experiment concluded Kashi Sheetal as O3 tolerant, as compared to Kashi Harittima. N amendments were effective in the partial amelioration of O3 stress, with N2 being the most effective nitrogen dose, at both ambient and elevated O3 concentrations. Kashi Sheetal has been determined to be O3 tolerant due to greater endogenous levels of H2O2 accumulation and enzymatic antioxidant contents with O3 exposure. The O3-sensitive variety, Kashi Harittima, responded more positively to N treatments, at both O3 concentrations. The positive effect of N amendments is attributed to the stimulated antioxidative enzyme activity, rather than the biophysical processes like stomatal conductance. Strengthened defense upon N amendments was attributed to the enhanced activities of APX and GR in Kashi Sheetal, while in Kashi Harittima, the two enzymes (APX and GR) were coupled by SOD and CAT as well, during the reproductive phase. Yield (weight of seeds plant-1) increments upon N (N2) amendments were higher in Kashi Harittima (O3 sensitive), as compared to Kashi Sheetal (O3 tolerant) at both ambient and elevated O3 concentration, due to higher antioxidant enzymatic response and greater rate of photosynthesis in the former.

Heavy Metal Contamination in the Aquatic Ecosystem: Toxicity and Its Remediation Using Eco-Friendly Approaches.

Urbanization and industrialization are responsible for environmental contamination in the air, water, and soil. These activities also generate large amounts of heavy metal ions in the environment, and these contaminants cause various types of health issues in humans and other animals. Hexavalent chromium, lead, and cadmium are toxic heavy metal ions that come into the environment through several industrial processes, such as tanning, electroplating, coal mining, agricultural activities, the steel industry, and chrome plating. Several physical and chemical methods are generally used for the heavy metal decontamination of wastewater. These methods have some disadvantages, including the generation of secondary toxic sludge and high operational costs. Hence, there is a need to develop a cost-effective and eco-friendly method for the removal of heavy metal ions from polluted areas. Biological methods are generally considered eco-friendly and cost-effective. This review focuses on heavy metal contamination, its toxicity, and eco-friendly approaches for the removal of heavy metals from contaminated sites.

Current status, and the developments of hosts and expression systems for the production of recombinant human cytokines.

Cytokines consist of peptides, proteins and glycoproteins, which are biological signaling molecules, and boost cell-cell communication in immune reactions to stimulate cellular movements in the place of trauma, inflammation and infection. Recombinant cytokines are designed in such a way that they have generalized immunostimulation action or stimulate specific immune cells when the body encounters immunosuppressive signals from exogenous pathogens or other tumor microenvironments. Recombinant cytokines have improved the treatment processes for numerous diseases. They are also beneficial against novel toxicities that arise due to pharmacologic immunostimulators that lead to an imbalance in the regulation of cytokine. So, the production and use of recombinant human cytokines as therapeutic proteins are significant for medical treatment purposes. For the improved production of recombinant human cytokines, the development of host cells such as bacteria, yeast, fungi, insect, mammal and transgenic plants, and the specific expression systems for individual hosts is necessary. The recent advancements in the field of genetic engineering are beneficial for easy and efficient genetic manipulations for hosts as well as expression cassettes. The use of metabolic engineering and systems biology approaches have tremendous applications in recombinant protein production by generating mathematical models, and analyzing complex biological networks and metabolic pathways via simulations to understand the interconnections between metabolites and genetic behaviors. Further, the bioprocess developments and the optimization of cell culture conditions would enhance recombinant cytokines productivity on large scales.

Deletion of the N-Terminal Domain of Yeast Eukaryotic Initiation Factor 4B Reprograms Translation and Reduces Growth in Urea.

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA recruitment. Recent evidence indicates yeast mRNAs have variable dependence on eIF4B under optimal growth conditions. Given the ability of eIF4B to promote translation as a function of nutrient conditions in mammalian cells, we wondered if eIF4B activities in translation could alter phenotypes in yeast through differential mRNA selection for translation. Here we compared the effects of disrupting yeast eIF4B RNA- and 40S-binding motifs under ∼1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but the 40S-binding N-terminal Domain (NTD) promoted growth in response to stressors requiring robust cellular integrity. In particular, the NTD conferred a strong growth advantage in the presence of urea, which may be important for pathogenesis of related fungal species. Ribosome profiling indicated that similar to complete eIF4B deletion, deletion of the NTD dramatically reduced translation, particularly of those mRNAs with long and highly structured 5-prime untranslated regions. This behavior was observed both with and without urea exposure, but the specific mRNA pool associated with ribosomes in response to urea differed. Deletion of the NTD led to relative increases in ribosome association of shorter transcripts with higher dependence on eIF4G, as was noted previously for eIF4B deletion. Gene ontology analysis indicated that proteins encoded by eIF4B NTD-dependent transcripts were associated with the cellular membrane system and the cell wall, while NTD-independent transcripts encoded proteins associated with cytoplasmic proteins and protein synthesis. This analysis highlighted the difference in structure content of mRNAs encoding membrane versus cytoplasmic housekeeping proteins and the variable reliance of specific gene ontology classes on various initiation factors promoting otherwise similar functions. Together our analyses suggest that deletion of the eIF4B NTD prevents cellular stress responses by affecting the capacity to translate a diverse mRNA pool.

Reconstitution and analyses of RNA interactions with eukaryotic translation initiation factors and ribosomal preinitiation complexes.

Control of translation initiation plays a critical role in the regulation of gene expression in all organisms, yet the mechanics of translation initiation in eukaryotic organisms are not well understood. Confounding studies of translation are the large number and overlapping functions of many initiation factors in cells, and a lack of cap-dependence in many in vitro systems. To shed light on intricate mechanisms that are often obscured in vivo, we use a fully reconstituted translation initiation system for analyzing RNA interactions with eukaryotic translation initiation factors and complexes from the model organism Saccharomyces cerevisiae. This system exhibits strong cap dependence, and dependence on translation factors varies with mRNA 5' UTR sequences as expected from genome-wide studies of translation. Here we provide optimized protocols for purification and analysis of the effects of labeled and unlabeled mRNA recruitment factors on both the rate and factor dependence of mRNA recruitment to the translation preinitiation complex in response to RNA sequence- and structure-changes. In addition to providing streamlined and detailed protocols, we describe a new construct for purification of higher yields of fluorescently labeled and unlabeled full-length eIF4G.

CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper-Division Undergraduate Laboratory Course.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society. Toward this goal, we developed a new hands-on laboratory exercise incorporating the use of CRISPR-Cas9 and homology directed repair (HDR) to edit two well-characterized genes in the budding yeast, Saccharomyces cerevisiae. The two genes edited in this exercise, Adenine2 (ADE2) and Sterile12 (STE12) affect metabolic and developmental processes, respectively. Editing the premature stop codons in these genes results in clearly identifiable phenotypes that can be assessed by students in a standard laboratory course setting. Making use of this basic eukaryotic model organism facilitates a laboratory exercise that is inexpensive, simple to organize, set up, and present to students. This exercise enables undergraduate students to initiate and follow-up on all stages of the CRISPR gene editing process, from identification of guide RNAs, amplification of an appropriate HDR fragment, and analysis of mutant phenotypes. The organization of this protocol also allows for easy modification, providing additional options for editing any expressed genes within the yeast genome to produce new mutations, or recovery of existing mutants to wild type. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):592-601, 2018.