Escherichia coli and Pichia pastoris: microbial cell-factory platform for -full-length IgG production.

Owing to the unmet demand, the pharmaceutical industry is investigating an alternative host to mammalian cells to produce antibodies for a variety of therapeutic and research applications. Regardless of some disadvantages, Escherichia coli and Pichia pastoris are the preferred microbial hosts for antibody production. Despite the fact that the production of full-length antibodies has been successfully demonstrated in E. coli, which has mostly been used to produce antibody fragments, such as: antigen-binding fragments (Fab), single-chain fragment variable (scFv), and nanobodies. In contrast, Pichia, a eukaryotic microbial host, is mostly used to produce glycosylated full-length antibodies, though hypermannosylated glycan is a major challenge. Advanced strategies, such as the introduction of human-like glycosylation in endotoxin-edited E. coli and cell-free system-based glycosylation, are making progress in creating human-like glycosylation profiles of antibodies in these microbes. This review begins by explaining the structural and functional requirements of antibodies and continues by describing and analyzing the potential of E. coli and P. pastoris as hosts for providing a favorable environment to create a fully functional antibody. In addition, authors compare these microbes on certain features and predict their future in antibody production. Briefly, this review analyzes, compares, and highlights E. coli and P. pastoris as potential hosts for antibody production.

Normal measurements of brainstem and related structures for all ages: An MRI-based morphometric study.

Background: Knowledge of the normal size of the brain stem and certain related structures play an important role in diagnosis of aging and neurodegenerative conditions affecting the brain. There is no well-established normative data for development and age-related changes pertaining to the brain stem and related structures in the Indian context. The objective of this study was to assess various linear and angle measurements of the brain stem, cerebral peduncles, middle cerebellar peduncles, and proximal cervical cord for all ages in patients who have undergone MRI brain for unrelated pathologies. Methods: A record-based retrospective cross-sectional and analytical study. T1WI axial and sagittal images were studied for the following variables: Cerebral peduncle transverse thickness, Interpeduncular angle, Middle cerebellar peduncle transverse thickness, Ventral midbrain anteroposterior thickness, Midbrain height, Mamillopontine distance (MPD), Pontomesencephalic angle (PMA), Pons anteroposterior thickness, Medulla anteroposterior thickness and Spinal cord anteroposterior diameter. Results: Significant differences (p = 0.001) were observed in nearly all the variables among various age groups. Males demonstrated significantly higher mean values (at 5% level of significance) for a majority of the variables. Most of the variables measured, e.g. Cerebral peduncle, Middle cerebellar peduncle, Ventral midbrain thickness, Midbrain height, Pons, Medulla, and Spinal Cord diameter, showed a steady and sharp increase in values from infancy and reached maximum values during the third decade, followed by a variable degree of decline in values. Conclusion: Magnetic Resonance Imaging (MRI) morphometry of brainstem and related structures is easily doable and is also reproducible. Present study lays down normative data for the brainstem and certain related structures for all ages, which can be referred to in day-to-day practice.

Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production.

This study explores the ability of methanotrophs to convert biogas into biopolymers, addressing H2S as a limitation in the utilization of biogas as a carbon source for bioconversion. Transcriptomic analysis was conducted to understand the growth and changes in the expression patterns of Type I and II methanotrophs under varying H2S concentrations. Results suggested that Type II methanotrophs can possess a native H2S utilization pathway. Both Type I and II methanotrophs were evaluated for their growth and polyhydroxybutyrate (PHB) production from biogas. Methylocystis sp. MJC1 and Methylocystis sp. OK1 exhibited a maximum biomass production of 4.0 and 4.5 gDCW/L, respectively, in fed-batch culture, aligning with the transcriptome data. Furthermore, Methylocystis sp. MJC1 produced 2.9 g PHB/L from biogas through gas fermentation. These findings underscore biogas-based biotechnology as an innovative solution for environmental and industrial challenges with further optimization and productivity enhancement research expected to broaden the potential in this field.

Methanotrophs as a reservoir for bioactive secondary metabolites: Pitfalls, insights and promises.

Methanotrophs are potent natural producers of several bioactive secondary metabolites (SMs) including isoprenoids, polymers, peptides, and vitamins. Cryptic biosynthetic gene clusters identified from these microbes via genome mining hinted at the vast and hidden SM biosynthetic potential of these microbes. Central carbon metabolism in methanotrophs offers rare pathway intermediate pools that could be further diversified using advanced synthetic biology tools to produce valuable SMs; for example, plant polyketides, rare carotenoids, and fatty acid-derived SMs. Recent advances in pathway reconstruction and production of isoprenoids, squalene, ectoine, polyhydroxyalkanoate copolymer, cadaverine, indigo, and shinorine serve as proof-of-concept. This review provides theoretical guidance for developing methanotrophs as microbial chassis for high-value SMs. We summarize the distinct secondary metabolic potentials of type I and type II methanotrophs, with specific attention to products relevant to biomedical applications. This review also includes native and non-native SMs from methanotrophs, their therapeutic potential, strategies to induce silent biosynthetic gene clusters, and challenges.

Biosynthesis of chiral diols from alkenes using metabolically engineered type II methanotroph.

Methanotrophs are environmentally friendly microorganisms capable of converting gas to liquid using methane monooxygenases (MMOs). In addition to methane-to-methanol conversion, MMOs catalyze the conversion of alkanes to alcohols and alkenes to epoxides. Herein, the efficacy of epoxidation by type I and II methanotrophs was investigated, and type II methanotrophs were observed to be more efficient in converting alkenes to epoxides. Subsequently, three (Epoxide hydrolase) EHs of different origins were overexpressed in the type II methanotroph Methylosinus trichosporium OB3b to produce 1,2-diols from epoxide. Methylosinus trichosporium OB3b expressing Caulobacter crescentus EH produced the highest amount of (R)-1,2-propanediol (251.5 mg/L) from 1-propene. These results demonstrate the possibility of using methanotrophs as a microbial platform for diol production and the development of a continuous bioreactor for industrial applications.

Thermostable phytase in feed and fuel industries.

Phytase with wide ranging biochemical properties has long been utilized in a multitude of industries, even so, thermostability plays a crucial factor in choosing the right phytase in a few of the sectors. Mesophilic phytases are not considered to be a viable option in the feed industry owing to its limited stability in the required feed processing temperature. In the recent past, inclusion of thermostable phytase in fuel ethanol production from starch based raw material has been demonstrated with economic benefits. Therefore, considerable emphasis has been placed on using complementary approaches such as mining of extremophilic microbial wealth, encapsulation and using enzyme engineering for obtaining stable phytase variants. This article means to give an insight on role of thermostable phytases in feed and fuel industries and methods for its development, highlighting molecular determinants of thermostability.

A substrate-based approach for the selection of oil-bearing heterotrophs from nitrogen-deficient soil for lipid production.

In this study, nine heterotrophic yeast isolates were tested for their ability to assimilate crude glycerol and consecutive conversion to triacylglycerides (TGAs). All the organisms were initially screened on crude glycerol-based selection media, and those producing lipid globules were further evaluated for lipid production. Sudan Black B staining of eight isolates showed lipid globules. These strains were further studied at different C/N ratio. The molecular identification revealed that the isolates belonged to the genera of Yarrowia and Candida. Among these isolates, SKY7 (Yarrowia lipolytica) produced up to 42.04 ± 0.11 % of lipid w/w) with a C/N ratio of 100 and fermentation time of 72 h. The other strains produced 5.82 ± 0.4 to 34.57 ± 0.44 % lipid (w/w). The GC-flame ionization detector (FID) lipid profile showed that the lipid produced by the strains had close resemblance with vegetable oil and could serve as a feedstock for biodiesel production. Biolog test of the isolates revealed a wide spectrum of carbon utilization.

Lipoglycans contribute to innate immune detection of mycobacteria.

Innate immune recognition is based on the detection, by pattern recognition receptors (PRRs), of molecular structures that are unique to microorganisms. Lipoglycans are macromolecules specific to the cell envelope of mycobacteria and related genera. They have been described to be ligands, as purified molecules, of several PRRs, including the C-type lectins Mannose Receptor and DC-SIGN, as well as TLR2. However, whether they are really sensed by these receptors in the context of a bacterium infection remains unclear. To address this question, we used the model organism Mycobacterium smegmatis to generate mutants altered for the production of lipoglycans. Since their biosynthesis cannot be fully abrogated, we manipulated the biosynthesis pathway of GDP-Mannose to obtain some strains with either augmented (∼1.7 fold) or reduced (∼2 fold) production of lipoglycans. Interestingly, infection experiments demonstrated a direct correlation between the amount of lipoglycans in the bacterial cell envelope on one hand and the magnitude of innate immune signaling in TLR2 reporter cells, monocyte/macrophage THP-1 cell line and human dendritic cells, as revealed by NF-κB activation and IL-8 production, on the other hand. These data establish that lipoglycans are bona fide Microbe-Associated Molecular Patterns contributing to innate immune detection of mycobacteria, via TLR2 among other PRRs.

Purification and biochemical characterization of methionine aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155.

The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.